JP2011008219A - Laser scanning microscope and control method - Google Patents

Abstract

To easily adjust an error between coordinates of a plurality of scanning systems.
A second scanning system 16-2 is applied to a fluorescent sample 34b arranged on a primary imaging surface 34 formed by a condenser lens 32 based on a coordinate system of the first scanning system 16-1. The coordinates of the irradiation spot of the laser beam irradiated via the are set. Then, after irradiating the irradiated portion with laser light through the second scanning system 16-2, the fluorescent sample 34b is scanned with laser light through the first scanning system 16-1, and scanning of the laser light is performed. An image of the region is acquired, and the coordinates of the location where the laser beam is actually irradiated through the second scanning system 16-2 are detected in the image. Thereby, based on the set coordinates and the detected coordinates, the correction for performing the correction to make the coordinate system of the second scanning system 16-2 coincide with the coordinate system of the first scanning system 16-1. A coefficient is determined. The present invention can be applied to, for example, a laser scanning microscope provided with a plurality of scanning systems.
[Selection] Figure 3

Description

  The present invention relates to a laser scanning microscope and a control method.

  2. Description of the Related Art Conventionally, there is a laser scanning microscope that includes a plurality of scanning units and observes a sample by irradiating the same observation surface with a laser beam scanned by each scanning unit.

  In general, the scanning means for scanning a laser beam is actually scanned with an assumed coordinate system even if an ideal coordinate system is set due to various mechanical or optical factors. Misalignment may occur with the physical coordinate system. Therefore, in a laser scanning microscope provided with a plurality of scanning means, it is difficult to make the coordinates of the respective scanning means completely coincide, and an error occurs between the coordinates of each scanning means.

  For example, in a laser scanning microscope including a scanning unit that scans the observation laser beam and a scanning unit that scans the stimulation laser beam, an image of the observation surface is first acquired using the observation laser beam. Then, when the stimulation location is set in the scanning means for the laser light for stimulation based on the coordinates of the scanning means for the observation laser light based on the image, it differs from the set stimulation location due to an error between the coordinates of the scanning means. The position is irradiated with stimulation laser light.

  For example, in Patent Document 1, even when an element (for example, a wavelength selection filter) that changes an optical condition is inserted in the optical path in the scanning unit, and the optical conditions among the plurality of scanning units are different, There has been disclosed a scanning laser microscope capable of reducing the influence caused by errors generated between the coordinates of the scanning means.

  However, in a laser scanning microscope having a plurality of scanning means, only the optical conditions in each scanning means do not cause an error. For example, it has different mechanical movable parts or different driving electric circuits. It is difficult to suppress the influence due to the error generated between the coordinates of each scanning means.

  For example, in a scanning means such as a galvano scanner or a resonant scanner configured to include an X scanner and a Y scanner, the error of each of the X scanner and the Y scanner, the error related to the optical arrangement of the X scanner and the Y scanner, the objective An error caused by the lens, an error caused by an optical axis shift between the scanning optical system and the objective lens optical system, an error related to combining optical paths of a plurality of scanning means, and the like cause errors between the coordinates of the respective scanning means. .

  As errors that each of the X scanner and Y scanner have, linearity error due to the relationship between the drive voltage and the rotation angle not being a linear function, magnification error due to the same change of the drive voltage range and the rotation angle range, and There is a misalignment due to the fact that the rotation axis of the scanner and the optical axis of the laser beam do not match. Further, as errors related to the optical arrangement of the X scanner and the Y scanner, the orthogonality error caused by the coordinate axes on the scanning plane not being orthogonal because the rotation axis of the X scanner and the rotation axis of the Y scanner are not precisely orthogonal. There is.

  The error due to the objective lens includes an error due to distortion of the lens and a positional shift due to a wavelength difference based on the aberration of the lens when using a light source having a different wavelength depending on the scanning system. Further, as errors related to combining the optical paths of a plurality of scanning means, there are errors due to reflection of a half mirror and a wavelength selection filter for optical path combining or tilting of the refractive surface.

  These factors act comprehensively, and an error occurs between the coordinates of each scanning means. For this reason, a laser scanning microscope is generally structured such that the mechanical mounting position of the scanner and electrical drive control can be adjusted, and an image acquired by laser light scanned by each scanning means. In comparison, mechanical adjustment is performed so that the error is minimized. Further, after mechanical adjustment, the error of the reference coordinate system with respect to the coordinates of each scanning means obtained from the image obtained by scanning the laser beam is retained in the laser scanning microscope as a correction coefficient, thereby correcting the error. Electrical adjustment using a coefficient is performed.

JP 2007-233241 A

  By the way, in general, in a laser scanning microscope, a scanning unit having a light source and scanning means and a microscope unit having a stage on which an objective lens and a sample are placed are configured as separate devices. And since it is necessary to adjust the error between the coordinates of a plurality of scanning systems based on the final image position, it is necessary to perform the adjustment in a state where the scanning unit and the microscope unit are combined.

  However, in a state where the scanning unit and the microscope unit are combined, an error caused by the scanning unit and an error caused by the microscope unit are combined, so that adjustment for eliminating the error becomes complicated.

  The present invention has been made in view of such a situation, and makes it possible to easily adjust an error between coordinates of a plurality of scanning systems.

