HK1219542B - Imaging systems, cassettes, and methods of using the same - Google Patents

Description

Imaging system, cartridge, and method of using the same

Technical Field

The present application relates to the field of imaging and, more particularly, to imaging systems, slide cassettes, and methods of processing biological samples on slides.

Background Art

Molecular imaging to identify changes in cellular structures that indicate disease is often crucial for a better understanding of medical science. Microscopy applications can be found in microbiology (e.g., Gram staining, etc.), plant tissue culture, animal cell culture (e.g., phase contrast microscopy, etc.), molecular biology, immunology (e.g., ELISA, etc.), cell biology (e.g., immunofluorescence, chromosome analysis, etc.), confocal microscopy, time-lapse and live cell imaging, frame-by-frame imaging, and three-dimensional imaging.

In manual microscopy, a sample carrier slide is manually loaded into a microscope and viewed through the eyepiece of the microscope. In medical applications, pathologists can view cell characteristics, infected cells compared to the count of uninfected cells or other characteristics of the sample. Due to the processing time of each slide, analyzing a large number of samples is often time-consuming. Long processing time may delay accurate diagnosis and treatment. Additionally, conventional microscopes often cannot capture high-resolution and high-quality images that are suitable for archiving and use later.

In automatic microscopy methods, digital images are often collected, viewed on high-resolution monitors, shared, and archived for later use. Unfortunately, conventional automatic slide handling systems often make mistakes, including misalignment of slide mishandling, microscope slides, and optical components (e.g., cameras, imaging capture devices, etc.), and breakage of microscope slides. By way of example, conventional reliable equipment often clamps the label end of a microscope slide. Exposed residual glue may exist at the label. If the holder contacts the edge of the slide adjacent to the label or the edge of the label, the exposed residual glue may stick to or adhere to the holder. This may cause the mishandling of the slide.

Summary of the Invention

In some embodiments, a microscope slide holding box includes a body, a plurality of shelves, and a plurality of support members. The body is configured to surround and protect the microscope slide and includes a first sidewall and a second sidewall. The shelves are capable of supporting the microscope slide and are positioned between the first and second sidewalls. When the microscope slide holding box is in an upright orientation, the shelves can be vertically separated from each other. The support members extend away from the respective shelves and include an elongated body and a clamp. The clamp protrudes upward from the elongated body and is capable of restricting movement of a positioned microscope slide along the elongated body.

In some other embodiments, a method of transporting microscope slides includes: using a pick-up device to pick up the microscope slide without contacting an edge of a label on the microscope slide and without contacting an edge of the microscope slide adjacent to the label. Using the pick-up device to transport the microscope slide to a desired location. In some embodiments, the microscope slide is transported between at least two processing stations (e.g., a staining unit, a coverslipper, an imaging system, an optical scanner, an optical imaging system) without contacting an edge of the label and without contacting an edge of the slide.

In some embodiments, a microscope slide pickup device includes an end effector comprising an elongated planar platform having an upper surface and a lower surface. A fluid line is positioned on the upper side of the platform. A head element includes a connector and a pipette tip. The connector is positioned on the upper side of the platform and coupled to the fluid line. The pipette tip is positioned on the lower side of the platform.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting and non-exhaustive embodiments are described with reference to the following figures. Throughout the various views, unless otherwise specified, like reference numerals refer to like parts or acts.

1 is a schematic illustration of an imaging system of a scanning microscope and/or other scanning device that may include various component devices used in connection with digital pathology sample scanning and imaging, according to various embodiments of the systems described herein.

FIG2 is a schematic diagram showing an imaging device including a focusing system according to an embodiment of the system described herein.

3A and 3B are schematic illustrations of embodiments of control systems illustrating that the control system may include appropriate electronics.

FIG4 is a schematic diagram illustrating a dither focus stage in more detail according to an embodiment of the system described herein.

5A-5E are schematic diagrams illustrating iterations of focusing operations according to the system described herein.

6A is a schematic diagram showing a plot of a command waveform and sharpness determination for dithered focus optics according to an embodiment of the system described herein.

FIG6B is a schematic illustration showing a plot of calculated sharpness ( _Zs ) values for a portion of a sinusoidal wave motion of a dithered lens.

7A and 7B are schematic illustrations showing focus determination and adjustment of a sample (tissue) according to an embodiment of the system described herein.

8 is a schematic diagram showing an example of a sharpness profile including a sharpness curve and a contrast ratio for each sharpness response at a plurality of points sampled by dithered focus optics according to an embodiment of the system described herein.

FIG9 shows a functional control loop block diagram illustrating the use of a control signal to control the contrast function of a slow focus stage.

10 is a schematic diagram showing a focus window split into regions in connection with focus processing according to an embodiment of the system described herein.

11 shows a graphical illustration of different sharpness values achievable at various points in time for an embodiment in accordance with the techniques herein.

12 is a flow chart illustrating dynamic instant focus processing during scanning of a sample under inspection according to an embodiment of the system described herein.

13 is a flow chart illustrating processing at a slow focus stage according to an embodiment of the system described herein.

14 is a flow chart illustrating an image capture process according to an embodiment of the system described herein.

FIG15 is a schematic illustration showing an alternative arrangement for focus processing according to an embodiment of the system described herein.

FIG16 is a schematic illustration showing an alternative arrangement for focus processing according to another embodiment of the system described herein.

17 is a flow chart illustrating a process for acquiring a mosaic image of tissue on a slide according to an embodiment of the system described herein.

18 is a schematic diagram illustrating an implementation of a precision stage (eg, the Y stage portion) of an XY stage according to an embodiment of the system described herein.

19A and 19B are more detailed views of a moving stage block of a precision stage according to an embodiment of the system described herein.

20 illustrates an implementation of an entire XY hybrid stage according to the precision stage features discussed herein and including a Y stage, an X stage, and a base plate according to an embodiment of the system described herein.

Figure 21 is a schematic diagram showing a slide caching device according to an embodiment of the system described herein.

Figure 22A is a flow diagram illustrating slide caching processing associated with a first slide according to an embodiment of the system described herein.

Figure 22B is a flow diagram illustrating slide caching processing associated with a second slide according to an embodiment of the system described herein.

23A and 23B show timing diagrams using slide caching techniques according to embodiments of the systems described herein and illustrate time savings according to various embodiments of the systems described herein.

Figure 24 is a schematic diagram showing a slide caching device according to another embodiment of the system described herein.

25A is a flow chart illustrating slide caching processing associated with a first slide according to an embodiment of the system described for a slide caching device having two XY hybrid stages for slide processing.

25B is a flow chart illustrating slide caching processing associated with a second slide according to an embodiment of the system described for a slide caching device having two XY hybrid stages for slide processing.

Figure 26 is a schematic diagram showing a slide caching device according to another embodiment of the system described herein.

FIG27 is a schematic illustration showing another view of the slide caching device according to FIG26.

Figures 28A-28J are schematic illustrations showing the slide caching operation of the slide caching device of Figures 26 and 27 according to an embodiment of the system described herein.

29 is a schematic diagram showing an illumination system for illuminating a slide using a light emitting diode (LED) illumination assembly according to an embodiment of the systems described herein.

30 is a schematic diagram showing a more detailed view of an embodiment for an LED lighting assembly according to the systems described herein.

31 is a schematic diagram illustrating an exploded view of a particular implementation of an LED lighting assembly according to an embodiment of the systems described herein.

32 is a schematic diagram illustrating a high-speed slide scanning apparatus that may be used in connection with digital pathology imaging according to an embodiment of the system described herein.

33 is a schematic illustration showing in greater detail the recesses on the tray of a high-speed slide scanning device according to an embodiment of the system described herein.

Figure 34 is a schematic illustration showing an imaging path starting at a first radial position relative to the slide for imaging a sample on a slide in a recess.

Figures 35A and 35B are schematic illustrations showing alternative arrangements of slides on a rotating slide holder according to another embodiment of the system described herein.

Figure 36 is a schematic diagram showing an imaging system including an objective lens configured to examine a sample on a slide according to an embodiment of the systems described herein.

Figure 37 is a flow chart illustrating high speed slide scanning using a rotatable tray according to an embodiment of the system described herein.

38 is a schematic diagram illustrating an optical double image system according to an embodiment of the systems described herein.

39A and 39B are schematic illustrations of an optical double image system showing reciprocating motion of a first tube lens and a second tube lens in front of an image sensor according to an embodiment of the system described herein.

40 is a front, top, and left side isometric view of an imaging system in accordance with one embodiment.

[00106] Figure 41 is a front, top, and left side isometric view of the imaging system of Figure 40. The outer protective housing is shown removed.

42 is a rear, top, and right side isometric view of the imaging system of FIG. 40 with the protective housing shown removed.

43 is a top plan view of the imaging system of FIG. 40 .

44 is an isometric view of a pickup device according to one embodiment.

45 is a top plan view of the pickup device of FIG. 44 .

46 is a cross-sectional view of the pickup device taken along line 46-46 of FIG. 45. FIG.

47 is an isometric view of the pickup device of FIG. 44 .

FIG. 48 is a detailed view of the pickup device of FIG. 46 .

Figure 49 is a front, top, right side isometric view of the slide holder assembly.

Figure 50 is a rear, top, left side isometric view of the wafer holder assembly of Figure 49.

Figure 51 is a front elevational view of the wafer holder assembly of Figure 49.

Figure 52 is a cross-sectional view of the slide holder apparatus taken along line 52-52 of Figure 51. The pick apparatus is positioned to insert a slide into the slide holder apparatus.

Figure 53 is a detailed view of the wafer holder device of Figure 52.

Figure 54 is a detailed view of the end of the upper shelf.

Figure 55 is a cross-sectional view of the wafer holder assembly taken along line 53-53 of Figure 51.

Figure 56A is a front elevational view of a slide positioned above the upper shelf of a slide holder assembly.

Figure 56B is a front elevational view of a slide placed on the upper shelf of Figure 56A.

DETAILED DESCRIPTION

FIG1 is a schematic illustration of an imaging system 5 for a scanning microscope and/or other scanning device, which may include various components and devices used in connection with digital pathology sample scanning and imaging, according to various embodiments of the systems described herein. The imaging system 5 may include, among other components and systems 50, an imaging device having a focusing system 10, a stage system 20, a slide buffer system 30, and an illumination system 40, as discussed in further detail elsewhere herein. It should also be noted that the systems described herein may be used in connection with the microscope slide scanning instrument architecture and techniques for image capture, stitching, and magnification described in U.S. Patent Application Publication No. 2008/0240613 A1 to Dietz et al., entitled “Digital Microscope Slide Scanning System and Methods,” which is incorporated herein by reference, including features associated with reconstructing images with magnification and displaying or storing the reconstructed images without a significant loss of accuracy.

FIG2 is a schematic diagram illustrating an imaging device 100 of an optical scanning microscope and/or other suitable imaging system according to an embodiment of the system described herein, including components of a focusing system for obtaining a focused image of a tissue sample 101 and/or other object disposed on a slide. The focusing system described herein provides for determining optimal focus for each snapshot as it is captured, which may be referred to as "dynamic, on-the-fly focusing." The apparatus and techniques provided herein result in a significant reduction in the time required to create a digital image of an area in a pathology slide. The system described herein integrates the steps of a conventional two-step approach and essentially eliminates the time required for pre-focusing. The system described herein provides for creating a digital image of a sample on a microscope slide using dynamic, on-the-fly processing for capturing snapshots, wherein the total time for capturing all snapshots is less than that required by methods that utilize a step of pre-determining the focus point for each snapshot before capturing the snapshot.

Imaging device 100 may include an imaging sensor 110, such as a charge-coupled device (CCD) and/or complementary metal-oxide semiconductor (CMOS) image sensor, which may be part of a camera 111 that captures digital pathology images. Imaging sensor 110 may receive transmitted light from microscope objective 120 via a tube lens 112, a beam splitter 114, and other components of a transmitted light microscope, including a condenser 116, a light source 118, and/or other suitable optical components 119. Microscope objective 120 may be infinity corrected. In one embodiment, beam splitter 114 may provide a distribution that distributes approximately 70% of the light beam source toward image sensor 110, with the remaining approximately 30% directed along a path toward a dither focus stage 150 and a focus sensor 160. A tissue sample 101 being imaged may be positioned on an XY translation stage 130 that is movable in the X and Y directions and may be controlled as further discussed elsewhere herein. The focusing stage 140 can control the movement of the microscope objective 120 in the Z direction to focus the image of the tissue 101 captured by the image sensor 110. The focusing stage 140 can include a motor and/or other suitable device for moving the microscope objective 120. The dither focusing stage 150 and the focus sensor 160 are used to provide fine focus control for dynamic instantaneous focusing according to the system described herein. In various embodiments, the focus sensor 160 can be a CCD and/or CMOS sensor.

The dithering focus stage 150 and focus sensor 160 provide dynamic, instantaneous focusing based on sharpness values and/or other metrics rapidly calculated during the imaging process to achieve optimal focus for each image snapshot as it is captured. As discussed in further detail elsewhere herein, the dithering focus stage 150 can be moved at a frequency, such as a sinusoidal motion, that is independent of and exceeds the frequency of movement feasible for slower motion of the microscope objective 120. The focus sensor 160 makes multiple measurements of focus information for viewing tissue within the range of motion of the dithering focus stage 150. The focus electronics and control system 170 may include electronics for controlling the focus sensor and dithering focus stage 150, a master clock, electronics for controlling the slow focus stage 140 (in the Z direction), the XY translation stage 130, and other components of embodiments of systems in accordance with the techniques herein. The focus electronics and control system 170 may be configured to perform sharpness calculations using information from the dithering focus stage 150 and focus sensor 160. The sharpness value may be calculated within at least a portion of a sinusoidal curve defined by the dithering motion. The focusing electronics and control system 170 can then use this information to determine the location of the best-focused image for the tissue and command the slow focus stage 140 to move the microscope objective 120 to the desired location (along the Z-axis, as shown) to obtain the best-focused image during the imaging process. The control system 170 can also use this information to control the speed of the XY stage 130, for example, the speed of movement of the stage 130 in the Y direction. In an embodiment, the sharpness value can be calculated by differencing the contrast values of adjacent pixels, squaring them, and adding those values together to form a score. Various algorithms for determining the sharpness value are further discussed elsewhere herein.

In various embodiments according to the systems described herein, and in accordance with components discussed elsewhere herein, an apparatus for creating a digital image of a sample on a microscope slide includes: an infinity-corrected microscope objective; a beam splitter; a camera focusing lens; a high-resolution camera; a sensor focusing lens assembly; a dither focus stage; a focus sensor; a coarse (slow) focus stage; and focusing electronics. The apparatus can allow the objective to be focused and each snapshot to be captured by the camera without requiring a predetermined focus point for all snapshots before the snapshots are captured, and wherein the total time for capturing all snapshots is less than that required by a system that requires a predetermined focus point for each snapshot before the snapshot is captured. The system may include computer controls for: i) determining a first focus point on the tissue to establish a nominal focus plane by moving the coarse focus stage through the entire z range and monitoring the sharpness value; ii) positioning the tissue in x and y starting at a corner of the region of interest; iii) setting a dithered fine focus stage into motion, wherein the dithered focus stage is synchronized to a master clock that also controls the speed of the xy stage; iv) commanding the stage to move from frame to adjacent frame, and/or v) generating a trigger signal to acquire a frame on the image sensor and trigger the light source to create a light pulse.