  The scanning laser microscope of the present invention is a scanning laser microscope that scans a laser beam on a sample and acquires an image of the sample using the light from the sample, and a plurality of scans that scan the laser beam. And an objective lens disposed on the sample side, which is disposed on a common optical path in which the optical paths of the laser beams emitted from the plurality of scanning units are combined. And a condensing means for forming a primary imaging plane, and for the fluorescent sample arranged on the primary imaging plane, based on a coordinate system of a specific scanning means among the plurality of scanning means, The coordinates of the irradiation spot of the laser beam irradiated through the scanning means are set, the laser beam is irradiated onto the irradiation spot via the other scanning means, and the fluorescence is passed through the specific scanning means. The laser beam is scanned over the material, and the laser The image of the scanning region of the light is acquired, and the coordinates of the portion where the laser beam is actually irradiated through the other scanning means in the image are detected, the set coordinates, and the detected coordinates Based on the above, a correction coefficient for performing correction for matching the coordinate system of the other scanning unit with the coordinate system of the specific scanning unit is obtained.

  The control method of the present invention is a control method of a scanning laser microscope that scans laser light on a sample and acquires an image of the sample using light from the sample, and is disposed on the primary imaging plane. The coordinates of the laser beam irradiation spot irradiated through other scanning means are set on the fluorescent sample based on the coordinate system of the specific scanning means among the plurality of scanning means, and the other The irradiation part is irradiated with the laser light through the scanning means, the fluorescent material is scanned through the specific scanning means, and an image of the scanning region of the laser light is acquired, The coordinates of the part where the laser beam is actually irradiated through the other scanning means in the image are detected, and the coordinates of the other scanning means are determined based on the set coordinates and the detected coordinates. The coordinates of the particular scanning means Characterized in that it comprises a step of obtaining a correction coefficient for correcting to match.

  In the scanning laser microscope and the control method of the present invention, the other scanning means is applied to the fluorescent sample arranged on the primary imaging plane based on the coordinate system of the specific scanning means among the plurality of scanning means. The coordinates of the irradiation spot of the laser beam irradiated via the laser beam are set, and the laser beam is irradiated to the irradiation spot via other scanning means. In addition, the fluorescent material is scanned with the laser beam through a specific scanning unit, and an image of the scanning region of the laser beam is acquired, and the portion of the image where the laser beam is actually irradiated through the other scanning unit is acquired. Coordinates are detected. Then, based on the set coordinates and the detected coordinates, a correction coefficient for correcting the coordinate system of the other scanning unit to the coordinate system of the specific scanning unit is obtained.

  According to the scanning laser microscope and the control method of the present invention, it is possible to easily adjust an error between coordinates of a plurality of scanning systems.

It is a figure which shows the structural example of a laser scanning microscope provided with a some scanning system. It is a figure explaining the difference | error of the coordinate of an attention location, and the coordinate of a detection location. It is a figure which shows the structural example of the scanning apparatus in the laser scanning microscope to which this invention is applied. It is a figure which shows the state by which the fluorescence sample was fixed using the coupling | bond part. It is the figure which expanded and showed the vicinity of the primary image formation surface. It is a figure which shows the coordinate system in the image acquired in the maximum scanning range in a 1st scanning system. It is a figure which shows the point which performs marking when a scanning magnification is 1 time. It is a figure which shows the point which performs marking when a scanning magnification is 1.5 times. It is a figure which shows the scanning range in each magnification. It is a figure which shows the example which combined the more complicated mark coordinate. It is a figure explaining the relationship between resolution, a scanning range, and a spot diameter. It is a figure which shows the example of the marking for every magnification. It is a figure which shows the example which performs marking by a some spot also to a Y direction. It is a flowchart explaining the process which optically stimulates an attention location and observes. It is a figure which shows the example of the image acquired before and behind light stimulation.

  Hereinafter, specific embodiments to which the present invention is applied will be described in detail with reference to the drawings.

  A laser scanning microscope having a plurality of scanning systems will be described with reference to FIG.

  A laser scanning microscope 11 shown in FIG. 1 scans a laser beam on an observation surface 12 of a sample, and acquires an image of the observation surface 12 using light from the observation surface 12. In the laser scanning microscope 11, the scanning unit 13 and the microscope unit 14 are configured separately, and the image of the observation surface 12 is displayed in a state where the scanning unit 13 and the microscope unit 14 are coupled by the coupling unit 15. It has a function to acquire.

  The laser scanning microscope 11 includes a first scanning system 16-1 and a second scanning system 16-2 in the scanning unit 13, and the first scanning system 16-1 and the second scanning system 16-. 2 scans the laser beam irradiated onto the observation surface 12.

  In the first scanning system 16-1, the laser light emitted from the laser light source 21-1 is converted into parallel light by the condenser lens 22-1 and transmitted through the dichroic mirror 35, and the X-axis scanning unit 23-1. The light is incident on a scanner (such as a galvano scanner or a resonant scanner) configured to include an X-axis scanning mirror 24-1, a Y-axis scanning unit 25-1, and a Y-axis scanning mirror 26-1.

  The X-axis scanning mirror 24-1 and the Y-axis scanning mirror 26-1 are attached to the drive shafts of the X-axis scanning means 23-1 and the Y-axis scanning means 25-1, respectively. The drive shaft and the drive shaft of the Y-axis scanning unit 25-1 are arranged so as to be orthogonal to each other. Accordingly, the X-axis scanning mirror 24-1 and the Y-axis scanning mirror 26-1 are rotated about the drive axis while reflecting the laser beam, so that the observation surface 12 is irradiated in the XY direction (irradiating the observation surface 12). The laser beam is scanned in a direction orthogonal to the Z direction, where the optical axis of the laser beam is Z direction.

  The laser beam scanned in the first scanning system 16-1 is reflected toward the observation surface 12 by the dichroic mirror 31, collected by the condenser lens 32, and once imaged, and then collected by the objective lens 33. It is illuminated to form a spot on the observation surface 12. The primary imaging surface 34 on which the laser light is imaged by the condenser lens 32 is actually a space where there is nothing, but since the scanning light is imaged at the position of the primary imaging surface 34, it is secondary. The light from the secondary light source is focused on the observation surface 12 by the objective lens 33.

  When the sample arranged on the observation surface 12 contains a substance that emits fluorescence in response to the wavelength of the laser light from the laser light source 21-1, the fluorescence generated on the observation surface 12 is reflected by the objective lens 33. As a result, an image is formed on the primary imaging surface 34, converted into parallel light by the condenser lens 32, reflected by the dichroic mirror 31, and introduced into the first scanning system 16-1. The fluorescence is descanned by the X-axis scanning mirror 24-1 and the Y-axis scanning mirror 26-1, then reflected by the dichroic mirror 35, condensed by the imaging lens 36, and incident on the photodetector 37. .

  The photodetector 37 outputs an intensity signal, which is an electrical signal corresponding to the intensity of the received fluorescence, and supplies it to an imaging circuit (not shown). The imaging circuit samples the intensity signal output from the light detector 37 in time-series order according to the scanning operation (angle) of the X scanning mirror 24-1 and the Y scanning mirror 26-1, and then scans the X scanning mirror 24. The image of the observation surface 12 is two-dimensionally imaged by associating the scanning position by the -1 and Y scanning mirrors 26-1 with the fluorescence intensity indicated by the intensity signal. The image of the observation surface 12 imaged by the imaging circuit is displayed on a display device (not shown), and measurement is performed thereby.

  Similarly to the first scanning system 16-1, the second scanning system 16-2 has a laser light source 21-2, a condenser lens 22-2, an X-axis scanning unit 23-2, and an X-axis scanning. The scanner includes a mirror 24-2, a Y-axis scanning unit 25-2, and a Y-axis scanning mirror 26-2. Then, the laser beam scanned in the second scanning system 16-2 is reflected toward the observation surface 12 by the mirror 38, passes through the dichroic mirror 31, and is collected on the primary imaging surface 34 by the condenser lens 32. After being focused and imaged, the light is condensed by the objective lens 33 to form a spot on the observation surface 12. The dichroic mirror 31 functions as an optical path combining unit that combines the optical path of the laser light from the first scanning system 16-1 and the optical path of the laser light from the second scanning system 16-2.

  As described above, the laser scanning microscope 11 includes the first scanning system 16-1 and the second scanning system 16-2, and scans the same sample with the laser light from each scanning system. be able to. Therefore, in the laser scanning microscope 11, for example, a laser beam having a certain wavelength is scanned by the first scanning system 16-1 to acquire an image of the observation surface 12, and at the same time, a laser beam having another wavelength is used for the second laser beam. It can be scanned by the scanning system 16-2.

  For example, the laser scanning microscope 11 performs raster scanning of laser light (excitation light) by the first scanning system 16-1 to form an image of a rectangular region of the observation surface 12, and based on the image, the laser light This is used for observation in which the location where the stimulation is performed is specified, vector scanning is performed by the second scanning system 16-2, and the location is irradiated with laser light (stimulation light).

  However, as described above, since an error occurs between the coordinates in the first scanning system 16-1 and the coordinates in the second scanning system 16-2, the first scanning system 16-1 is used. Even if the coordinates of the acquired image are set in the second scanning system 16-2 as they are, a deviation occurs between the set coordinates and the coordinates where the laser beam is actually irradiated. That is, an attention spot that is focused on giving a stimulus in an image acquired using the first scanning system 16-1 and the coordinates of the attention spot are set in the second scanning system 16-2, and the stimulation light is set. The position where the irradiation is performed becomes a position different from the detection position detected by acquiring the image again.

  With reference to FIG. 2, an error between the coordinates of the target location and the coordinates of the detection location will be described. In FIG. 2A, five points of interest are indicated by white circles (◯), and in FIG. 2B, five points of detection are indicated by black circles (●).

  Based on the image obtained using the first scanning system 16-1, as shown in FIG. 2A, attention is paid to five locations on the X axis and the Y axis in the coordinates of the first scanning system 16-1. Assume that the places P0 to P4 are specified. Then, with respect to the second scanning system 16-2, the points of interest P0 to P4 are set by the coordinates of the first scanning system 16-1, and the laser passes through the second scanning system 16-2 at an appropriate time. When stimulation with light is performed, the amount of fluorescent material changes due to the excitation light emission of the fluorescent material. Thereafter, when an image is acquired again using the first scanning system 16-1, the fluorescence emission decreases at the position irradiated with the laser light from the second scanning system 16-2. 'To P4' are detected.

  If there is no error between the coordinates of the first scanning system 16-1 and the coordinates of the second scanning system 16-2, the points of interest P0 to P4 coincide with the detection points P0 ′ to P4 ′. As shown in FIG. 2, the detection points P0 ′ to P4 ′ are detected at different positions from the attention points P0 to P4. Although the actual error is a slight amount, the error is exaggerated in FIG.

  Such a difference between the attention points P0 to P4 and the detection points P0 ′ to P4 ′ reflects a difference in physical position with respect to the same coordinate between the first scanning system 16-1 and the second scanning system 16-2. Therefore, by analyzing the difference, for example, the relationship of the coordinates of the second scanning system 16-2 with respect to the coordinates of the first scanning system 16-1 can be obtained.