Furthermore, according to another embodiment, the system described herein can provide a computer-implemented method for creating a digital image of a sample on a microscope slide. The method can include determining a scan area that includes an area of the microscope slide (the area including at least a portion of the sample). The scan area can be divided into a plurality of snapshots. The snapshots can be captured using a microscope objective and a camera, wherein the objective and microscope can be focused for each snapshot and each snapshot can be captured by the camera without requiring a predetermined focus point for all snapshots before capturing the snapshots. The total time required to capture all snapshots can be less than the time required for methods that require a predetermined focus point for each snapshot before capturing the snapshot.

FIG3A is a schematic illustration of an embodiment of a focus electronics and control system 170 including focus electronics 161, a master clock 163, and stage control electronics 165. FIG3B is a schematic illustration of an embodiment of the focus electronics 161. In the illustrated embodiment, the focus electronics 161 may include suitable electronics such as a suitably fast A/D converter 171 and a field programmable gate array (FPGA) 172 with a microprocessor 173 that can be used to perform sharpness calculations. The A/D converter 171 may receive information from the focus sensor 160, which is coupled to the FPGA 172 and microprocessor 173 and used to output sharpness information. A master clock included in the system 170 may supply a master clock signal to the focus electronics 161, the stage control electronics 165, and other components of the system. The stage control electronics 165 may generate control signals used to control the slow focus stage 140, the XY translation stage 130, the dither focus stage 150, and/or other control signals and information, as discussed further elsewhere herein. Among other information, the FPGA 172 can supply a clock signal to the focus sensor 160. Measurements in the laboratory have shown that sharpness calculations on a 640x32 pixel frame can be made in 18 microseconds, easily fast enough for proper operation of the system described herein. In an embodiment, the focus sensor 160 can include a monochrome CCD camera windowed into 640x32 stripes, as discussed further elsewhere herein.

Scanning microscopes can acquire 1D or 2D pixel arrays that include contrast and/or intensity information in RGB or some other color space, as discussed further elsewhere herein. The system finds the optimal focus within a large field of view, such as on a 25 mm × 50 mm glass slide. Many commercial systems sample the scene produced by a 20x, 0.75 NA microscope objective with a CCD array. Given an NA of 0.75 for the objective and condenser and a wavelength of 500 nm, the lateral resolution of the optical system is approximately 0.5 microns. To sample this resolution element at the Nyquist frequency, the pixel size at the object is approximately 0.25 microns. For a 4 Mpixel camera (e.g., the Dalsa Falcon 4M30/60) with a pixel size of 7.4 microns and running at 30 fps, the magnification from the object to the imaging camera is 7.4/0.25 = 30x. Thus, one frame at 2352×1728 can cover an area of 0.588 mm×0.432 mm at the subject, which is equivalent to approximately 910 frames for a typical tissue section defined as 15 mm×15 mm in area. The system described herein is ideally used in situations where the tissue spatial variation in focus size is much lower than the frame size at the subject. In practice, focus changes occur over larger distances, and most focus adjustments are made to correct for deflections. These deflections are typically in the range of 0.5-1 micrometer per frame size at the subject.

The resulting time for current scanning systems (e.g., the BioImagene iScan Coreo system) is approximately 3.5 minutes for pre-scanning and scanning a 20x 15 mm x 15 mm field and approximately 15 minutes for a 40x scan of a 15 mm x 15 mm field. The 15 mm x 15 mm field is scanned by running 35 frames in 26 passes. Scanning can be performed unidirectionally with a retracement time of 1 second. Using the techniques of the system described herein, the scanning time can be approximately 5 seconds to find the nominal focus plane, 1.17 seconds per pass (25 passes), for a total of 5 + 25 × (1.17 + 1) = 59.25 seconds (approximately 1 minute). This is a significant time savings compared to conventional methods. Other embodiments of the system described herein may allow even faster focus times, but may be limited by the amount of light required for the short illumination times to avoid motion blur on consecutive scans. This problem can be alleviated by pulsing or strobing the light source 118, which can be an LED light source as discussed further elsewhere herein, to allow for high peak illumination. In an embodiment, the pulsing of the light source 118 can be controlled by the focus electronics and control system 170. Additionally, running the system bidirectionally eliminates retrace time, saving approximately 25 seconds for a 20x scan, resulting in a scan time of 35 seconds.

It should be noted that components used in connection with the focus electronics and control system 170 may also be more generally referred to as electrical components used to perform a variety of different functions in connection with embodiments of the technology described herein.

FIG4 is a schematic diagram illustrating a dither focus stage 150 in greater detail, according to an embodiment of the system described herein. The dither focus stage 150 can include a dither focus lens 151 that can be moved by one or more actuators 152a, b, such as voice coil actuators, and can be mounted in a rigid housing 153. In embodiments, the lens can be a commercially available achromatic lens with a 50 mm focal length, such as those available from Edmund Scientific, NT32-323. Alternatively, the dither focus lens 151 can be constructed of plastic, aspheric, and shaped to minimize lens weight (extremely low mass). A flexure structure 154 can be attached to the rigid housing 153 and to a rigid ground point and can only permit translational motion of the dither lens 151 over a small distance, for example, approximately 600-1000 microns. In embodiments, the flexure structure 154 can be constructed from a suitable stainless steel sheet approximately 0.010" thick in the flexure direction and form a four-bar linkage. The flexure 154 may be designed from a suitable spring steel at a working stress far from its fatigue limit (less than one fifth) to operate over many cycles.

The moving mass of the dither focus lens 151 and flexure 154 can be designed to provide a first mechanical resonance of approximately 60 Hz or above. A suitable high-bandwidth (e.g., greater than 1 kHz) position sensor 155, such as a capacitive sensor or eddy current sensor, can be used to monitor the moving mass to provide feedback to the control system 170 (see Figure 2). For example, KLA Tencor's ADE division manufactures a capacitive sensor 5 mm 2805 probe suitable for this application, with a 1 kHz bandwidth, a 1 mm measurement range, and a 77 nm resolution. A dither focus and control system, such as that represented by the functionality included in system 170, can maintain the amplitude of the dither focus lens 151 within a specified focus range. The dither focus and control system can rely on well-known gain-controlled oscillator circuits. When operating resonantly, the dither focus lens 151 can be driven with low current, dissipating low power in the voice coil windings. For example, using a BEI Kimco LAO8-10 (Winding A) actuator, the average current can be less than 180 mA and the dissipated power can be less than 0.1 W.

It should be noted that other types of motion of the dither lens and other types of actuators 152a, b can be used in connection with various embodiments of the system described herein. For example, piezoelectric actuators can be used as actuators 152a, b. Further, the motion of the dither lens can be at a different resonant frequency than that which remains independent of the motion of the microscope objective 120.

A sensor 155, such as the capacitive sensor mentioned above, which may be included in embodiments according to the present technology, can provide feedback regarding the position of the dithered focus lens (e.g., regarding the sine wave or cycle corresponding to the lens movement). As will be described elsewhere herein, a determination can be made regarding which image frame acquired using the focus sensor produces the best sharpness value. For that frame, the position of the dithered focus lens can be determined relative to the position of the sine wave as indicated by sensor 155. The position indicated by sensor 155 can be used by control electronics 170 to determine appropriate adjustments for focus stage 140. For example, in one embodiment, the movement of microscope objective 120 can be controlled by a slow stepper motor of slow focus stage 140. The position indicated by sensor 155 can be used to determine a corresponding amount of movement (and corresponding control signal(s)) to position microscope objective 120 in the Z direction at the best focus position. The control signal(s) can be transmitted to the stepper motor of slow focus stage 140 to cause any necessary repositioning of microscope objective 120 at the best focus position.

Figures 5A-5E are schematic diagrams illustrating iterations of a focusing operation according to the system described herein. The diagrams depict image sensor 110, focus sensor 160, dither focus stage 150 with a dither lens, and microscope objective 120. Tissue 101 is illustrated as being moved along the Y axis, i.e., on XY stage 130, while a focusing operation is performed. In this example, dither focus stage 150 can move the dither lens at a desired frequency, such as 60 Hz or higher (e.g., 80 Hz, 100 Hz), though it should be noted that in other embodiments, the system described herein can also operate with the dither lens moving at a lower frequency (e.g., 50 Hz), depending on applicable conditions. XY stage 130 can be commanded to move, for example, in the Y direction from frame to frame. For example, stage 130 can be commanded to move at a constant rate of 13 mm/sec, which corresponds to an acquisition rate of approximately 30 frames/sec for a 20x objective. Because the dither focus stage 150 and the XY stage 130 can be phase-locked, the dither focus stage 150 and sensor 160 can make focus calculations 60 times per second, or 120 focus points per second, or 4 focus points per frame, running bidirectionally (reading about the upward and downward motion of the sine wave). For a frame height of 1728 pixels, this equates to one focus point every 432 pixels, or one focus point every 108 microns for a 20x objective. Because the XY stage 130 is moving, focus points should be captured in very short time periods, such as 330 μsec (or less), to minimize changes in the scene.

In various embodiments, as discussed further elsewhere herein, this data can be stored and used to extrapolate the focus position for the next frame, or alternatively, no extrapolation can be used and the last focus point can be used as the focus position for the active frame. With a 60 Hz dither frequency and a frame rate of 30 frames per second, a focus point is obtained at a position no more than 1/4 of the frame from the center of the frame being snapped. Generally, tissue height does not vary enough in 1/4 of the frame to make the focus point inaccurate.

A first focus point can be found on the tissue to establish a nominal focus plane or reference plane 101'. For example, the reference plane 101' can be determined by initially moving the microscope objective 120 through the entire Z range, say +1/-1 mm, using a slow focus stage 140 and monitoring the sharpness values. Once the reference plane 101' is found, the tissue 101 can be positioned in X and Y starting at a corner and/or other specific location of the region of interest and the dither focus stage 150 can be set to move and/or the movement of the dither focus stage 150 can be otherwise continuously monitored, starting in FIG5A.

The dither focus stage 150 can be synchronized to a master clock in the control system 170 (see FIG2 ), which can also be used in conjunction with controlling the speed of the XY stage 130. For example, if the dither focus stage 150 is moved at 60 Hz using a sinusoidal motion of 0.6 mm p-v (peak to valley), with a 32% duty cycle to utilize the more linear range of the sine wave, eight points can be collected through the focus range in a 2.7-millisecond period. In FIG5B-5D , the dither focus stage 150 moves the dither lens sinusoidally and carries the focus sample through at least a portion of the sinusoid. Thus, the focus sample is acquired every 330 μsec, or at a rate of 3 kHz. At a magnification of 5.5x between the object and the focus sensor 160, a 0.6 mm p-v motion at the dither lens is equivalent to a 20-micron p-v motion at the objective lens. This information is used to calculate the position of highest sharpness, i.e., best focus, and communicate it to the slower stepper motor of the slow focus stage 140. As shown in FIG5E , the slow focus stage 140 is commanded to move the microscope objective 120 to the optimal focus position (illustrated by the range of motion 120 ′) in time for the image sensor 110 to capture the optimally focused image 110 ′ of the region of interest of the tissue 101. In embodiments, the image sensor 110 can be triggered, for example by the control system 170, to take a snapshot of the image after a specific number of cycles of the dither lens motion. The XY stage 130 moves to the next frame, the cyclical motion of the dither lens in the dither focus stage 150 continues, and the focusing operation of FIG5A-5E is repeated. Sharpness values can be calculated at a rate that does not hinder processing, for example, 3 kHz.

FIG6A is a schematic illustration of a plot 200 showing a command waveform and sharpness determination for dithered focus optics according to an embodiment of the system described herein. In the time-based embodiment discussed in connection with the examples of FIG5A-5E:

T=16.67 msec, /*Period of the dither lens sine wave if the lens resonates at 60 Hz*/

F=300 μm, /*positive range of focus value*/

N=8, /*Number of focus points obtained in cycle E*/

Δt=330 μsec, /*focus point samples acquired every 330 μsec*/

E=2.67 msec, /*the period during which N focus points are obtained*/

At the center of the focusing stroke, Δf = 1.06 μm. /*Step size of the focusing curve*/

Therefore, with this duty cycle of 32%, 8.48 μm (8×1.06 μm=8.48 μm) is sampled by the focusing process.

6B is a schematic illustration of a plot 210 showing calculated sharpness (Zs) values for a portion of the sinusoidal motion of the dithered lens shown in plot 210. The position (z) for each focal plane sampled as a function of each point i is given by Equation 1:

Equation 1.

Windowing the CCD camera downward can provide a high frame rate suitable for the systems described herein. For example, Dalsa, a company in Waterloo, Ontario, Canada, produces the Genie M640-1/3 640×480 black-and-white camera. The Genie M640-1/3 will operate at 3,000 frames per second with a frame size of 640×32. The pixel size on the CCD array is 7.4 microns. At 5.5x magnification between the object and the focal plane, one focused pixel is equivalent to approximately 1.3 microns at the object. While some averaging of approximately 16 object pixels (4×4) per focused pixel may occur, sufficient high-frequency contrast variation is retained to obtain good focus information. In one embodiment, the optimal focus position can be determined based on the peak of the sharpness calculation plot 210. In additional embodiments, it should be noted that other focus calculations and techniques can be used to determine the optimal focus position based on other metrics, including the use of contrast metrics, as discussed further elsewhere herein.

Figures 7A and 7B are schematic diagrams illustrating focus determination and adjustment of a sample (tissue) according to an embodiment of the system described herein. In Figure 7A, diagram 250 is a view of a sample shown in an approximate image frame associated with movement of the sample along the Y-axis according to movement of the XY stage 130 as discussed herein. Diagram 250 illustrates a single pass or traverse of the sample associated with movement of the sample along the Y-axis (e.g., according to movement of the XY stage). Diagram 250' is a magnified version of a portion of diagram 250. One frame of diagram 250' is designated as a DTP, referring to a distinct tissue point of the sample. In the example of diagram 250', the sample boundary is shown, and during a scan thereof, multiple focus calculations are performed according to the system described herein. In frame 251, and by way of example, a best focus determination is illustrated after four focus calculations (shown as focus positions 1, 2, 3, and 0*) are performed in association with imaging the sample, although many more focus calculations may be performed in association with the system described herein. 7B shows a schematic diagram 260 showing a plot of the Z-axis position of a microscope objective relative to the Y-axis position of a sample being inspected. The diagram positions 261 show positions determined along the Z-axis for adjusting the microscope objective 120 to achieve optimal focus according to an embodiment of the system described herein.

It should be noted that the system described herein offers significant advantages over conventional systems, such as those described in U.S. Patent Nos. 7,576,307 and 7,518,642, incorporated herein by reference, in which the entire microscope objective moves through focus in a sinusoidal or triangular pattern. The system provided herein is advantageous in that it is suitable for use with microscope objectives and accompanying stages that are heavy (especially if additional objectives are added via a turret) and cannot move at the higher frequencies described using dither optics. The dither lens described herein can have a tuned mass (e.g., making it lighter, requiring less glass) and the imaging demands on the focus sensor are less than those imposed by the microscope objective. As described herein, focus data can be acquired at a high rate to minimize scene changes when calculating sharpness. By minimizing scene changes, the system described herein reduces discontinuities in the sharpness metric caused by the system shifting in focus and defocus as tissue moves under the microscope objective. In conventional systems, such discontinuities add noise to the best focus calculation.