  Here, a method for matching the coordinate system of the second scanning system 16-2 with the first scanning system 16-1 with reference to the coordinate system of the first scanning system 16-1 will be described. Note that the same method may be used to make the coordinate systems of the first scanning system 16-1 and the second scanning system 16-2 coincide with the virtual coordinate system. In addition, hereinafter, a laser beam is irradiated to a spot of interest on the observation surface 12 using the second scanning system 16-2 based on the coordinates of the first scanning system 16-1, and the color fading position is formed. This is called marking.

  Then, by comparing the coordinates of the target location and the coordinates of the detection location, the X-axis expansion coefficient Sx, the Y-axis expansion coefficient Sy, the shift amount 0x in the X direction about the center of the coordinate system, and the Y direction The X axis deviation angle θx and the Y axis deviation angle θy with respect to the deviation amount 0y and the orthogonality of the coordinate system are obtained.

  The X-axis expansion / contraction coefficient Sx is the distance in the X-axis direction between two points on the first scanning system 16-1 and the distance in the X-axis direction on the second scanning system 16-2 corresponding to these two points. It is a coefficient which shows the expansion-contraction rate. The Y-axis expansion coefficient Sy is the distance in the Y-axis direction between two points on the first scanning system 16-1 and the distance in the Y-axis direction on the second scanning system 16-2 corresponding to these two points. It is a coefficient which shows the expansion-contraction rate.

  A shift amount 0x in the X direction with respect to the center of the coordinate system is a value indicating a difference in distance in the X direction between the origin of the first scanning system 16-1 and the origin of the second scanning system 16-2. . The amount of deviation 0y in the Y direction with respect to the center of the coordinate system is a value indicating the difference in distance in the Y direction between the origin of the first scanning system 16-1 and the origin of the second scanning system 16-2. .

  The X-axis deviation angle θx for the orthogonality of the coordinate system is a line segment obtained by linear interpolation of the detection points on the X-axis of the second scanning system 16-2 (straight lines passing through the detection points P1 ′ and P2 ′ in FIG. 2B). ) Of the first scanning system 16-1 with respect to the X axis. The Y axis deviation angle θy for the orthogonality of the coordinate system is a line segment obtained by linear interpolation of the detection points on the Y axis of the second scanning system 16-2 (straight lines passing through the detection points P3 ′ and P4 ′ in FIG. 2B). ) Of the first scanning system 16-1 with respect to the Y axis.

  Then, the coordinates (X1, Y1) of the target location designated based on the image acquired by the first scanning system 16-1 and the coordinates (X2, Y2) of the detected location measured from the image acquired again after marking. The relationship with Y2) is expressed by the following equations (1) and (2). Note that the expansion / contraction rate is a constant that does not depend on the coordinate position, and the inclination angle of each axis is fixed.

X2 = Sx * X1 * cos ([theta] x) + Sy * Y1 * sin ([theta] y) + 0x
... (1)
Y2 = Sx * X1 * sin ([theta] x) + Sy * Y1 * cos ([theta] y) + 0y
... (2)

  And in order to make the detection location marked by the 2nd scanning system 16-2 correspond to an attention location in the coordinate system in the 1st scanning system 16-1, Formula (1) and Formula (2) ), Conversion coefficients X ′ and Y ′ for converting the coordinates (X2, Y2) of the detected location into the coordinates (X1, Y1) of the location of interest may be obtained. That is, conversion coefficients X ′ and Y ′ that satisfy the following expressions (3) and (4) may be obtained.

X1 = Sx × X ′ × cos (θx) + Sy × Y ′ × sin (θy) + 0x
... (3)
Y1 = Sx × X ′ × sin (θx) + Sy × Y ′ × cos (θy) + 0y
... (4)

  Here, the X-axis expansion coefficient Sx, the Y-axis expansion coefficient Sy, the shift amount 0x in the X direction about the center of the coordinate system and the shift amount 0y in the Y direction, and the X axis regarding the orthogonality of the coordinate system The deviation angle θx and the Y-axis deviation angle θy can be measured by appropriately processing the two images acquired by the first scanning system 16-1, and can be handled as known constants. it can.

  Therefore, Sx × cos (θx) is a constant a, Sy × sin (θy) is a constant b, 0x is a constant c, Sx × sin (θx) is a constant d, and Sy × cos (θy) is a constant e. Assuming that 0y is a constant f, equations (3) and (4) are expressed by the following equations (5) and (6).

X1 = a × X ′ + b × Y ′ + c
... (5)
Y1 = d × X ′ + e × Y ′ + f
... (6)

  In the equations (5) and (6), since the coordinates (X1, Y1) of the target location are also known values (values designated as the target location by the user), the primary values for the transform coefficients X ′ and Y ′ It can be solved as a set of equations, and conversion coefficients X ′ and Y ′ can be obtained. By using the conversion coefficients X ′ and Y ′ thus obtained, the coordinate system of the first scanning system 16-1 and the coordinate system of the second scanning system 16-2 can be matched.

  Thus, in order to make the coordinate system of the first scanning system 16-1 coincide with the coordinate system of the second scanning system 16-2, attention is paid on the coordinate system of the first scanning system 16-1. It is necessary to designate the coordinates (X1, Y1) of the place, set the coordinates (X1, Y1) of the noticed place in the coordinate system of the second scanning system 16-2, and perform marking.