FIG8 is a schematic diagram 300 illustrating an example of a sharpness profile resulting from movement through focus positions, including sharpness curves and contrast ratios for each sharpness response at multiple points sampled by the dithered focus optics, according to an embodiment of the system described herein. Plot 310 shows dither lens amplitude in micrometers along the x-axis and sharpness units along the y-axis. As illustrated, the dither lens motion can be centered around representative points A, B, C, D, and E; however, it should be noted that the calculations described herein can be applied to every point on the sharpness curve. Plots 310 a-e show the sharpness response generated from the focus sensor 160 for a half-cycle of the dither lens sine wave when the dither lens motion is centered around each of points A, B, C, D, and E, respectively. Based on this, the contrast ratio for each sharpness response having a corresponding one of points A-E is calculated according to the contrast function = (max-min)/(max+min). In association with a contrast function determined for one of points A-E (e.g., about which the dither lens motion is centered) and a corresponding one of sharpness response curves 310a-e, max represents the maximum sharpness value obtained from the sharpness response curve, and min represents the minimum sharpness value obtained from the sharpness response curve. The resulting contrast function plot 320 is shown below sharpness curve plot 310 and plots contrast ratio values corresponding to movement of the dither lens according to the dither lens amplitude. The minimum value of the contrast function in plot 320 is the best focus position. Based on the contrast function and the best focus position determination, a control signal can be generated that is used to control slow focus stage 140 to move microscope objective 120 to the best focus position before image sensor 110 captures image 110′.

FIG9 shows a functional control loop block diagram 350 illustrating the use of control signals to control the contrast function of the slow focus stage 140. For example, _Ud can be considered a disturbance in the focus control loop and can represent slide deflection or varying tissue surface height. Function block 352 illustrates the generation of sharpness vector information, which can be generated by the focus sensor 160 and communicated to the focus electronics and control system 170. Function block 354 illustrates the generation of a contrast number (e.g., the value of the contrast function) at the point where the dither lens is sampling focus. This contrast number is compared to a setpoint or reference value (Ref) generated at an initial step where best focus was previously established. The error signal generated from this comparison (at function block 356) with the appropriately applied gain _K1 corrects the slow focus motor, which acts (at function block 358) to keep the scene in focus. It should be noted that embodiments can adjust the position of the microscope objective 120 according to a minimum or threshold movement amount. Thus, such embodiments can avoid making adjustments less than the threshold.

FIG10 is a schematic diagram illustrating the division of focus window 402 into zones in connection with focus processing according to an embodiment of the system described herein. In the illustrated embodiment, the focus window is subdivided into eight zones (402′); however, fewer or more zones may be used in connection with the system described herein. A first subset of the zones may be within snapshot n, and a second subset of the zones may be within snapshot n+1. For example, zones 2, 3, 4, and 5 may be within image frame 404, which was snapped at time t1. Zones 6 and 7 may be completely within the next image frame to be snapped as XY stage 130 traverses from bottom to top in the figure, and/or zones 0 and 1 may be completely within the next image frame to be snapped as stage 130 traverses from top to bottom in the figure. Focus positions 0, 1, 2, and 3 may be used to extrapolate the optimal focus position for the next snapshot frame at position 0*. Tissue coverage may be established, for example, by performing a serpentine pattern across the entire region of interest.

The rectangular window 404 of the image sensor can be oriented along the direction of travel of the stage 130, such that the columns of frames acquired during imaging are aligned with the rectangular focus window 402. Using a 30x tube lens, the size of an object in image frame 406 using, for example, a Dalsa 4M30/60 CCD camera is 0.588 mm × 0.432 mm. The array size can be (2352 × 7.4 microns/30) × (1720 × 7.4 microns/30). The wider dimension of image frame 406 (0.588 mm) can be oriented perpendicular to the focus window 402 and allow for the minimum number of columns traversed across a section of tissue. In the focus leg, using 5x magnification, the focus sensor is 0.05 mm × 0.94 mm. The rectangular focus window 402 can be (32 × 7.4 microns/5.0) × (640 × 7.4 microns/5.0). Thus, the focus sensor frame 402 can be approximately 2.2x taller than the image sensor frame 404 and can be advantageously used in conjunction with look-ahead focus techniques involving multiple zones, as discussed further elsewhere herein. According to an embodiment of the system described herein, 120 best focus determinations can be made per second, with acuity calculations performed every 333 μsec, resulting in 8 acuities calculated in 2.67 msec, which is equivalent to a duty cycle of approximately 32% for an 8.3 msec half-dither period for dithering the lens motion.

A sharpness metric can be calculated and stored for each zone. When multiple zones are used to calculate a sharpness metric for a single focus point, a sharpness metric can be determined for each zone and combined, for example, such as by summing all sharpness metrics for all zones considered at such a single point. An example of a sharpness calculation for each zone is shown in Equation 2 (e.g., based on the use of a camera windowed into 640×32 strips). For row i, dimension n up to 32, and column j, dimension m up to 640/z, where z is the number of zones, the sharpness for the zone can be expressed by Equation 2:

Sharpness Equation 2

Where k is an integer between 1 and 5 or equal to 1 and 5. Other sharpness metrics and algorithms can also be used in conjunction with the system described herein. As the XY stage 130 moves along the y-axis, the system acquires sharpness information for all zones 0-7 in the focus window 402. It is ideal to know how the height of the tissue slice changes as the stage 130 moves. By calculating a sharpness curve (maximum sharpness is best focus), zones 6 and 7 can, for example, provide information before moving the next frame above where the next best focus plane will be, by changing the focus height. If a large focus change is expected from this look-ahead, the stage 130 can be slowed down to provide more closely spaced points to better track the height transition.

During the scanning process, it may be advantageous to determine whether the system is transitioning from empty space (no tissue) to darker space (tissue). By calculating the sharpness in zones 6 and 7, for example, it is possible to predict whether this transition is imminent. While scanning the column, if zones 6 and 7 show increasing sharpness, the XY stage 130 can be commanded to slow down to create more closely spaced focus points at the tissue boundary. If, on the other hand, a transition from high to low acuity is detected, it can be determined that the scanner field of view is entering empty space, and slowing down the stage 130 to create more closely spaced focus points at the tissue boundary may be desirable. In areas where these transitions do not occur, the stage 130 can be commanded to move at a higher constant speed to increase the overall throughput of slide scanning. This approach can advantageously allow for rapid tissue scanning. According to the system described herein, snapshots can be taken while focus data is being collected. Furthermore, all focus data can be collected and stored in the first scan, and snapshots can be taken at the optimal focus point during subsequent scans. Embodiments may use contrast ratios or function values in a manner similar to that as described herein with sharpness values to detect focus changes and thus determine transitions into or out of regions containing tissue or empty space.

For example, for a 15 mm x 15 mm 20x scan, at an image frame size of 0.588 x 0.432 mm, there are 26 columns of data, each with 35 frames. At an imaging rate of 30 fps, each column is scanned in 1.2 seconds, or approximately 30 seconds. Because the focus sensor 160 calculates 120 (or more) focus points per second, the system described herein can obtain four focuses per frame (120 focuses/second divided by 30 fps). At an imaging rate of 60 fps, the scan time is 15 seconds, with two focuses per frame (120 focuses/second divided by 60 fps).

In another embodiment, a color camera can be used as the focus sensor 160, and a chromaticity metric can be determined alternatively and/or additionally to the sharpness contrast metric. For example, a Dalsa color version of a 640×480 Genie camera can be suitably used as the focus sensor according to this embodiment. The chromaticity metric can be described as the chromaticity relative to the luminance of a similarly illuminated white. In equation form (Equations 3A and 3B), chromaticity (C) can be a linear combination of the R, G, and B color measurements:

Equation 3A

Equation 3B

Note that R = G = B, and _CB = _CR = 0. Based on _CB and _CR , a value for C representing the total chromaticity may be determined (e.g., such as by adding _CB and _CR ).

As the XY stage 130 moves along the y-axis, the focus sensor 160 can acquire color (R, G, B) information, as in bright-field microscopy. It is desirable to know how the height of the tissue section is changing as the stage moves. As with contrast techniques, the use of RBG color information can be used to determine whether the system is transitioning from blank space (no tissue) to colored space (tissue). By calculating the chromaticity in regions 6 and 7, for example, it is possible to predict whether this transition is imminent. If, for example, very little chromaticity is detected, then C = 0, and it can be determined that no tissue boundary is approaching. However, while scanning the focus column, if regions 6 and 7 show increasing chromaticity, the stage 130 can be commanded to slow down to create more closely spaced focus points at the tissue boundary. If, on the other hand, movement from high to low chromaticity is detected, it can be determined that the scanner is entering blank space, and it may be desirable to slow down the stage 130 to create more closely spaced focus points at the tissue boundary. In areas where these transitions do not occur, the stage 130 can be commanded to move at a higher, constant speed to increase the overall throughput of slide scanning.

Processing changes can be implemented in conjunction with using sharpness values, contrast ratio values, and/or chromaticity values to determine when the field of view or upcoming frame(s) are entering or leaving a region of the slide containing tissue. For example, when entering a region containing tissue from a clear region (e.g., between tissue regions), movement in the Y direction can be reduced and the number of focus points achieved can also be increased. When viewing clear space or regions between tissue samples, movement in the Y direction can be increased and less focus can be determined (e.g., by increasing chromaticity and/or sharpness values) until movement over a region containing tissue is detected.

FIG11 shows a graphical illustration of different sharpness values that can be obtained at various points in time in accordance with an embodiment of the technology herein. The top portion 462 includes a curve 452 corresponding to a half-sine wave cycle of a dither lens motion (e.g., half of a single peak-to-peak cycle or period). The X-axis corresponds to the dither lens amplitude value during this cycle, and the Y-axis corresponds to the sharpness value. Each point, such as point 462a, represents a point at which a frame was acquired using a focus sensor, where each frame was acquired at a dither lens amplitude represented by the point's X-axis value and had a sharpness value represented by the point's Y-axis value. Elements 465 in the bottom portion 464 represent curves fitted to the set of acquired sharpness values, as represented in portion 462 for the illustrated data points.

FIG12 is a flow chart 500 illustrating a dynamic on-the-fly focus process during scanning of a sample under inspection, according to an embodiment of the system described herein. At step 502, a nominal focal plane, or reference plane, can be determined for the sample under inspection. Following step 502, processing proceeds to step 504, where the dither lens is set to move at a specific resonant frequency, according to the system described herein. Following step 504, processing proceeds to step 506, where the XY stage is commanded to move at a specific velocity. It should be noted that, as with other steps of the process discussed herein, the order of steps 504 and 506 can be modified appropriately according to the system described herein. Following step 506, processing proceeds to step 508, where a sharpness calculation for the focus point relative to the sample under inspection is performed in conjunction with the motion (e.g., sinusoidal) of the dither lens, according to the system described herein. The sharpness calculation can include the use of contrast, chromaticity, and/or other appropriate measurements, as discussed further elsewhere herein.

After step 508, processing proceeds to step 510, where a best focus position is determined for the position of a microscope objective lens used in conjunction with an image sensor to capture images according to the system described herein. Following step 510, processing proceeds to step 512, where a control signal related to the best focus position is sent to a slow focus stage that controls the position (z-axis) of the microscope objective lens. Step 512 may also include sending a trigger signal to a camera (e.g., an image sensor) to capture an image of the portion of the sample beneath the objective lens. The trigger signal may be a control signal that causes the image sensor to capture the image, such as after a specified number of cycles (e.g., as associated with a dither lens movement). Following step 512, processing proceeds to test step 514, where a determination is made as to whether the speed of the XY stage holding the sample under scanning should be adjusted. As discussed in further detail elsewhere herein, this determination can be made based on look-ahead processing techniques using the sharpness of multiple regions within the focused field of view and/or other information. If, at test step 514, it is determined that the speed of the XY stage should be adjusted, processing proceeds to step 516, where the speed of the XY stage is adjusted. After step 516, processing proceeds back to step 508. If, at test step 514, it is determined that no adjustments to the velocity of the XY stage will be made, then processing proceeds to test step 518 where it is determined whether the focusing process will continue. If the process will continue, then processing returns to step 508. Otherwise, if the process will not continue (e.g., the scan of the current sample has completed), then the focusing process is terminated and the process is complete.

FIG13 is a flow chart 530 illustrating processing at a slow focus stage according to an embodiment of the system described herein. At step 532, the slow focus stage, which controls the position of a microscope objective (e.g., along the Z axis), receives a control signal containing information for adjusting the position of the microscope objective of a sample being examined. Following step 532, processing proceeds to step 534, where the slow focus stage adjusts the position of the microscope objective according to the system described herein. Following step 534, processing proceeds to a wait step 536, where the slow focus stage waits to receive another control signal. Following step 536, processing returns to step 532.

FIG14 is a flow chart 550 illustrating an image capture process according to an embodiment of the system described herein. At step 552, the image sensor of the camera receives a trigger signal and/or other instructions that trigger the process of capturing an image of a sample undergoing microscopic examination. In various embodiments, according to the system described herein, the trigger signal may be received from a control system that controls the triggering of the image sensor image capture process after a specified number of cycles of movement of a dither lens used in the focusing process. Alternatively, the trigger signal may be provided by a position sensor on the XY stage. In one embodiment, the position sensor may be a Renishaw linear encoder model No. T1000-10A. After step 552, the process proceeds to step 554, where the image sensor captures an image. As discussed in detail herein, according to the system described herein, the image captured by the image sensor may be in focus, depending on the operation of the focusing system. The captured images may be stitched together in accordance with other techniques referenced herein. After step 554, the process proceeds to step 556, where the image sensor waits for another trigger signal. After step 556, the process returns to step 552.

FIG15 is a schematic diagram 600 illustrating an alternative arrangement for focus processing according to an embodiment of the system described herein. A windowed focus sensor can have a frame field of view (FOV) 602 that can be tilted or otherwise positioned to diagonally scan a line substantially equal to the width of the imaging sensor frame FOV 604. As described herein, the window can be tilted along the direction of travel. For example, the tilted focus sensor's frame FOV 602 can be rotated 45 degrees, which would have an effective width of 0.94 × 0.707 = 0.66 mm at the object (tissue). The imaging sensor's frame FOV 604 can have an effective width of 0.558 mm, so that as the XY stage holding the tissue moves under the objective lens, the tilted focus sensor frame FOV 602 sees the edge of the line observed by the image sensor. In the diagram, multiple frames of the tilted focus sensor are shown at intermediate positions (0, 1, 2, and 3) in time, superimposed on the image sensor frame FOV 604. Focus points can be taken at three points between the centers of adjacent frames in the focus column. Focus positions 0, 1, 2, and 3 are used to extrapolate the optimal focus position for the next snapshot frame at position 0*. The scan time for this method will be similar to the method described elsewhere herein. Although the skewed focus sensor's frame FOV 602 has a shorter lookahead, in this case 0.707×(0.94-0.432)/2=0.18 mm, or the skewed focus sensor encroaches 42% of the next frame to be acquired, the skewed focus sensor's frame FOV 602 is skewed relative to the image sensor frame FOV 604, viewing tissue at the edge of the scan line, which can be advantageous in some cases for providing edge focus information.

FIG16 is a schematic diagram 650 illustrating an alternative arrangement for focus processing according to another embodiment of the system described herein. As in diagram 650, a skewed focus sensor frame FOV 652 and an image sensor frame FOV 654 are shown. The skewed sensor frame FOV 652 can be used to acquire focus information for a forward pass across the tissue. In the backward pass, the imaging sensor snaps frames while the focus stage adjusts using focus data from the previous forward pass. If one were to acquire focus data at each image frame skipping intermediate positions 0, 1, 2, and 3 in the previous approach, assuming a high rate of focus point acquisition, the XY stage could move at 4x speed in the forward pass. For example, for a 15 mm x 15 mm image at 20x magnification, one column of data would be 35 frames. Because focus data is acquired at 120 points per second, the forward pass can be performed in 0.3 seconds (35 frames per second / 120 focus points). In this example, the number of columns is 26, so the focusing portion can be completed in 26 × 0.3, or 7.6 seconds. Image acquisition at 30 fps is approximately 32 seconds. Therefore, the focusing portion of the total scan time is only 20%, which is very efficient. Furthermore, if the focus is allowed to jump every other frame, the focusing portion of the scan time can be further significantly reduced.