  Here, when marking is performed by arranging a fluorescent material on the observation surface 12 in a state where the scanning unit 13 and the microscope unit 14 are combined, as described above, the error caused by the scanning unit 13 and the microscope unit 14 are detected. Are combined with the error caused by. This makes it difficult to define simple conversion coefficients. Therefore, in the laser scanning microscope 11, marking for correcting the error caused by the scanning unit 13 is performed by separating the error caused by the scanning unit 13 from the error caused by the microscope unit 14.

  Next, a method for realizing marking in the laser scanning microscope 11 will be described with reference to FIGS.

  FIG. 3 is a diagram illustrating a configuration example of the scanning unit 13 in the laser scanning microscope 11 to which the present invention is applied.

  As shown in FIG. 3, in the scanning unit 13, a fluorescent sample 34 b for marking is fixed to a sample fixing adapter 34 a and is arranged on a primary imaging plane 34 on which laser light is collected by a condenser lens 32. The Then, the coordinates (X1, Y1) of the point of interest on the coordinate system of the first scanning system 16-1 are set with respect to the second scanning system 16-2, via the second scanning system 16-2. Thus, marking is performed on the fluorescent sample 34b. Thereafter, an image irradiated on the fluorescent sample 34b is acquired via the first scanning system 16-1, a portion where the laser beam is actually irradiated is detected, and conversion coefficients X 'and Y' are obtained.

  FIG. 4 is a diagram showing a state in which the fluorescent sample 34 b is fixed using the coupling portion 15. As shown in FIG. 4, the sample fixing adapter 34 a is arranged inside the housing 13 ′ by using a coupling portion 15 provided at an end of the housing 13 ′ that houses the scanning unit 13.

  FIG. 5 is an enlarged view showing the vicinity of the primary imaging plane 34.

  The coupling portion 15 is, for example, a flange-shaped hardware, and is attached to the outside of the housing 13 '. The sample fixing adapter 34 a has a substantially cylindrical structure that can be inserted into the coupling portion 15, and the fluorescent sample 34 b can be attached to the tip portion thereof. Further, the sample fixing adapter 34a is formed with an outer diameter portion 34a ′ having a part of the outer diameter opposite to the tip portion larger than the inner diameter of the coupling portion 15, and the sample fixing adapter 34a is attached to the housing 13 ′. The length is set such that the fluorescent sample 34b is arranged on the primary imaging surface 34 when the distal end side surface of the outer diameter portion 34a ′ comes into contact with the coupling portion 15 after being inserted into the coupling portion 15 from the outside. It is formed and functions as a positioning mechanism for determining the position of the fluorescent sample 34b. As a result, the fluorescent sample 34b can be easily matched with the primary imaging plane 34.

  Further, the fluorescent sample 34b is coated with an adhesive on the back surface thereof, and is attached to the front end surface of the sample fixing adapter 34a so that it can be easily replaced. Accordingly, the fluorescent sample 34b can be replaced and the sample fixing adapter 34a can be used repeatedly, and the fluorescent sample 34b can be replaced in a short time.

  Next, selection of coordinates (coordinate arrangement) for marking will be described with reference to FIGS.

  FIG. 6 shows a coordinate system in an image acquired in the maximum scanning range in the first scanning system 16-1. The scanning unit 13 performs marking at a plurality of locations within the maximum scanning range.

  Here, in order to perform marking on the fluorescent sample 34b arranged on the primary imaging plane 34, although it depends on the material of the fluorescent sample 34b, an inexpensive fluorescent seal can be adopted. It takes several seconds to mark one point. In addition, when the scanning magnification at the time of acquiring an image by raster scanning by the first scanning system 16-1 is changed, various characteristics usually change by changing the amplitude for driving the scanner. The conversion coefficients X ′ and Y ′ obtained from (Expressions (5) and (6)) also change.

  Therefore, it is necessary to obtain conversion coefficients X ′ and Y ′ to be adapted for each single scanning magnification. Therefore, for example, a process of individually obtaining conversion coefficients X ′ and Y ′ is performed for a typical scanning magnification for observation.

  The scanning magnification is the reciprocal of the ratio of the scanning range at the time of observation to the maximum scanning range that is the maximum range that can be scanned by the scanning system. For example, when the scanning range at the time of observation is half of the maximum scanning range, an image obtained by observation is acquired (displayed) with the same size as the image obtained in the maximum scanning range, and is observed at a magnification of 2 times. Since the same effect as that obtained is obtained, it is generally called a scanning magnification.

  Further, in addition to obtaining conversion coefficients X ′ and Y ′ for each scanning magnification, for example, conversion coefficients X ′ and Y ′ are obtained for each optical characteristic of the scanning unit 13. That is, the scanning unit 13 is configured so that the dichroic mirrors 31 and 35 can be exchanged. Depending on the combination of the wavelength of the laser beam used for observation and the fluorescent material, the dichroic mirrors 31 and 35 are arranged. Can be switched. As the dichroic mirrors 31 and 35 are changed, the optical characteristics of the scanning unit 13 change, and the coordinate system of the first scanning system 16-1 and the coordinate system of the first scanning system 16-2 are changed. Therefore, it is necessary to obtain conversion coefficients X ′ and Y ′ for each optical characteristic of the scanning unit 13.

  Specifically, the number of detection points is five, and six types of wavelength selection filters are provided at each of seven magnifications (1 ×, 1.5 ×, 2 ×, 3 ×, 4 ×, 6 ×, and 8 ×). When the conversion coefficients X ′ and Y ′ are acquired at the time of use, the marking is performed at 210 locations (210 = 5 × 7 × 6). If the time required for marking one point is 3 seconds, 620 seconds are required for marking 210 locations.

  Further, when marking is performed by exchanging the fluorescent sample for each type of scanning magnification and wavelength selection filter, 42 (42 = 7 × 6) replacement operations are required. The time required for all markings, 620 seconds, and the time and effort of exchanging the fluorescent sample 42 times cause a reduction in manufacturing efficiency. Furthermore, when the processing for obtaining the conversion coefficients X ′ and Y ′ is performed at the user's site where the laser scanning microscope 11 is installed for maintenance or the like, there is a possibility that it becomes a big problem. That is, reducing the marking effort is a practically important requirement.

  Therefore, in the coordinate system as shown in FIG. 6, all the magnifications (1 ×, 1.5 ×, 2 ×, 3 ×, 4 ×, 6 ×, 8 ×) are marked one fluorescent sample at a time. Thus, only the measurement is performed by changing the magnification, so that it is not necessary to replace the fluorescent sample by the magnification. In addition, when there are coordinates that are common to each magnification, the number of coordinate points also contributes to the reduction of the total number of markings, so that the marking time can be reduced.

  As shown in FIG. 6, when the maximum scanning range is expressed by coordinates (x, y), the first scanning system 16 − has a range of −1.0 ≦ x ≦ + 1.0 and −1.0 ≦ y ≦ + 1.0. Set the coordinate system at 1.

  And the coordinate which performs marking is determined so that the arrangement | positioning of the detection location in each magnification may overlap on the coordinate system in the 1st scanning system 16-1. In the following, points corresponding to five points of interest as shown in FIG. 2, that is, point P0 corresponding to the origin of the coordinate system, points P1 and P2 near both ends of the scanning range on the X axis, and Y The points P3 and P4 in the vicinity of both ends of the scanning range on the axis will be described as marking points.

  FIG. 7 shows five points P0 to P4 for marking when the scanning magnification is 1. FIG. As shown in FIG. 7, the coordinates of the point to be marked when the scanning magnification is 1 are the point P0 (0.0, 0.0) corresponding to the origin and the two points P1 (-0.9, 0.0) on the X axis. And point P2 (0.9, 0.0), two points P3 (0.0, -09) and point P4 (0.0, 0.9) on the Y axis.

  When the scanning magnification is 1.5, the coordinates of the points P0 to P4 at the magnification of 1 are determined by dividing the coordinates by the magnification (1.5). The points P0 to P4 are arranged in the same manner as the points P0 to P4 at 1 × magnification.

  FIG. 8 shows five points P0 to P4 for marking when the scanning magnification is 1.5. As shown in FIG. 8, the coordinates for marking when the scanning magnification is 1.5 are the point P0 (0.0, 0.0) corresponding to the origin and the two points P1 (-0.6, 0.0) on the X axis. And point P2 (0.6, 0.0), two points P3 (0.0, -0.6) and point P4 (0.0, 0.6) on the Y-axis.

  By determining the coordinates for marking in this way, the arrangement of the locations to be marked for the scanning range T1 at a magnification of 1 (FIG. 7) and the marking for the scanning range T1.5 at a magnification of 1.5 times The arrangement of the places to perform (FIG. 8) is relatively the same arrangement.

  Similarly, the coordinates for marking when the scanning magnification is doubled are the point P0 (0.0, 0.0) corresponding to the origin, the two points P1 (-0.45, 0.0) and the point P2 (on the X axis). 0.45,0.0), two points P3 (0.0, -0.45) and point P4 (0.0,0.45) on the Y-axis, and the arrangement of locations for marking for the scanning range T1 at a magnification of 1.times. The arrangement of the places where marking is performed with respect to the scanning range T2 (FIG. 9) at double magnification is relatively the same arrangement.

  The coordinates for marking when the scanning magnification is set to 3 are the point P0 (0.0, 0.0) corresponding to the origin, the two points P1 (-0.3, 0.0) on the X axis, and the point P2 (0.3, 0.0). ), By setting two points P3 (0.0, -0.3) and point P4 (0.0, 0.3) on the Y-axis, the arrangement of the location for marking for the scanning range T1 at a magnification of 1 and 3 times The arrangement of the places where marking is performed with respect to the scanning range T3 (FIG. 9) at the magnification is relatively the same.

  The coordinates for marking when the scanning magnification is set to 4 are the point P0 (0.0,0.0) corresponding to the origin, the two points P1 (-0.225,0.0) and the point P2 (0.225,0.0) on the X axis. ), By setting two points P3 (0.0, -0.225) and P4 (0.0,0.225) on the Y-axis, the arrangement of the location for marking for the scanning range T1 at a magnification of 1x and 4x The arrangement of the places where marking is performed with respect to the scanning range T4 (FIG. 9) at the magnification is relatively the same.

  In addition, the coordinates for marking when the scanning magnification is set to 6 are the point P0 (0.0,0.0) corresponding to the origin, the two points P1 (-0.15,0.0) and the point P2 (0.15,0.0) on the X axis. ), By setting two points P3 (0.0, -0.15) and point P4 (0.0,0.15) on the Y-axis, the arrangement of the location for marking for the scanning range T1 at a magnification of 1x, and 6x The arrangement of the portions to be marked with respect to the scanning range T6 (FIG. 9) at the magnification is relatively the same arrangement.