It should be noted that in other embodiments, the focus bar of the focus sensor may be positioned at other locations within the field of view and in other orientations to sample adjacent data columns to provide additional look-ahead information that may be used in connection with the systems described herein.

The XY stage that transfers the slide can repeat the best focus points produced on the forward travel relative to those produced on the backward travel. For a 20x 0.75 NA objective lens with a focus depth of 0.9 microns, repeatability to about 0.1 microns would be ideal. The stage can be constructed to meet 0.1 micron forward/backward repeatability, and therefore, this requirement is technically feasible, as further discussed elsewhere herein.

In embodiments, tissue or smears on glass slides being examined according to the systems described herein can cover the entire slide or an area of approximately 25 mm x 50 mm. Resolution depends on the numerical aperture (NA) of the objective, the coupling medium to the slide, the NA of the condenser, and the wavelength of the light. For example, at 60x, for a 0.9 NA microscope objective, Plan Apochromat (Plan APO), the lateral resolution of the microscope is approximately 0.2 μm in air under green light (532 nm), with a depth of focus of 0.5 μm.

In connection with the operation of the systems described herein, a digital image can be obtained by moving a limited field of view over an area of interest via a line scan sensor or CCD array and assembling the limited fields of view or frames or tiles together to form a mosaic. Ideally, the mosaic appears seamless, with no visible stitching, focus, or illumination irregularities, as the viewer navigates across the image.

Figure 17 is a flowchart 700 illustrating a process for obtaining a mosaic image of tissue on a slide according to an embodiment of the system described herein. At step 702, a thumbnail image of the slide can be obtained. The thumbnail image can be a low resolution image at approximately 1x or 2x magnification. If a barcode is present on the slide label, the barcode can be decoded at this step and attached to the slide image. After step 702, the process proceeds to step 704, where standard image processing tools can be used to find tissue on the slide. The tissue can be delimited to narrow the scan area to a given area of interest. After step 704, the process proceeds to step 706, where an XY coordinate system can be attached to the tissue surface. After step 706, the process proceeds to step 708, where one or more focus points can be generated for the tissue at regular X and Y intervals, and focusing techniques can be used to determine optimal focus, such as one or more of the dynamic instant focusing techniques discussed elsewhere herein. After step 708, processing may proceed to step 710 where the coordinates of the desired focus point and/or other appropriate information may be saved and referred to as an anchor point. Note that the focus point may be interpolated if the frame lies between anchor points.

After step 710, processing may proceed to step 712, where the microscope objective is positioned at an optimal focus position according to techniques discussed elsewhere herein. After step 712, processing proceeds to step 714, where images are collected. After step 714, processing proceeds to test step 716, where a determination is made as to whether the entire region of interest has been scanned and imaged. If not, processing proceeds to step 718, where the XY stage moves the tissue in the X and/or Y directions according to techniques discussed elsewhere herein. After step 718, processing returns to step 708. If, at test step 716, it is determined that the entire region of interest has been scanned and imaged, processing proceeds to step 720, where the collected image frames are stitched or otherwise combined to create a mosaic image according to the systems described herein and using techniques discussed elsewhere herein (e.g., see U.S. Patent Application Publication No. 2008/0240613). After step 720, processing is complete. It should be noted that other suitable sequences for acquiring one or more mosaic images may also be used in connection with the systems described herein.

For favorable operation of the systems described herein, z-position repeatability must be repeatable to a fraction of the objective's depth of focus. Small errors in z-position returned by the focus motor are readily apparent in mosaic systems (2D CCD or CMOS) and adjacent columns of line-scan systems. For the aforementioned resolution at 60x, a z-peak repeatability of approximately 150 nanometers or less is ideal, and such repeatability would be correspondingly suitable for other objectives, such as 4x, 20x, and/or 40x objectives.

Further in accordance with the systems described herein, various embodiments of a slide stage system including an XY stage for pathology microscopy applications are provided, which can be used in connection with the features and techniques for digital pathology imaging discussed herein, including, for example, serving as the XY translation stage 130 discussed elsewhere herein in connection with dynamic instant focus techniques. According to embodiments, and as discussed in further detail elsewhere herein, the XY stage can include a rigid base block. The base block can include a flat glass block supported on a raised boss and a second glass block having a triangular cross-section supported on the raised boss. The two blocks can be used as a smooth and straight track or path for guiding the moving stage block.

FIG18 is a schematic diagram illustrating an embodiment of a precision stage 800 (e.g., the Y stage portion) of an XY stage according to an embodiment of the system described herein. For example, precision stage 800 can achieve a z-peak repeatability of approximately 150 nanometers or less over a 25 mm x 50 mm area. As further discussed elsewhere herein, precision stage 800 can be used in conjunction with features and techniques discussed elsewhere herein, including, for example, in conjunction with XY translation stage 130 discussed with respect to the dynamic live focus technique. Precision stage 800 can include a rigid base block 810 in which a flat glass block 812 is supported on raised bosses. The spacing of these bosses minimizes sagging of the glass block on a simple support due to the weight of precision stage 800. A second glass block 814 having a triangular cross-section is supported on the raised bosses. Glass blocks 812, 814 can be adhesively bonded to base block 810 using a semi-rigid epoxy that does not damage the glass blocks. Glass blocks 812, 814 can be straight and polished to one or two wavelengths of light at 500 nm. A low thermal expansion material such as Zerodur can be used as the material for glass blocks 812, 814. Other suitable types of glass can also be used in conjunction with the system described herein. Cutout 816 can allow light from the microscope condenser to illuminate the tissue on the slide.

Two glass blocks 812, 814 can be used as smooth and straight tracks or roads in order to guide the mobile stage block 820. The mobile stage block 820 can include hard plastic spherical buttons (e.g., 5 buttons) of the contact glass blocks, as shown in positions 821a-e. Because these plastic buttons are spherical, the contact surface can be confined to a very small area (<<0.5 mm) determined by the elastic modulus of the plastic. For example, PTFE or other thermoplastic mixtures from British GGB Bearing Technology Co., Ltd. can be added with other lubricant additives and cast into the shape of contact buttons with a diameter of about 3 mm. In an embodiment, the friction coefficient between the plastic button and the polished glass should be as low as possible, but it may be ideal to avoid using liquid lubricants to save instrument maintenance. In an embodiment, a friction coefficient between 0.1 and 0.15 can be easily achieved when no lubrication is run.

Figures 19A and 19B are more detailed views of a moving stage block 820 according to an embodiment of the system described herein, showing spherical buttons 822a-e contacting glass blocks 810, 812 at locations 821a-e. The buttons can be positioned to allow for excellent stiffness in all directions except the actuation direction (Y). For example, two plastic buttons can face each other to contact the sides of triangular glass block 814 (i.e., four buttons 822b-e), with one plastic button 822a positioned to contact flat glass block 812. Moving stage block 820 can include one or more holes 824 that are lightweight and shaped to place the center of gravity at the center of mass 826 of the triangle formed by the positions of the plastic support buttons 822a-e. In this way, each plastic button 822a-e at the corner of triangle 828 can have equal weight at all times during the motion of stage 800.

Referring back to FIG. 18 , the slide 801 is clamped via a spring-loaded arm 830 in a nest 832. The slide 801 can be placed manually in the nest 832 and/or robotically with an auxiliary mechanism. A rigid cantilever 840 supports and rigidly clamps the end of a small diameter flexure rod 842, which can be made of high fatigue strength steel. In one example, this diameter can be 0.7 mm. The other end of the rod flexure 842 can be attached to a center of mass location 826 on the moving stage 820. The cantilever 840 can be attached to a bearing block 850, which can run on a hardened steel guide rail 852 via a recirculating bearing design. A guide screw assembly 854 can be attached to the bearing block 850 and can be rotated by a stepper motor 856. Suitable components for the above-mentioned elements can be obtained through several companies such as THK of Japan. The guide screw assembly 854 drives the carrier block 850 on the guide rail 852 , which pulls or pushes the moving stage block 820 via the rod flexure portion 842 .

The bending stiffness of the rod flexure 842 can be less than the stiffness of the moving stage block 820 on its plastic pad (this is the stiffness opposing the force normal to the plane of the moving stage in the z-direction) by a factor greater than 6000x. This effectively isolates the moving stage block 820 from the up and down motion of the bearing block 850/cantilever 840 caused by bearing noise.

The careful mass balance and attention to geometry in the design of the precision stage 800 described herein minimize the moments on the moving stage block 820 that would produce small rocking motions. Additionally, because the moving stage block 820 travels on polished glass, the moving stage block 820 has a z-position repeatability of less than 150 nanometers peak, which is sufficient for scanning at 60x magnification. Since the 60x condition is the most stringent, other lower magnification high NA objectives such as 20x and 40x also show suitable performance similar to that obtained under the 60x condition.

FIG20 illustrates an embodiment of an entire XY hybrid stage 900, including a Y stage 920, an X stage 940, and a base plate 960, in accordance with the precision stage features discussed herein and in accordance with an embodiment of the system described herein. In this case, the base block for the Y stage 920 becomes the X stage 940, which acts as a translation stage in the X direction. The base block for the X stage 940 is the base plate 960, which can be secured to a bottom surface. According to the system described herein, the XY hybrid stage 900 provides repeatability of approximately 150 nanometers in the Z direction and repeatability of approximately 1-2 microns (or less) in the X and Y directions. Submicron accuracy can be achieved according to the system described herein if the stage includes position feedback via a tape scale, such as those manufactured by Renishaw of Gloucestershire, England.

A stage design according to the system described herein can be superior to spherical bearing supported moving stages because an XY stage according to the system described herein does not suffer from repeatability errors caused by non-spherical ball bearings or non-cylindrical cross roller bearings. Additionally, when repeatedly cycling the bearing design, the replenishment of new balls at different sized balls can cause non-repeatable motion. An additional benefit of the embodiments described herein is the cost of the stage. The glass elements utilize standard grinding and polishing techniques and are not prohibitively expensive. The bearing block and guide screw assembly do not need to be of particularly high quality because the rod flexure decouples the moving stage from the bearing block.

Further in accordance with the systems described herein, it is advantageous to reduce and/or otherwise minimize scan time during scanning of digital pathology slides. In a clinical setting, an ideal workflow is to place a rack of slides into a robotic slide scanning microscope, close the door, and command the system to scan the slides. Ideally, no user intervention is required until all slides are scanned. The batch size can include multiple slides (e.g., 160 slides) and the time to scan all slides is called the batch time. Slide throughput is the number of slides processed per hour. Cycle time is the time between each available slide image being ready for viewing.

Cycle time in acquiring an image may be affected by the following steps: (a) robotically picking up a slide; (b) creating a thumbnail or overview image of the slide tissue area and labeling; (c) computationally defining a region of interest on the slide tissue; (d) pre-scanning the bounded tissue area to find a regular array of optimal focus points on the tissue; (e) scanning the tissue based on stage and/or sensor movement; (f) creating a compressed output image ready for viewing; and (g) depositing the slide in preparation for the next slide. It should be noted that if dynamic focusing or "dynamic on-the-fly" focusing is performed according to the system described herein, step (d) may not be necessary, and accordingly, scanning/image acquisition time may be reduced due to the use of dynamic on-the-fly focusing techniques.

The systems described herein can further involve eliminating or significantly reducing the time to perform steps (a), (b), (c), and (g). According to various embodiments of the systems described herein, these gains can be achieved, for example, by using a buffering concept in which the above-mentioned steps (a), (b), (c), and (g) for one slide are overlapped in time with steps (d), (e), and (f) for another slide, as discussed in further detail herein. In various embodiments, the overlap of steps (a), (b), and (c) for one slide with steps (d), (e), and (f) for another slide can provide gains of 10%, 25%, or even 50% compared to a system in which steps (a), (b), and (c) for one slide do not overlap with steps (d), (e), and (f) for another slide.

Figure 21 is a schematic diagram of a slide caching device 1000 according to an embodiment of the system described herein. A slide pick head 1002 can be positioned to pick up a slide 1001. The pick head 1002 can use a mechanical device and/or a vacuum device to pick up the slide 1001. The slide 1001 can be one of a collection of slides in a batch, for example a batch of 160 slides. This collection of slides can be set in a slide holder 1003. The pick head 1002 is attached to a carrier car or block 1004 that travels on rails 1005. The carrier block 1004 is moved by rotating guide screws 1006. A rotary encoder 1007 can be used to detect the motor count and convert it into linear movement to control the slide position in the Y direction. Elements 1002-1007 can include a moving assembly called a slide loader/unloader 1008. The slide loader/unloader 1008 also moves in the x-direction on rails 1010 on a motorized carriage or block 1009, which allows the slide loader/unloader 1008 to move in both the X and Y directions.

In operation, the slide, still held on the pick head 1002, can be placed under the low-resolution camera 1011 to obtain a thumbnail or overview of the slide's organizational areas and labels (e.g., step (b) above). Once this operation is complete, step (c) can be performed and the slide can be placed into position on the slide buffer 1012. The slide buffer 1012 can include two (or more) buffer slots or positions 1018a, 1018b and is shown as including a slide 1017 in buffer position 1018a.

In an embodiment, a hybrid XY stage 1013 may include a platen 1014 that moves in the Y direction and is mounted to a platen 1015 that moves in the x-direction. The XY stage 1013 may have similar features and functionality as discussed elsewhere herein, including, for example, the features of the hybrid XY stage 900 discussed herein. The platen 1014 may also include an additional slide pick head 1016. The pick head 1016 may be similar to the pick head 1012 described above. The pick head 1016 may use a mechanical device and/or a vacuum device to pick up a slide.

The pickup head 1016 of the hybrid XY stage 1013 can move to buffer position 1018a and pick up slide 1017. Slide 1017 can now proceed to one or more of the steps mentioned above, including the steps of (d) prescanning, (e) scanning, and (f) creating an output image. While this process is being performed, the slide loader/unloader 1008 can pick up another slide (e.g., slide 1001), use the camera 1011 to obtain a thumbnail view of slide 1001, and place slide 1001 in an empty position 1018b in the slide buffer 1012, schematically indicated by the dashed line 1001'. When the scan on the previous slide (slide 1017) is completed, the slide pickup head 1016 of the hybrid XY stage 1013 can place slide 1017 in buffer position 1018a and pick up the next slide (slide 1001) ready for scanning from buffer position 1018b. The hybrid XY stage 1013 can be moved in a regular back-and-forth scanning pattern to acquire high-resolution images of biological tissues under the high-resolution optical system microscope optics and camera 1019 in accordance with features and techniques discussed elsewhere herein. It should be further noted that the movement and slide selection of the hybrid XY stage 1013 and/or the slide loader/unloader 1008 can be controlled by one or more processors in the control system.

The slide loader/unloader 1008 can move to the buffer position 1018a and pick up slide 1017 and deposit it into the slide rack 1003. This slide 1017 has completed all the steps listed above. The slide loader/unloader 1008 can then proceed to pick up another slide and load it into the slide buffer 1012, and finally pick up slide 1001 and return it to the slide rack 1003. A similar process as described above can continue until all slides in the slide rack 1003 have been scanned.

The slide caching technology according to the system described herein provides advantageous time savings. For example, in a system at a 20x 15 mm × 15 mm field, the pick-up time is approximately 25 seconds, the thumbnail acquisition is approximately 10 seconds, the pre-scan time is approximately 30 seconds, and the scan time is 90 seconds. Output file generation is completed simultaneously with the scanning process and can increase by approximately 5 seconds. The storage of the slide is approximately 20 seconds. Adding all these times together indicates a 180-second cycle time. The XY hybrid stage still needs time to pick up and store the scanned slide, which can account for approximately 10 seconds. Therefore, the reduction in scan time is approximately 1-(180-55+10)/180 = 25%. For systems using dynamic focusing technology, such as the dynamic instant focus discussed further elsewhere herein, the pre-scan time can be eliminated, and with a high data rate camera, the time not associated with picking up and storing can be reduced to 20-30 seconds. The reduction in scanning time when using a slide buffer can in this case be approximately 1- (75-55+ 10)/75 = 50%.

Figure 22A is a flowchart 1100 illustrating slide buffering processing in accordance with an embodiment of the system described herein, relating to a first slide. At step 1102, the first slide is picked up from a slide holder. Following step 1102, processing proceeds to step 1104, where a thumbnail image is obtained and/or other thumbnail processing is performed on the first slide, which may include determining a region of interest of tissue on the slide. Following step 1104, processing proceeds to step 1106, where the first slide is deposited into a slide buffer. Following step 1106, processing proceeds to step 1108, where the first slide is picked up from the slide buffer. Following step 1108, processing proceeds to step 1110, where the first slide is scanned and imaged according to the same techniques discussed further elsewhere herein. It should be noted that in various embodiments, the scanning and imaging techniques may include a pre-scan focusing step and/or use a dynamic focusing technique, such as a dynamic instant focus technique. Following step 1110, processing proceeds to step 1112, where the first slide is deposited into the slide buffer. After step 1112, processing proceeds to step 1114, where a first slide is picked up from the slide buffer. After step 1114, processing proceeds to step 1116, where the first slide is deposited in a slide holder. After step 1116, processing with respect to the first slide is complete.

Figure 22B is a flowchart 1120 illustrating slide buffer processing associated with a second slide according to an embodiment of the system described herein. As further discussed herein, the various steps of flowchart 1120 can be performed in parallel with the steps of flowchart 1100. At step 1122, a second slide is picked up from the slide holder. After step 1102, processing proceeds to step 1124, where a thumbnail image is obtained and/or other thumbnail processing is performed on the second slide, which may include determining a region of interest of tissue on the slide. After step 1124, processing proceeds to step 1126, where the second slide is deposited into a slide buffer. After step 1126, processing proceeds to step 1128, where the second slide is picked up from the slide buffer. After step 1128, processing proceeds to step 1130, where the second slide is scanned and imaged according to the same techniques discussed further elsewhere herein. It should be noted that in various embodiments, the scanning and imaging techniques may include a pre-scan focusing step and/or use of dynamic focusing techniques, such as dynamic instant focusing techniques. After step 1130, processing proceeds to step 1132, where the second slide is deposited in the slide buffer. After step 1132, processing proceeds to step 1134, where the second slide is picked up from the slide buffer. After step 1134, processing proceeds to step 1136, where the second slide is deposited in the slide holder. After step 1136, processing with respect to the second slide is complete.

According to embodiments of the system described herein that address slide buffering, the steps of flowchart 1100 for a first slide can be performed by a slide buffering device in parallel with the steps of flowchart 1120 for a second slide to reduce cycle time. For example, steps 1122, 1124, and 1126 of flowchart 1120 for the second slide (e.g., steps associated with picking up the second slide from the slide holder, thumbnail image processing, and depositing the second slide into the slide buffer) can overlap with steps 1108, 1110, and 1112 of flowchart 1100 for the first slide (e.g., steps associated with picking up the first slide from the slide buffer, scanning and imaging the first slide, and depositing the first slide into the slide buffer). Furthermore, steps 1134 and 1136 (e.g., steps associated with picking up the second slide from the slide buffer and depositing the slide into the slide holder) can also overlap with the scanning steps of the first slide. According to the systems described herein, time gains of up to 50% can be achieved based on parallel slide processing techniques compared to processing one slide at a time, with possible additional gains using other aspects of the systems and techniques described herein.

23A and 23B show timing diagrams for using slide caching techniques according to embodiments of the systems described herein and illustrate time savings according to various embodiments of the systems described herein.

FIG23A shows a timing diagram 1150 for a scenario in which a pre-scan step is used. The timing diagram shows the timing for three slides (slides 1, 2, and 3) over a span of approximately 300 seconds associated with using a slide buffer to perform slide processing steps, including picking a slide from a slide rack, thumbnail image processing, depositing the slide in a buffer, picking from the buffer, pre-scanning, scanning the slide and outputting a file, depositing into a buffer, and depositing into a slide rack. As illustrated, in an embodiment, the cycle time for the illustrated process can be approximately 150 seconds.

FIG23B shows a timing diagram 1160 for a scenario where dynamic live focus technology is used (without pre-scanning). The timing diagram shows the timing for three slides (slides 1, 2, and 3) over a span of approximately 150 seconds associated with using a slide buffer to perform slide movement and scanning steps, including picking a slide from a slide holder, thumbnail image processing, depositing the slide in a buffer, picking from the buffer, scanning the slide and outputting a file, depositing into a buffer, and depositing into a slide holder. As illustrated, in an embodiment, the cycle time for the illustrated process can be approximately 50 seconds.

FIG24 is a schematic diagram illustrating a slide buffer 1200 according to another embodiment of the system described herein. In the illustrated embodiment, no buffer is required, and using the slide buffer 1200, the pick, thumbnail, and storage times can be eliminated from the cycle time. The slide buffer 1200 can include two independently operated XY hybrid stages 1210, 1220. Each of the XY hybrid stages 1210, 1220 can have similar features to those discussed herein with respect to the XY hybrid stage 1013. A first slide holder 1211 can be positioned at the end of the stage 1210, and a second slide holder 1221 can be positioned at the end of the stage 1220. It should be noted that in connection with another embodiment of the system described herein, the first slide holder 1211 and the second slide holder 1211 can alternatively be referred to as parts of one slide holder. Two thumbnail cameras 1212, 1222 can serve each of the XY hybrid stages 1210, 1220. Each of the slide holders 1211 and 1221 can provide a slide to its associated XY hybrid stage 1210 or 1220 using a corresponding pickup head. A single microscope optical train 1230 can service both XY hybrid stages 1210 and 1220. For example, while one XY hybrid stage (e.g., stage 1210) is scanning a slide, the other (e.g., stage 1220) can perform its pickup, thumbnail, and deposit functions on another slide. These functions can overlap with the scanning time. Therefore, the cycle time can be determined by the slide scanning time, and according to the illustrated embodiment of the system described herein, the pickup, thumbnail, and deposit times are eliminated from the cycle time.

Figure 25A is a flow chart 1250 showing slide caching processing associated with a first slide for an embodiment of the system described for a slide caching device having two XY hybrid stages for slide processing. At step 1252, the first slide is picked up from the slide holder. After step 1252, processing proceeds to step 1254, where thumbnail processing is performed on the first slide. After step 1254, processing proceeds to step 1256, where the first slide is scanned and imaged according to the same techniques discussed further elsewhere herein. It should be noted that in various embodiments, the scanning and imaging techniques may include a pre-scan focusing step and/or use a dynamic focusing technique, such as a dynamic instant focusing technique. After step 1256, processing proceeds to step 1258, where the first slide is deposited back into the slide holder. After step 1258, processing with respect to the first slide is complete.

Figure 25B is a flow chart 1270 showing slide caching processing associated with a second slide for an embodiment of the system described for a slide caching device having two XY hybrid stages for slide processing. At step 1272, the second slide is picked up from the slide holder. After step 1272, processing proceeds to step 1274, where thumbnail processing is performed on the second slide. After step 1274, processing proceeds to step 1276, where the second slide is scanned and imaged according to techniques similar to those discussed further elsewhere herein. It should be noted that in various embodiments, the scanning and imaging techniques may include a pre-scan focusing step and/or use a dynamic focusing technique, such as a dynamic instant focusing technique. After step 1276, processing proceeds to step 1278, where the second slide is deposited back into the slide holder. After step 1278, processing with respect to the second slide is complete.

According to embodiments of the systems described herein that involve slide caching, the steps of flowchart 1250 for a first slide can be performed by the slide caching device in parallel with the steps of flowchart 1270 for a second slide to reduce cycle time. For example, steps 1272, 1274, and 1278 for the second slide (e.g., picking, thumbnail processing, and depositing) can overlap with step 1256 for the first slide (e.g., scanning/imaging of the first slide), and vice versa, such that the time for picking, thumbnail processing, and depositing is eliminated from the cycle time. Thus, according to embodiments of the systems described herein, the cycle time is determined solely by the time it takes to scan the slides.

Figure 26 is a schematic illustration of a slide caching device 1300 according to another embodiment of the system described herein. The slide caching device 1300 may include a slide holder configured as a carousel 1310, a slide transporter 1320, a buffer 1330, and an XY stage 1340. The carousel 1310 may include one or more slide holder devices (e.g., cassettes) defining positions 1312, 1312', 1312" at which slides, such as slide 1301, may be placed before and/or after being imaged by an imaging device 1350, which may have similar features and functionality as discussed elsewhere herein.

Positions 1312, 1312', 1312" are shown as an array of wedges (e.g., 8 wedges), and as discussed further elsewhere herein, the carousel 1310 can have a height such that multiple carrier positions extend below each of the top wedge positions 1312, 1312', 1312" shown. The carrier handler 1320 can include an arm 1322 that acts as a pick head and can include mechanical and/or vacuum devices to pick up carriers. The arm 1322 on the carrier handler 1320 can move between positions 1322a-d to move carriers between the carousel 1310, buffer 1330, and XY stage 1340.

The buffer 1330 may include a plurality of buffer positions 1332, 1334. One buffer position 1332 may be designated as a return buffer position 1332, where slides returned from the imaging device 1350 via the XY stage 1340 may be placed before being moved back to the carousel 1310 by the slide handler 1320. Another buffer position 1334 may be designated as a camera buffer position 1334, where slides to be sent to the imaging device 1350 may first have a thumbnail image of the slide captured according to the techniques discussed elsewhere herein. After the thumbnail image of the slide has been captured at the camera buffer position 1334, the slide may be moved to position 1342 on the XY stage 1340, which transfers the slide to the imaging device 1350 for scanning and imaging according to the techniques discussed elsewhere herein.

FIG27 is a schematic diagram illustrating another view of slide caching device 1300. Components of slide caching device 1300 may be capable of operating in various motions and with multiple degrees of freedom. For example, carousel 1310 may be rotatable along direction 1311 and may include multiple slide positions 1312a-d at multiple heights at each rotational position to accommodate multiple slides (shown as slides 1, 2, 3, and 4). In an embodiment, the plurality of slide positions 1312a-d in each of the wedge positions 1312, 1312', 1312" may include positions for, for example, 40 slides equidistantly positioned within the height of the carousel 1310, which may measure 12 inches in one embodiment. Further, the carousel 310 may also include a user tray 1314 having one or more slide positions 1314a, b where a user may insert slides to be imaged in addition to other slides in the carousel 1310. Interaction of a slide into the user tray 1314 (e.g., lifting the lid of the user tray 1314 and/or inserting a slide into one of the positions 1314a, b of the user tray 1314) may be used to trigger a bypass mode in which a slide from the user tray 1314 is processed instead of the next slide from the wedge position of the carousel 1310.

13. The arm 1322 of the slide handler 1320 is shown having at least three degrees of freedom in motion. For example, the arm 1322 can rotate along direction 1321a to engage each of the carousel 1310, the buffer 1330, and the XY stage 1340. Additionally, the arm 1322 can be adjustable along direction 1321b corresponding to different heights of positions 1312a-d of the carousel 1310. Additionally, the arm 1322 can extend along direction 1321c in connection with loading and unloading slides from the carousel 1310, the buffer 1330, and the XY stage 1340. In embodiments, it may be advantageous to minimize the arc of rotation of the arm 1322 and/or minimize other distances traversed by the arm 1322 and/or the slide handler 1320 in order to minimize idle time of the slide caching device 1300, as discussed further below. In various embodiments, the motion of the carousel 1310, slide handler 1320, and XY stage 1340 can be controlled by the same control system as discussed elsewhere herein. It should also be noted that in embodiments, the buffer 1330 and XY stage 1340 can be at the same height.

Slides can carry different types of biological samples. A biological sample can be a tissue sample (e.g., any cell collection) removed from a subject plant. In some embodiments, biological samples include, without limitation, tissue sections, organs, tumor sections, smears, frozen sections, cytological smears, or cell lines. Samples can be obtained using incisional biopsy, core biopsy, excisional biopsy, fine needle aspiration biopsy, core needle biopsy, stereotactic biopsy, open biopsy, or surgical biopsy. In some embodiments, the sample can be a section of a plant or plant tissue culture.

A slide can generally be a flat, transparent backing capable of carrying a sample for examination using an instrument such as an optical instrument (e.g., a microscope or other optical device). For example, slide 1301 of FIG. 26 can generally be a rectangular sheet of transparent material having a front surface for supporting a sample. Microscope slide 1301 can take the form of a standard microscope slide made of glass or other transparent material. In some embodiments, slide 1301 has a length of approximately 3 inches (75 mm), a width of approximately 1 inch (25 mm), and a thickness of approximately 1 mm.

Slide 1301 may include a label. The label may include a machine-readable code (such as a one-dimensional or multi-dimensional barcode or infoglyph, an RFID tag, a Bragg diffraction grating, a magnetic stripe, or a nanobarcode) with encoded instructions (e.g., imaging instructions), subject information, or tracking information. The label may be analyzed by a reader located at various locations in slide caching device 1300.

The slide can include a cover slip for protecting the sample. If the slide 1301 is a standard microscope slide, the cover slip can have a length ranging from about 0.5 inches (13 mm) to about 3 inches (76 mm), a width ranging from about 0.5 inches (13 mm) to about 1 inch (25.5 mm), and a thickness ranging from about 0.02 inches (0.5 mm) to about 0.08 inches (2 mm). In some embodiments, a standard cover slip having a length of about 50 mm, a width of about 24 mm, and a thickness of about 0.2 mm is used. Other sizes are possible if needed or desired. The cover slip can be made in whole or in part from one or more polymers, plastics, composites, glass, combinations thereof, or other suitable materials that can be generally rigid, semi-rigid, or flexible.

Figures 28A-28J are schematic illustrations of the slide buffering operation of the slide buffering device of Figures 26 and 27 according to an embodiment of the system described herein. According to an embodiment, the slide operations discussed herein minimize the system's dead time (i.e., the time during slide pickup and transfer operations that does not overlap with slide scanning and imaging operations). Dead time can include, for example, the dwell time during which the XY stage 1340 moves to a position that allows the slide handler 1320 to pick up a slide. Other contributions to dead time include moving the slide to the return position of the buffer 1330 and reloading the XY stage 1340 with a slide.