  The coordinates for marking when the scanning magnification is set to 8 are the point P0 (0.0,0.0) corresponding to the origin, the two points P1 (-0.1125,0.0) and the point P2 (0.1125,0.0) on the X axis. ), By setting two points P3 (0.0, -0.1125) and P4 (0.0,0.1125) on the Y-axis, the arrangement of the location for marking for the scanning range T1 at a magnification of 1x, and 8x The arrangement of the portions to be marked with respect to the scanning range T8 (FIG. 9) at the magnification is relatively the same arrangement.

  In this way, by determining the coordinates of the points P0 to P4 at a magnification of 1 by dividing by the respective magnifications, the markings are arranged so that the arrangement of the portions to be marked on the scanning range is the same at each magnification. The location to perform is set. Thus, the arrangement | positioning which becomes the same can simplify the process which measures the marked position. Note that such a relationship is not necessarily required.

  Therefore, in the laser scanning microscope 11, when obtaining the conversion coefficients X ′ and Y ′, marking at each magnification can be performed on one fluorescent sample, so that the trouble of replacing the fluorescent sample for each magnification can be reduced. This saves the work efficiency.

  In addition, in each magnification, the number of places to be marked is not limited to the five places of points P0 to p4, and in order to perform correction accurately, marking is performed in various combinations on more places. May be. That is, the description in FIGS. 6 to 9 is an example when the process is simplified and simplified, and such an example does not affect the gist of the present invention. For example, FIG. 10 shows an example in which more complicated mark coordinates are combined.

  Thus, by marking a plurality of locations on the fluorescent sample 34b, the coordinate system of the first scanning system 16-1 and the coordinate system of the second scanning system 16-2 are used in the scanning unit 13 of the laser scanning microscope 11. A process for eliminating the error is performed.

  That is, first, the fluorescent sample 34b is attached to the tip of the sample fixing adapter 34a, the sample fixing adapter 34a is inserted into the coupling portion 15, and the fluorescent sample 34b is placed on the primary imaging plane 34. Next, the coordinates of a predetermined portion (coordinates in the coordinate system of the first scanning system 16-1) for each scanning magnification as described with reference to FIGS. 6 to 10 are set, and the second scanning system 16 is set. -2 is driven, and the marking is performed by irradiating the fluorescent sample 34b with the laser light output from the laser light source 21-2. Thereafter, the first scanning system 16-1 is driven, and an operation of acquiring an image in the scanning range for each scanning magnification is performed.

  Then, for each scanning magnification image, the marking used in the correction of the scanning magnification is detected, and an operation for obtaining the coordinates of the marking in the coordinate system of the first scanning system 16-1. Based on the coordinates thus detected (the coordinates of the detected location) and the coordinates set in the marking (the coordinates of the target location), the coordinate system of the second scanning system 16-2 is changed to the first scanning system. An operation for calculating a conversion coefficient that matches the coordinate system 16-1 is performed and stored in a control device that controls the laser scanning microscope 11.

  As described above, in the laser scanning microscope 11, the coordinate system of the first scanning system 16-1 and the coordinate system of the second scanning system 16-2 are used by using the fluorescent sample 34b disposed on the primary imaging plane 34. A conversion coefficient capable of eliminating an error with the system can be obtained. Therefore, the coordinates of the point of interest input based on the coordinate system of the first scanning system 16-1 are converted using the conversion coefficient, and the laser is transmitted by the second scanning system 16-2 according to the converted coordinates. By irradiating with light, it is possible to reliably irradiate the input target portion with laser light via the second scanning system 16-2.

  In addition, such processing for obtaining the conversion coefficient can be performed only on the scanning unit 13 side, and the conversion coefficient can be obtained more easily than the case of obtaining the conversion coefficient in a state where the scanning unit 13 and the microscope unit 14 are combined. That is, the error can be easily adjusted.

  That is, in the state where the scanning unit 13 and the microscope unit 14 are combined, errors caused by the respective components are combined, and the coordinate system of the first scanning system 16-1 and the coordinate system of the second scanning system 16-2 are combined. It is difficult to define the relationship between and with a simple coefficient. On the other hand, by obtaining the conversion coefficient only on the scanning unit 13 side, the conversion coefficient can be defined with a simple coefficient, and the error can be easily adjusted. In this way, among various error factors, errors other than those caused by the microscope unit 14 including the objective lens 33 are corrected by the scanning system alone, and the error factors are simplified, so that the final image obtained by the laser scanning microscope 11 is used. The correction amount can be minimized, and correction can be simplified or unnecessary.

  In addition, in order to perform marking in a state where the scanning unit 13 and the microscope unit 14 are combined, the sample must be placed on the slide and the thickness should be the same as the actual observation sample. There were many restrictions. On the other hand, in the present embodiment, since the fluorescent sample 34b disposed on the primary imaging plane 34 is used, there is no such limitation, and the operation can be facilitated by adopting the fluorescent seal as described above. Can be done.

  In general, the scanning unit 13 and the microscope unit 14 are combined when the laser scanning microscope 11 is installed. Conventionally, the user who receives the laser scanning microscope 11 adjusts the error by obtaining a conversion coefficient. It was necessary to carry out processing. On the other hand, since it is possible to perform the process of obtaining the conversion coefficient and adjusting the error only by the scanning unit 13, for example, the process can be performed in a factory or the like before the laser scanning microscope 11 is shipped.

  In addition, when performing the process which calculates | requires the position of marking from the image of the fluorescence sample 34b, the coordinate of the marked location can be calculated | required more correctly by performing marking of an appropriate magnitude | size for every scanning magnification.