FIG28A begins the illustrated sequence, in which slide 2 is currently being scanned and imaged at imaging device 1350. Slides 1, 3, and 4 are waiting in carousel 1310 to be scanned and imaged, and slide handler 1320 is in position for delivering slide 2 to XY stage 1340. FIG28B shows slide handler 1320 rotating and lowering to load the next slide (slide 3) to be scanned and imaged, while slide 2 continues to be scanned and imaged. FIG28C shows slide handler 1320 transporting slide 3 to camera buffer position 1334 of buffer 1330 to obtain a thumbnail image of slide 3. FIG28D shows slide handler 1320 positioned to unload slide 2 from XY stage 1340 after scanning of slide 2 is complete, having returned it from imaging device 1350. Note that the time it takes for XY stage 1340 to move to the position to be unloaded is an example of slack time. The time after the XY stage 1340 is in the position to be unloaded with the slide 2 waiting thereon to be unloaded, and the slide 3 waits to be loaded onto the XY stage 1340 is an example of the docking time.

FIG28E shows slide 2 being transported by slide handler 1320 from XY stage 1340 to return position 1332 of buffer 1330. Slide handler 1320 then moves to a position to pick up slide 3 from camera buffer position 1334. FIG28F shows slide 3 being picked up from camera buffer position 1334 and unloaded onto XY stage 1340. FIG28G shows slide 3 currently being scanned while slide 2 is being picked up by slide handler 1320 from return buffer position 1332. FIG28H shows slide handler 1320, which has been rotationally and translationally moved to the appropriate position, returning to its position in carousel 1310. FIG28I shows slide handler 1320 translationally moved to the appropriate position to pick up slide 1 from carousel 1310. 28J shows slide handler 1320 transporting and unloading slide 1 at the camera buffer location where a thumbnail image of slide 1 is obtained, while slide 3 is still currently being scanned. Similar further iterations discussed in relation to the illustrated sequence may be performed with respect to any remaining slides on carousel 1310 (e.g., slide 4) and/or for any user slides inserted by the user into user tray 1314 to initiate bypass mode operation as discussed herein.

Further in accordance with the systems described herein, an illumination system can be used in conjunction with microscopy embodiments applicable to the various techniques and features of the systems described herein. It is known that microscopes can generally utilize Köhler illumination for brightfield microscopy. A key feature of Köhler illumination is that both the numerical aperture and area of the illumination are controllable via an adjustable iris, allowing the illumination to be adapted to machine a wide range of microscope objectives with varying magnifications, fields of view, and numerical apertures. Köhler illumination provides desirable results but can require multiple components that occupy a significant volume of space. Thus, various embodiments of the systems described herein further provide features and techniques for advantageous illumination in microscopy applications that maintain the advantages of Köhler illumination while avoiding certain disadvantages of known Köhler illumination systems.

FIG29 is a schematic diagram illustrating an illumination system 1400 for illuminating a slide 1401 using a light-emitting diode (LED) illumination assembly 1402, according to an embodiment of the system described herein. LED illumination assembly 1402 can have various features according to various embodiments discussed further herein. Light from LED illumination assembly 1402 is transmitted to condenser 1406 via a reflector 1404 and/or other suitable optical components. Condenser 1406 can be any condenser with a suitable working distance (e.g., at least 28 mm) to accommodate any required working distance of an XY stage 1408, as discussed further elsewhere herein. In one embodiment, the condenser can be the SG03.0701 condenser manufactured by Motic, which has a 28 mm working distance. Condenser 1406 can include an adjustable iris diaphragm to control the numerical aperture (cone angle) of the light illuminating the sample on slide 1401. Slide 1401 can be positioned on XY stage 1408 beneath microscope objective 1410. LED illumination assembly 1402 may be used in connection with scanning and imaging a sample on slide 1401, in accordance with features and techniques of the systems described herein, including, for example, operations with respect to XY stage movement, slide caching, and/or dynamic focusing.

The LED illumination assembly 1402 can include an LED 1420, such as a bright white LED, a lens 1422 that can be used as a collector element, and an adjustable iridescent field aperture 1424 that can control the illumination area on the slide 1401. The emitting surface of the LED 1420 can be imaged by the lens 1422 onto an entrance pupil 1406a of the condenser 1406. The entrance pupil 1406a can be co-located with an NA adjustment aperture 1406b of the condenser 1406. The lens 1422 can be selected to collect a large portion of the output light of the LED 1420 and also to focus the image of the LED 1420 onto the NA adjustment aperture 1406b of the condenser 1406 with an appropriate magnification such that the image of the LED 1420 fills the aperture of the NA adjustment aperture 1406b of the condenser 1406.

Condenser 1406 can be used with NA adjustment aperture 1406b to focus light from LED 1420 onto slide 1401. The illuminated area on slide 1401 can be controlled by field aperture 1424 mounted in LED illumination assembly 1402. The field aperture and/or the spacing between condenser 1406 and field aperture 1424 can be adjusted to image light from LED 1420 onto the face of slide 1401, such that field aperture 1424 can control the area of slide 1401 that is illuminated.

Because the image sensor acquires frames while the Y-stage containing the slide is moving, the LED 1420 can be pulsed on and off (e.g., strobed) to allow very high brightness for short periods of time. For example, for a Y-stage moving at approximately 13 mm/sec, to maintain a blur of no more than 0.5 pixels (0.250 microns/pixel), the LED 1420 can be pulsed on for 10 microseconds. In accordance with the focusing systems and techniques discussed further elsewhere herein, the LED light pulses can be triggered by a master clock that is locked to the dither lens resonant frequency.

30 is a schematic illustration showing a more detailed side view of an embodiment of an LED lighting assembly 1402′ in accordance with the systems described herein and corresponding to features described herein with respect to LED lighting assembly 1402. The implementation and configuration of LED 1430, lens 1432, and field aperture 1434 are shown relative to and in relation to other structural support and alignment components 1436.

Figure 31 is a schematic illustration of an exploded view of a particular embodiment of an LED lighting assembly 1402" having features and functions similar to those discussed with respect to LED lighting assembly 1402 in accordance with an embodiment of the system described herein. Adapter 1451, base 1452, clamp 1453, and base 1454 can be used to securely mount and position LED 1455 in LED lighting assembly 1402" so as to be securely positioned relative to lens 1462. The LED lighting assembly 1402" may further be secured and mounted using appropriate screw and washer assemblies 1456-1461. In various embodiments, the LED 1455 may be a Luminus, PhlatLight White LED CM-360 series, a bright white LED with an optical output of 4,500 lumens and a long life of 70,000 hours, and/or a suitable LED manufactured by Luxeon. The lens 1462 may be an MG9P6mm, 12mm OD (outer diameter) lens. The lens 1462 may be positioned and mounted relative to the adjustable field aperture assembly 1465 using a tube lens assembly 1463, an adapter 1464, a stacking tube lens assembly, and a retaining ring 1467. The adjustable field aperture assembly 1465 may be a ring activated iris diaphragm manufactured by Thor Labs, part number SM1D12D. The stacking tube lens 1466 may be a P3LG stacking tube lens manufactured by Thor Labs. The tube lens 1463 may be a tube lens assembly 1464 manufactured by Thor Labs. Labs manufactured P50D or P5LG tube lens. Additional washers 1468 and screw components 1469 may be used, where appropriate, to further secure and mount components of the LED lighting assembly 1402".

Further in accordance with the systems described herein, apparatus and techniques are provided for high-speed slide scanning for digital pathology applications according to various embodiments of the systems described herein. In one embodiment, a slide holder for a pathology microscope may include: (i) a tray in the form of a circular disk and (ii) a plurality of recesses formed in the tray, wherein each recess is adapted to accommodate a slide and the recesses are circumferentially arranged in the tray. The tray may include a central axis hole and two locking holes, wherein the locking holes are adapted to engage a drive adapted to rotate at high speed about an axis orthogonal to the tray. The recesses may be milled into the tray at different angular positions. The recesses may have semicircular protrusions to contact the slide without excessively constraining it, thereby allowing the slide to be substantially strain-free. The recesses may also have cutouts that allow a fingerhole for an operator to place and extract a slide from the recesses. In various embodiments, the slide holder and its operation may be used in conjunction with the features and techniques discussed elsewhere herein with respect to imaging systems.

FIG32 is a schematic diagram illustrating a high-speed slide scanning device 1500 that can be used in connection with digital pathology imaging according to an embodiment of the system described herein. A slide holder 1510 can include a tray 1512 having recesses 1514a, b, ...n disposed at angular locations on a circumference or annular ring 1515 of the tray 1512. Each recess 1514a-n can be sized to hold a slide 1501. The tray 1512 is illustrated as a circular disk and can be manufactured to hold a desired number of slides. For example, to hold 16 slides, the tray 1512 can measure approximately 13 inches in diameter. It should be noted that other configurations of slide and tray sizes and shapes can be used in connection with the system described herein (where appropriate), and the orientation and configuration of the recesses 1514a-n can be modified appropriately. A slide can be placed in each recess 1514a-n of the tray 1512, such as slide 1501 in recess 1514a, and the tray 1512 can be placed into the high-speed slide scanning apparatus 1500. The tray 1512 can include a central axis hole 1516c and two locking holes 1516a and 1516b that can engage a drive that rotates the slide holder 1510 at high speed in a rotational direction 1519 about an axis 1518. The tray 1512 can be placed into a low-profile drawer, representatively shown as 1502, which can retract the tray 1512 into the apparatus 1500.

FIG33 is a schematic illustration of a recess 1520 on a tray of a high-speed slide scanning apparatus according to an embodiment of the system described herein in greater detail. Recess 1520 can be any of recesses 1514a-n. Recess 1520 can include a plurality of semicircular protrusions, such as three protrusions 1522a-c, to touch the slide 1501 but not overly constrain the slide 1501, thereby allowing the slide 1501 to be substantially strain-free. A cutout 1523 allows a finger hole to be placed and removed from the recess 1520 by an operator. The centripetal acceleration, schematically shown by arrow 1521, generated by the slide holder 1510/tray 1512 as it rotates about axis 1518 can apply a small retaining force to the slide 1501 to hold it in place while imaging occurs. The holding force can be designed to be at least 0.1 g's by initially rotating the tray 1512 at a rate greater than 100 rpm to align the slide 1501 against the semicircular protrusions 1522a-c. Once the slide 1501 is aligned, the rotation rate can be reduced according to the imaging rate of the system similar to the imaging rates discussed elsewhere herein. At lower rates, even a slight holding force will stabilize the slide 1501 against the protrusions 1522a-c.

Referring again to FIG. 32 , a microscope imaging system 1530, similar to those discussed in detail elsewhere herein, can be positioned above the rotating tray 1512 to image the area of the circumferential ring 1515 in which the slide is positioned. The imaging system 1530 can include a high-NA microscope objective 1532, such as a 0.75 NA with a large working distance, an intermediate lens 1534, and a CCD or CMOS 2D array image sensor 1536 positioned at an appropriate distance to magnify the object on the slide 1501 to the image sensor 1536. The image sensor 1536 can have a high frame rate, such as greater than 100 frames per second. For example, the image sensor 1536 can be a portion of a Dalsa Falcon 1.4M100 camera operating at 100 frames per second, or an equivalent. The imaging system 1530 can be rigidly mounted to a 2-axis motorized drive, which can be constructed from components such as a DC motor or stepper motor, ball or lead screws, and/or linear guides. One axis, radial axis 1531a, can move imaging system 1530, or at least one component thereof, radially with small movements, for example, in 1 mm steps with a resolution of 10 microns, to image one or more rings on the underlying spinning tray 1512. Another axis, focus axis 1531b, can move with small movements of 5-10 microns with a resolution of 0.1 microns. The focus axis can be configured to perform movements at high speeds, for example, in milliseconds. Movement of microscope objective 1534 can be controlled by a control system and can be used in conjunction with the same dynamic focus techniques discussed elsewhere herein.

An illumination system 1540 can be placed beneath the rotating tray 1502 and include a light source 1542, such as a high-brightness white LED, one or more optical path components, such as a reflector 1544, and a condenser 1546 similar to the illumination assemblies discussed elsewhere herein. In embodiments, the condenser and imaging path of the microscope can be coupled together and moved as a rigid body, with the direction 1541 of movement of the illumination system 1540 being in the same direction as the radial direction 1531a of the imaging system 1530. In the focus direction 1531b, the imaging path can be decoupled from the condenser path such that one or more components of the imaging system 1530 can include independent movement along the focus direction 1531b to perform high-speed focus movement.

FIG34 is a schematic illustration of an imaging path starting at a first radial position relative to slide 1501 to image sample 1501′ on slide 1501 in recess 1520. Recess 1520 with slide 1501 rotates along rotational direction 1524 with slide holder 1510. Images can be captured for frames (e.g., frame 1525) according to image capture techniques discussed elsewhere herein. As shown, as tray 1512 rotates under imaging system 1530, images are captured for a row of frames (e.g., frame 1525) for each slide on slide holder 1510. After one full rotation of tray 1512, the radial position of imaging system 1530 is increased to capture another row of frames for each slide. Each frame is acquired at high speed, temporarily freezing the underlying scene. Brightfield illumination can be sufficiently luminous to allow for such short exposures. These exposures can be within a timeframe of tens to hundreds of microseconds. This process continues until the entire region of interest has been imaged for each slide in the slide holder 1510. As relevant to the present embodiment, the processing of the collected images into a mosaic image of the region of interest requires appropriate organization and/or image labeling to correctly correlate the multiple rows of frames between the multiple slides rotating on the tray 1512. Appropriate image processing techniques can be used to label the images so that the captured images are associated with the appropriate slides, as the arcuate motion of the image slice collection can be resolved using known stitching software and transformed into a view that a pathologist will understand when viewing under a standard microscope.

As an example, with a tray in the form of a 13.2-inch diameter disc rotating at 6 rpm, a 20x microscope objective with NA=0.75 produces a field of view of approximately 1 mm ^2. This arcuate field of view is traversed in approximately 10 msec. For tissue sections within a 15 mm ² active area and assuming a 25% overlap between fields of view, 20 additional fields would need to be acquired along the axis of rotation. If the frame transfer is short enough not to limit acquisition time, 20 complete rotations would be sufficient to image 16 slides on the disc. This would occur in 200 seconds at 6 rpm, or a throughput of 1 slide every 12.5 seconds.

Figures 35A and 35B are schematic illustrations showing an alternative arrangement of slides on a rotating slide holder according to another embodiment of the system described herein. Figure 35A shows a tray 1512' having a recess 1514' configured so that the longer dimension of slide 1501 is oriented along the radius of the disk-shaped tray 1512' rotated in direction 1519'. In this configuration, more slides (e.g., 30 slides) can fit on tray 1512'. Figure 35B is a schematic illustration showing the imaging path for slide 1501 in recess 1520' configured as mentioned above. In the illustrated embodiment, slide 1501 is held in recess 1520' by a centripetal force indicated by direction 1521' and protrusions 1522a'-c'. The direction of rotation 1524' on which image processing is performed is shown for an image set of frame 1525' of sample 1501'. The radial position of the imaging system 1530 is increased in increments along the length of the slide to capture images for successive rows of frames for each slide. In this example, for a 15 mm x 15 mm active area and assuming a 25% overlap between fields, twenty additional fields would need to be added along the radial axis. Again, 20 rotations at 6 rpm would provide complete imaging in 200 seconds, but given the more efficient scanning, slide orientation and therefore throughput would increase to one slide every 6.67 seconds.

FIG36 is a schematic diagram illustrating an imaging system 1550 including an objective lens 1552 configured to inspect a sample 1551′ on a slide 1551, according to an embodiment of the system described herein. In embodiments, the focus position can be predetermined by prior, slower rotation of the disk prior to image acquisition. A budget of up to 20 seconds per slide for autofocus would result in a total scan time per slide of under 30 seconds—an order of magnitude faster than the current state of the art systems. As the tray 1560, on which the slides 1551 are positioned, rotates in direction 1561, the objective lens 1552 can undergo slight movement in direction 1562 to be positioned at the optimal focus determined according to the system described herein. Different autofocus values need not be set for each field of view 1553, but rather applied to different larger regions 1554 on the slide 1551, such as a 3×3 field of view or subframe due to the greater spatial frequency of slide warpage or tissue thickness. The autofocus values will be interpolated using optimal focus as the slide moves beneath the camera in its arcuate path.

Alternatively, dynamic focusing techniques, such as the dynamic instantaneous focusing techniques described elsewhere herein, can be advantageously employed in connection with the high-speed scanning systems provided herein. It should be noted that the time required to acquire a focus point (e.g., 120 focus points per second) enables the use of dynamic instantaneous focusing in conjunction with the high-speed rotational scanning techniques discussed above. It should further be noted that it is well within the scope of a control system to control the speed of the rotating disk to within 1/10,000, allowing open-loop sampling of each image without relying on feedback from the rotation of the disk.

Typically, a low resolution thumbnail image of the slide is produced. This can be accomplished by placing a low resolution camera at an angular position on the disc so as not to interfere with the high resolution microscope just described. For very high volume applications, the disc format facilitates robotic handling. A semiconductor wafer robot can be used to handle 300 mm (~12") discs to move the discs from a buffer stock to a high speed scanner. In addition, most technologies position the slide under the microscope objective using a linear stage in a step and repeat motion. These motions dictate the image acquisition times. The system described herein using rotational motion is efficient and highly repeatable. Autofocus and image acquisition times are an order of magnitude less than the current state of the art products.

Most systems also require a clamping mechanism or spring hold-down to hold the slide in place during the stopping and starting motion of the stage. The system described herein does not require a hold-down mechanism because the rotational motion generates centripetal acceleration that pushes the slide into a predetermined position in the recess cut into the disc. This makes the construction of the slide holder simpler and more reliable. Additionally, a slide hold-down could warp or strain the slide, complicating the autofocus process and is advantageously avoided according to the system described herein.

Current systems have a peak speed of 2-3 minutes for an active area of 15 mm per slide. For the examples listed above, the systems and methods provided herein allow the same active area to be scanned in 30 seconds. Many pathology laboratories expect to scan from 100 to 200 slides per day. With these high rates of image acquisition, an operator can work through a daily inventory of slides in under an hour, including the additional steps of loading and unloading discs, barcode reading, and pre-focusing. This allows for faster time to results and enhanced economic benefits for the laboratory.

FIG37 is a flowchart 1600 illustrating high-speed slide scanning using a rotatable tray according to an embodiment of the system described herein. At step 1602, a slide is positioned in a recess of the rotatable tray. Following step 1602, processing proceeds to step 1604, where the rotatable tray is moved relative to the scanning and imaging system to a slide scanning position. Following step 1604, processing proceeds to step 1606, where rotation of the rotatable tray is initiated. As discussed above, the rotation of the rotatable tray causes a centripetal force to act on the slides to maintain the slides in the desired imaging position. Following step 1606, processing proceeds to step 1608, where the imaging system captures an image for a row of frames for each slide on the circumferential ring of the rotatable tray, in accordance with the systems and techniques described herein and including dynamic focusing techniques. Following step 1608, processing proceeds to a test step 1610, where a determination is made as to whether the desired region of interest on each slide on the rotatable tray has been scanned and imaged. If not, then processing proceeds to step 1612, where the imaging system and/or certain components thereof are moved one increment along the radial direction of the rotatable tray. After step 1612, processing proceeds back to step 1608. If at test step 1610, it is determined that the region of interest on each slide has been scanned and imaged, then processing proceeds to step 1614, where one or more mosaic images are created corresponding to the region of interest imaged for each slide. After step 1614, processing is complete.

Further in accordance with the systems described herein, optical doubling devices and techniques can be provided and used in conjunction with the imaging system features described herein. In one embodiment, the system described herein can sample the resolution element produced by a 20x 0.75 NA Plan Apo objective lens. This resolution element is approximately 0.5 microns at a wavelength of 500 nm. To achieve further sampling of this resolution element, the tube lens in front of the imaging sensor can be changed. Given the objective lens, an approximate calculation for calculating the focal length of the tube lens (f_tube lens = the focal length of the tube lens in front of the image sensor) is:

pix_sensor = pixel size on the CCD or CMOS image sensor

pix_object = pixel size of the object or tissue

f_tube lens = pix_object/pix_sensor*9 mm.

To achieve a pixel size of 0.25 micron for a Dalsa Falcon 4M30/60 (7.4 micron sensor pixels), the focal length of the tube lens should be approximately 266 mm. For a pixel size of 0.125 micron, the focal length of the tube lens should be approximately 532 mm. It may be desirable to switch between these two object pixel sizes, and this can be achieved by mounting two or more tube lenses to a stage that shuttles back and forth in front of the imaging sensor. Given the different path lengths associated with each new focal length, a fold mirror would also need to be added to fold the path for a fixed image sensor position.

FIG38 is a schematic diagram illustrating an optical frequency doubling imaging system 1700 according to an embodiment of the systems described herein. As described elsewhere herein, optical frequency doubling imaging system 1700 may include an image sensor 1710, a camera 1711, and a microscope objective 1720. It should be noted that other components associated with the systems and techniques discussed herein, such as a dynamic instant focus system, may also be used with the illustrated optical frequency doubling imaging system 1700. To achieve two or more object pixel sizes, multiple tube lenses, such as a first tube lens 1740 and a second tube lens 1750, may be provided in connection with the systems described herein. A stage 1730 may shuttle first and second tube lenses 1740 and 1750, respectively, in front of the imaging sensor. In an embodiment, stage 1730 may be a linearly actuated stage that moves along direction 1731, however, it should be noted that other types of stages and their movements may also be used in connection with the systems described herein. A mirror assembly 1752 is shown relative to the second tube lens 1750 , which may include one or more folding mirrors to adjust the optical path from the second tube lens 1750 to the image sensor 1710 .

Figures 39A and 39B are schematic diagrams illustrating an optical doubling imaging system 1700 that shuttles a first tube lens 1740 and a second tube lens 1750 in front of an image sensor 1710, according to an embodiment of the system described herein. Figure 39A illustrates an optical path 1741 for first tube lens 1740 positioned in front of image sensor 1710 on stage 1730. Figure 39B illustrates an optical path 1751 for second tube lens 1750 after being shuttled in front of image sensor 1710 via stage 1730. As illustrated, optical path 1751 has been increased using one or more mirrors of mirror assembly 1752. In both figures, it should be noted that optical doubling imaging system 1700 may include other suitable structure and optical components 1760 similar to those discussed in detail elsewhere herein.

FIG40 illustrates an imaging system 2000 having many components generally similar to those shown in FIG1 , 2 and 26 . An entry door 2002 can be opened to load a cassette onto the carousel. The cassette can carry a covered slide. After loading the cassette, the entry door 2002 can be closed. A controller 2004 can be used to operate the imaging system 2000. After imaging the sample, the door 2002 can be opened and the cassette removed. Advantageously, slides can be imaged while the user is loading and unloading the carousel to increase laboratory throughput. This also avoids unnecessary waiting time and ensures that slides are always ready for processing.

FIG41 shows the protective housing 2010 of FIG40 removed. FIG41 shows a slide buffering device 2011 including a carousel 2012 having nine upper and nine lower receptacles. Each receptacle is capable of receiving and holding a cassette or other type of slide holder device (e.g., a rack, magazine, etc.). The carousel 2012 rotates to position the cassettes at the front of the system 2000 to facilitate cassette loading and unloading.

41-43 , a slide can be moved to a buffer 2020 having a return 2022 and a camera 2024. The slide can be fed by a slide handler 2028 to a slide handler in the form of an XY stage 2026. Imaging optics 2030 can capture an image of a sample on a slide loaded in the stage 2026. Imaging optics 2030 can include, without limitation, one or more lenses, cameras, mirrors, filters, or light sources. A pickup device or end effector 2034 carried by an arm 2036 can transport the slide between various components.

FIG44 shows a pickup device 2034 transporting a slide 2050 without contacting the periphery of the label 2051 and without contacting the edge of the microscope slide adjacent to the label 2051, thereby avoiding contact with exposed residual glue (if any) used to mount the label 2051. The slide 2050 can reliably and efficiently transport the slide without problems caused by glue sticking to the pickup device 2034. As used herein, the term "slide" includes both covered microscope slides and microscope slides without a cover slip. The illustrated slide 2050 includes a cover slip. If the pickup device 2034 is part of a slide mounting apparatus, the pickup device 2034 can transport microscope slides without a cover slip.

The pickup device 2034 includes a connector unit 2038 and an end effector 2040. The connector unit 2038 fluidically couples a head element 2044 to a pressurized source (e.g., a pump or other device capable of drawing a vacuum). The head element 2044 is capable of maintaining a vacuum with respect to the slide 2050. Sufficient vacuum can be maintained to securely hold the slide 2050 and to transport the slide 2050 between workstations. The vacuum can be reduced or eliminated to release the slide 2050.

Connector unit 2038 includes a mounting body 2055 and a pair of mounting arms 2056a, 2056b that can be coupled to arm 2036 (see Figures 42 and 43). Fluid control components 2060 provide fluid communication with end effector 2040. Fluid components include, but are not limited to, fluid lines (e.g., hoses, tubing, conduits, etc.), valves, couplers (e.g., fluid line couplers), and other components for controlling or establishing fluid flow. In Figures 44 and 45, fluid component 2060 includes a delivery line 2062 that is fluidically coupled to end effector 2040.

The connector unit 2038 can allow the end effector 2040 and/or microscope slide 2050 to contact an object and allow the end effector 2040 to deflect and return to its original position to inhibit, limit, or substantially prevent damage to the end effector 2040 and/or microscope slide 2050. A protective head 2058 can serve as a collision protection feature. The head 2058 is movably coupled to the mounting body 2055. If the microscope slide 2050 or the end effector 2040, or both, contact an object, the head 2058 can rotate as indicated by arrows 2059a, 2059b in FIG. 46 to inhibit, limit, or prevent damage to the end effector 2050 and/or microscope slide 2050. A biasing member 2061 can urge the head 2058 into a fixed position. When sufficient force is applied to the end effector 2040, the biasing force provided by the biasing member 2061 is overcome, and the head 2058 moves relative to the mounting body 2055. When the applied force is sufficiently low or substantially eliminated, the biasing member 2061 pulls the head 2058 back to the fixed position. The biasing member 2061 may include, without limitation, one or more springs (including helical springs, coil springs, etc.), pneumatic biasing members, or other components capable of providing a biasing force.

44 and 45 , the end effector 2040 includes a platform 2070, a fluid line 2072, and a head element 2044. The platform 2070 has an upper surface 2074 and a lower surface 2076 ( FIG. 46 ) and can be made in whole or in part of a rigid material (e.g., metal, rigid plastic, etc.). The illustrated platform 2070 is mounted in a cantilevered manner to the connector unit 2038 and can be made of sheet metal, such as stainless steel.

47 , the sensor 2089 may include one or more circuits (e.g., flexible circuits) having a relatively low profile to analyze the microscope slide. To detect the presence of a slide, the sensor 2089 may be an optical sensor capable of analyzing a reflective surface. By way of example, the sensor 2089 may detect the presence of a slide by evaluating the reflection of a slide label (if any). The sensor 2089 may send a signal indicating the presence of a slide. Based on the signal, the controller 2004 may determine whether the pickup device 2034 holds a slide. Alternatively, the sensor 2089 may take the form of a barcode reader, a scanner, a contact sensor, a proximity sensor, or a combination thereof. Any number of sensors may be used to determine information associated with the microscope slide 2050.

42, the spacers 2090a-d cooperate to hold the microscope slide 2050 in a substantially horizontal orientation and substantially parallel to the platform 2070. The front and rear spacers 2090c, 2090a are generally positioned along the longitudinal axis 2118 of the platform 2070. The lateral compensating spacers 2090b, 2090d are positioned against the longitudinal sides 2091, 2093 of the platform 2070. Any number of spacers defined in different types of patterns may be used based on the desired slide interaction.

To avoid sticking to excess residual label glue, spacers 2090 can be positioned so that spacers 2090a-d do not come into contact with such glue. Spacer 2090a contacts the top of label 2051 and is well spaced from the edge of the label to ensure that it does not come into contact with residual exposed glue. Alternatively, spacer 2090a can be moved out of the label area toward pickup 2044 and still provide stability to the slide. Spacers 2090b, 2090d can be positioned so as to contact the top surface of the microscope slide well spaced from the edge of label 2051.

Referring again to Figures 44-46, the fluid line 2072 is positioned on the upper surface 2074 and extends between the connector unit 2038 and the head element 2044. To enhance the rigidity of the end effector 2040, the fluid line 2072 may comprise a hypodermic tube made in whole or in part from steel (including stainless steel), a nickel alloy, or other suitable rigid material. In some embodiments, the outer diameter of the hypodermic tube may be less than approximately 0.3 inches. The hypodermic tube gauge may range from 7 to 32 based on a Stubs needle gauge and may be selected based on the desired vacuum to be drawn and the desired mechanical properties of the pickup device 2034. Other tube sizes may also be used. The position of the fluid line 2072 relative to the platform 2070 may be selected to achieve a desired moment of inertia and overall profile. The fluid line 2072 may be moved away from the platform 2070 to enhance the rigidity of the end effector 2040 by increasing the moment of inertia. In order to reduce the maximum height of the end effector 2040, the fluid line 2072 can be moved against or in contact with the platform 2070.

The clamp assembly 2077 includes a clamp body 2078 for holding the fluid line 2072 and fixtures 2080a, 2080b for opening and closing the clamp assembly 2077. To reduce the number of parts of the pickup device 2034, the fluid line 2072 can be directly coupled (eg, adhered, glued, welded) to the platform 2070.

48 , the head element 2044 includes a connector 2100 and a pipette tip 2102. The connector 2100 is positioned on the upper side of the platform 2070 and is coupled to the fluid line 2072. The pipette tip 2102 is positioned on the lower side of the platform 2070 and is capable of maintaining a vacuum with the covered microscope slide 2050. In some embodiments, the pipette tip 2102 can be made of a material that is sufficiently flexible (e.g., rubber, silicon, polymer, etc.) to maintain an airtight seal when the outer edge 2103 of the head 2102 covers the edge of the coverslip. This allows for consistent and reliable pickup even if the pipette tip 2102 is misaligned with the coverslip.

The locator 2110 is keyed to the platform 2070 and can serve as an anti-rotation feature to maintain the head element 2044 properly aligned with the platform 2070 even after a relatively long service life. The illustrated locator 2110 is a protrusion positioned in a complementary notch 2112. Additionally or alternatively, the head element 2044 can be coupled to the platform 2070 using one or more pieces of glue or adhesive. In yet another embodiment, the nozzle head 2102 can be mechanically coupled to the platform 2070 using mechanical fasteners (e.g., screws, pins, etc.).

The end effector 2040 can have a relatively low profile to facilitate access into relatively confined spaces. FIG48 illustrates the maximum height H of the end effector 2040, which can range from approximately 0.15 inches to approximately 0.19 inches. This allows the end effector 2040 to be inserted between microscope slides held in a wide variety of different slide holders, including slide holders with vertically spaced shelves. Such slide holders can take the form of storage racks, magazines, and portable cases, among others. The end effector 2040 can have various configurations, sizes, and dimensions, as well as component arrangements selected based on the desired application. The width W of the end effector 2040 (see FIG45 ) can be equal to or less than the width of the slide 2050. In some embodiments, including the illustrated embodiment of FIG45 , the end effector 2040 has a width W that is substantially less than the width of the slide 2050. This allows the end effector 2040 to be positioned from side to side relative to the slide 2050.

FIG49 illustrates a microscope slide holder in the form of a cassette 2400. The cassette 2400 includes a main body 2410 and vertically spaced shelves 2414. Covered slides 2422 (illustrated in dashed lines) are supported by the uppermost shelf 2414a. If the cassette 2400 is manually transported between workstations, the user can hold the cassette 2400 in a generally vertical orientation, as illustrated. Even if the cassette 2400 is tilted from the vertical orientation, the slides can be conveniently retained on the shelves 2414. The cassette 2400 is portable for easy transport between workstations. The latches help prevent the slides from slipping out of the cassette 2400 during transport (e.g., when transporting the cassette 2400 between laboratory equipment) or from slipping out of the cassette due to mechanical vibrations, such as those caused by motor-driven equipment on or adjacent to the slide holder.

49 and 50 , the body 2410 includes a front opening 2430, an upper wall 2444, a lower wall 2446, a rear wall 2448, and sidewalls 2452, 2454. The upper wall 2444 extends between the sidewalls 2452, 2454 and defines an access opening 2460. The lower wall 2446 extends between the sidewalls 2452, 2454. A pick-up device can pass through the access opening 2460 to load and unload slides. The lower wall 2446 can be placed on a support surface to hold the cassette 2400 in a generally upright orientation. The handles 2447, 2449 can be gripped to handle the cassette 2400.

The rear wall 2448 includes ventilation openings 2470 through which fluids (e.g., air, mounting fluid, etc.) can pass. The illustrated openings 2470 are adjacent to the spaces between adjacent shelves 2414. If the slides are wet, air can pass through the openings 2470 for air drying, dredging, or both. The length of the openings 2470 can be less than the width of the slides to prevent the slides from passing through the rear wall 2448. Additionally or alternatively, the ventilation openings can be located elsewhere, such as along one or both of the side walls 2452, 2454.

The body 2410 can be made in whole or in part of plastic, polymer, metal, or combinations thereof and can have a one-piece or multi-piece construction. Non-limiting example plastics include, without limitation, acrylonitrile butadiene styrene (ABS), polyurethane, polyester, polypropylene, or combinations thereof. Fillers may also be utilized to enhance performance. In some embodiments, the body 2410 is made of glass-filled (e.g., 30% glass by volume or weight) ABS plastic. The surface of the shelf 1412 can have a relatively low coefficient of friction to facilitate slide placement. In cast embodiments, two cast shelves can be welded, bonded, or otherwise coupled together to form the cassette 2400. Any number of cast sections can be assembled and coupled together. Alternatively, machined components can be assembled together to form the cassette 2400. Mechanical fasteners, glue, or welding can be used.

50 is positioned behind the cartridge 2400. If the workstation has a magnet (e.g., a permanent magnet or an electromagnet), the coupler 2474 can be made in whole or in part of a ferromagnetic material or other material that is attracted to the magnet to help retain the cartridge 2400 in the workstation. Alternatively, the coupler 2474 can include a magnet for coupling to a ferromagnetic component of the workstation.

FIG50 also shows a pair of protruding keying features 2482, 2484. When the cassette 2400 is moved into the docking station, the keying features 2482, 2484 can be inserted into corresponding receptacles (e.g., slots, openings, etc.) of the docking station. If the cassette 2400 is inserted upside down, the keying features 2482, 2484 will not be received by the receptacles, thereby preventing docking. The illustrated keying features 2482, 2484 are generally U-shaped parallel members. The position, configuration, and orientation of the keying features can be selected based on the cassette size, desired cassette orientation, and design of the docking station. The tang 2485 of FIG49 can trigger a mechanism (e.g., a mechanical interlock) or sensor that sends a signal indicating that the cassette 2400 is properly installed. Additionally or alternatively, contact sensors, proximity sensors, or other types of sensors may be placed at various locations (eg, within the carousel 2012) to determine whether the cartridge 2400 is properly loaded.

Figures 51 and 52 illustrate 20 vertically spaced shelves 2414 for simulating 20 slides. Each shelf 2414 is positioned between sidewalls 2452, 2454 and is oriented to support microscope slides in a substantially horizontal orientation when the cassette 2400 is in an upright orientation. The shelves 2414 can be spaced evenly or unevenly from one another. In evenly spaced embodiments, the average distance between adjacent shelves 2414 can range from approximately 0.25 inches to approximately 0.38 inches. The average distance can be selected to achieve a desired clearance for the pickup head. The size, pitch, and other dimensions of the shelves can be selected to facilitate transfer between slide holders. By way of example, the pitch of the shelves 2414 can be generally similar to the pitch of the slots or shelves of a dip and dunk type basket, thereby facilitating the insertion of slides from the basket into the cassette. If slides are transferred from a slide rack to the cassette 2400, the shelves 2414 can have a pitch that generally corresponds to the pitch of the shelves of the rack. Thus, the spatial arrangement of the shelves 2414 can be selected based on different processing parameters and criteria.

51, the openings are labeled to facilitate positioning of the slides. The uppermost opening 2490a is labeled with the letter "A," and the lowermost opening 2490t is labeled with the letter "T." Alternatively, the openings may be labeled with numbers or other "markers."

Figures 51 and 52 show a covered slide 2422 (illustrated in dashed lines) supporting a sample on a rack 2414a. A pair of support members 2500a, 2500b supports the label end 2504 of the slide 2422. Each of the support members 2500a, 2500b extends away from the rack 2414a. In a one-piece implementation, the support members 2500a, 2500b are integrally formed with the rack 2414a. In a non-one-piece embodiment, the support members 2500a, 2500b are coupled to the rack 2414a using adhesives, fasteners, male/female connectors, and the like.

Support members 2500a, 2500b can be generally similar to each other, and therefore, unless otherwise indicated, a description of one also applies to the other. Figures 53 and 54 illustrate support member 2500a including an elongated body 2508a and a clip 2510a. Elongated body 2508a includes a mounting end 2516a coupled to shelf 2414, an opposite free end 2518a carrying clip 2510a, and a central portion 2519a.

A shoulder 2522a of the clamp 2510a protrudes upward from the elongated body 2508a and can restrict movement of the microscope slide 2422 relative to the support member 2500a. The shoulder 2522a can hinder sliding of the slide 2422. The height _Hs of the shoulder 2522a can be less than the thickness _Ts defined by the mounting surface and the bottom surface of the slide 2422. In some embodiments, the height _Hs is equal to or less than approximately 60%, 50%, or 40% of the thickness _Ts . In one embodiment, the shoulder height _Hs is equal to approximately half the thickness Ts. Other shoulder heights _Hs are possible if desired or required. The distance between the clamp 2510a and the back wall 2532 can generally be equal to or slightly greater than the longitudinal length of the slide 2422. This can help minimize movement of the slide 2422 during transport.

The abutment surface 2528a can be generally perpendicular to the support surface of the elongated body 2508a. In other embodiments, the catch 2518 can be a barb, a protrusion (e.g., a partially spherical protrusion, a tang, etc.), or other features for limiting (e.g., inhibiting or preventing) movement of the slide 2504.

55 , shelf 2414 has a generally H-shaped plate. The plate may generally have a flat upper surface, and in some embodiments may be textured or polished to enhance loading and unloading of microscope slides. The illustrated shelf 2414 is connected to and spans between sidewalls 2452 and 2454. In other embodiments, shelf 2414 is cantilevered from one of the sidewalls.

The elongated body 2508a has a longitudinal axis 2540a that is substantially parallel to the longitudinal axis 2540b of the elongated body 2508b of the support member 2520b. The longitudinal axes 2540a, 2540b may lie in a plane and may define an angle, if any, of less than approximately 5 degrees. The illustrated embodiment has two support members, but additional support members and any number of racks may be used.

Figures 56A and 56B show a microscope slide 2422 (illustrated in dashed lines) placed on the upper shelf 2414. Guides 2560a, 2560b can help accommodate changes in slide placement from one side to the other. As shown in Figure 56A, the slide 2422 is positioned to the left of the opening 2490a. Due to gravity, the edge 2561a of the slide 2422 can slide along the surface 2562a to allow the slide 2422 to fall into the desired position. The inclined surfaces 2562a, 2562b are inclined to allow the microscope slide to slide smoothly downward and onto the shelf 2414a. In some embodiments, the angles of inclination of the surfaces 2562a, 2562b can be generally equal to each other.

40 , to operate the imaging system 2000, a user can open the access door 2002 to insert a cartridge into one of the 18 receptacles (e.g., slots) of the carousel 2012. The carousel 2012 can be rotated sequentially for loading and unloading. Advantageously, the carousel 2012 can have a relatively compact design to reduce the overall footprint of the system 2000.

Once the cartridge is loaded into the dock, knob 2602 (see FIG. 41 ) can be rotated counterclockwise (e.g., approximately 90 degrees counterclockwise) to lock the cartridge in place. Additionally, rotation of knob 2602 can cause rotation of a corresponding identifier (see identifier 2607) that is analyzed by sensor 2606. The position of the identifier corresponds to the position of knob 2602. Thus, sensor 2606 can determine whether the knob is in the open or closed position.

Sensor 2606 may be an optical sensor capable of analyzing the position of the three upper boxes and the three aligned lower boxes.

In the illustrated embodiment, sensor 2606 determines the presence of cartridges 2400a-c based on the position of markers attached to knobs 2602a-c. Cartridges 2400d-f are analyzed by another sensor. Advantageously, the optical sensor/marker combination allows a fixed rod within the center of the carousel to analyze the presence of a cartridge, thereby eliminating the need for wiring across the rotational boundary and eliminating the use of complex cable retainers. This can help increase the reliability of carousel 2012. In some embodiments, tangs 2485 (see FIG. 50 ) are used to trigger a mechanical interlock that allows knob 2602 to rotate, while the knob of an empty socket cannot be rotated.

Referring again to FIG. 42 , the slide manipulator 2028 moves the pickup device 2034 to transport slides between the various components of the imaging system 2000. FIG. 52 shows the low-profile end effector 2034 positioned to be inserted into the opening 2490a (as indicated by arrow 2616). Once the end effector 2034 is positioned over the microscope slide 2422, it can be placed on the microscope slide 2422. A vacuum can be drawn between the suction head 2102 and the microscope slide. The end effector 2034 can move the slide 2422 vertically through the clamp 2510a. The raised slide 2422 can be removed from the opening 2490a. The end effector 2034 can transport the slides 2422 to the desired location without contacting the adhesive (e.g., adhesive at the periphery or adjacent edges of labeled microscope slides). The pick-up device can move the slides to any number of processing stations, including (without limitation) a carousel, a buffer, an XY stage, a slide manipulator, a slide buffer, a rack, an imaging device, or a docking station. After imaging the slides, the slides can be moved from the slide buffer 2020a to an empty shelf of the cassette. Once the cassette is filled with imaged slides, the cassette can be rotated to the access area for easy removal using the access door 2002.

The various embodiments discussed herein can be combined with one another in appropriate combinations in connection with the systems described herein. Additionally, in some instances, the order of steps in the flowcharts, program diagrams, and/or described process flows can be modified where appropriate. Furthermore, various aspects of the systems described herein can be implemented using software, hardware, a combination of software and hardware, and/or other computer-implemented modules or devices having the described features and performing the described functions. Software implementations of the systems described herein can include executable code stored in a computer-readable storage medium and executed by one or more processors. Computer-readable storage media can include computer hard drives, ROMs, RAMs, flash memories, portable computer storage media such as CD-ROMs, DVD-ROMs, flash drives, and/or other drives having, for example, a Universal Serial Bus (USB) interface, and/or any other suitable tangible storage medium or computer memory on which executable code can be stored and executed by a processor. The systems described herein can be used in connection with any suitable operating system.

Other embodiments of the present invention will be apparent to those skilled in the art from consideration of the specification or practice of the invention disclosed herein. It should be noted that the specification and examples are to be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims (18)

1. A low-profile microscope slide picking device, comprising: End effector, including: An elongated platform having an upper surface and a lower surface. Fluid piping, which is positioned on the upper side of the platform, and The head element includes a connector and a suction head, the connector being positioned on the upper side of the platform and coupled to the fluid line, and the suction head being positioned on the lower side of the platform. 2. The low-profile microscope slide pickup device according to claim 1, wherein the suction tip is sufficiently flexible to engage a covered microscope slide and maintain an airtight seal with the covered microscope slide, while maintaining sufficient vacuum to transport the microscope slide. 3. The low-profile microscope slide pickup device according to claim 1, wherein the suction tip is configured to maintain a seal with the covered microscope slide when the suction tip covers the edge of the cover and physically contacts the microscope slide under the cover. 4. The low-profile microscope slide picking device according to claim 1, wherein the head element has an anti-rotation feature that is housed by a storage feature of a complementary shape of the platform. 5. The low-profile microscope slide picking device according to claim 1, wherein the head element is adhered to the platform. 6. The low-profile microscope slide picking device of claim 1, wherein the end effector has a maximum height equal to or less than approximately the width of the end effector, and a longitudinal length longer than both the height and the width. 7. The low-profile microscope slide pickup device according to claim 1, further comprising: A connector unit includes a mounting body and a feed line, wherein the platform is coupled to the mounting body and the fluid line is fluidly coupled to the feed line. 8. The low-profile microscope slide picking device according to claim 1 further includes a connector unit, wherein the connector unit includes a collision protection head. 9. The low-profile microscope slide pickup device according to claim 1, wherein the fluid conduit includes a hypodermal injection tube. 10. The low-profile microscope slide picking device of claim 1, wherein the end effector includes a plurality of protrusions for contacting a covered slide held by the end effector when the platform is horizontally oriented to hold the covered slide in a substantially horizontal orientation. 11. The low-profile microscope slide picking device of claim 10, wherein the suction head is positioned between two protrusions relative to the longitudinal axis of the end effector. 12. A method for transporting a labeled microscope slide, the method comprising: The microscope slide being transported is picked up using the low-profile microscope slide pickup device of any one of claims 1-11 without touching the edge of the label on the microscope slide and without touching the edge of the microscope slide adjacent to the label; and The microscope slide is transported to the desired location using the low-profile microscope slide pick-up device. 13. The method of claim 12, further comprising: Microscope slides are transported between at least two processing stations without touching the edges of the labels and without touching the edges of the microscope slides adjacent to the labels. 14. The method of claim 12, further comprising: Position the pickup device on the microscope slide; and A vacuum is drawn between the pick-up device tip and the microscope slide to hold the microscope slide when the tip is positioned directly on the microscope slide. 15. The method of claim 12, further comprising: Position the pick-up device's suction tip on the microscope slide; A vacuum is drawn between the suction tip and at least a portion of the coverslip on the microscope slide; and Transporting microscope slides while maintaining a vacuum. 16. The method of claim 12, further comprising: The microscope slide is held in place by the suction tip of the pickup device, while at least one spacer physically contacts the upper surface of the microscope slide to hold the slide in the desired orientation. 17. The method of claim 12, wherein the microscope slide carries a coverslip covering the tissue sample. 18. The method of claim 12, further comprising: The pickup device is used to transport microscope slides between at least two processing stations without touching any part of the slide within 1 mm of the label.