  With reference to FIGS. 11 to 13, auxiliary marking coordinates for performing marking of an appropriate size for each scanning magnification will be described.

  In order to recognize the scanning range of the laser beam scanned in the first scanning system 16-1 as an image, the scanning position on the observation surface 12 and the fluorescence luminance indicated by the intensity signal of the photodetector 37 are temporally related. Processing is performed. Then, based on the relationship between the scanning speed at which the laser beam is scanned and the resolution of the acquired image, the luminance of the fluorescence emitted from the observation surface 12 is detected as an integral value of the luminance from a certain range.

  Further, the laser light scanned on the observation surface 12 has a certain size (hereinafter, the size of the laser light irradiated onto the observation surface 12 is appropriately referred to as a spot diameter). The spot diameter is determined according to the optical relationship with 21. Therefore, there is a limit of resolving the spot position on the observation surface 12 based on the relationship between the resolution, the scanning range, and the spot diameter.

  With reference to FIG. 11, the relationship between the resolution, the scanning range, and the spot diameter will be described.

  FIG. 11 shows a luminance integration range of one pixel determined by a temporal change and resolution due to scanning of the X axis, and a luminance value change obtained for each luminance integration range. The horizontal axis represents the pixel position, and the vertical axis represents the luminance of the fluorescence. 11A and 11B show an example in which spots are located at different positions within the luminance integration range corresponding to one pixel, and FIG. 11C shows spots at positions over the luminance integration range of two pixels. An example is shown.

  The luminance integration range in the X-axis direction is determined according to the resolution of the image, and when the spot diameter is smaller than the luminance integration range, as shown in FIGS. 11A and 11B, different positions within the luminance integration range corresponding to one pixel. Even if marking is performed, since the detected luminance value is the same, the position information of the marking within the luminance integration range corresponding to one pixel does not change. That is, even if marking is performed at different positions within the luminance integration range corresponding to one pixel, it is detected that marking has been performed at the same position.

  On the other hand, as shown in FIG. 11C, even if the spot is the same size as the spot shown in FIG. 11A and FIG. 11B, marking is performed at a position across the luminance integration range of two pixels. When this occurs, a difference occurs in the luminance value detected by the two pixels. That is, the luminance value detected by each pixel changes depending on the size of the spot in each luminance integration range. Therefore, a minute spot position difference can be calculated from the ratio of the luminance values.

  In this way, by performing marking so that the spot due to marking always crosses over a plurality of pixels, for example, marking with a plurality of spots so as to be adjacent to each other, from the difference in luminance values detected by the plurality of pixels, The center position of the marking can be calculated accurately.

  FIG. 12 shows an example of marking for each magnification.

  For example, when the length in the X direction of the luminance integration range when the magnification is 1 is not less than 2 times and not more than 3 times the spot diameter, three spots are adjacent at a magnification of 1 Marking is performed. As a result, the marking consisting of three spots always extends over the two luminance integration ranges, and the center position of the marking (in this case, the center position of the central spot) from the difference of the luminance integration ranges over which the marking extends. Is required.

  When the magnification is 1.5 times and 2 times, by performing marking such that two spots are adjacent to each other, the central position of the marking (in this case 2 Between the two spots). In addition, when the magnification is 3 times and 8 times, even if marking is performed by one spot, the marking always extends over two luminance integration ranges. The center position of the marking (the middle position of the spot) is obtained.

  Although the X-axis direction has been described with reference to FIG. 12, the center position of the marking can be accurately calculated by marking with a plurality of spots so as to be adjacent to each other in the Y-axis direction as well.

  FIG. 13 shows an example of marking with a plurality of spots in the Y direction.

  For example, when the magnification is 1, marking is performed with five spots such that the spots are adjacent to the top and bottom and to the left and right of the center spot. When the magnification is 1.5 times and 2 times, marking is performed with four spots such that two spots are adjacent vertically and horizontally around the point where the spot touches.

  By performing such marking, the center position of the marking can be accurately calculated in the X direction and the Y direction. In other words, even if the spot diameter by marking is a constant size, by continuously forming spots so as to be an appropriate size according to the magnification and resolution, the size of the size across multiple pixels Marking can be performed. Such marking is important for measuring highly accurate marking position information.

  Next, FIG. 14 is a flowchart for explaining processing for performing observation by applying light stimulation to a point of interest using the laser scanning microscope 11 of FIG.

  As described above, the control device of the laser scanning microscope 11 stores the correction coefficient obtained from the marking for the fluorescent sample 34b arranged on the primary imaging plane 34. In step S <b> 11, the laser light source 21-1 emits laser light for observation, the first scanning system 16-1 scans the laser light on the observation surface 12, and observation is performed based on the output from the light detector 37. An image of surface 12 is acquired. Thereby, for example, a visual field image 51 as shown in FIG. 15 is acquired and displayed on a display device (not shown).

  Then, when the user designates a spot of interest to be stimulated by laser light in the observation object 52 shown in the image 51, the coordinates (Xt, Yt) of the spot of interest are specified in step S12. At this time, since the point of interest is represented by a coordinate system associated with the image 51, the coordinates (Xt, Yt) of the point of interest are the coordinate system of the measured image 51, that is, the first scanning system 16. Obtained according to the coordinate system of -1.

  In step S13, calculation is performed to correct the coordinates to the coordinates (Xt, Yt) of the point of interest in the coordinate system of the second scanning system 16-2 by correction calculation using a correction coefficient stored in advance. The corrected coordinates after the correction are acquired.

  In step S14, the correction coordinates acquired in step S13 are given as scanning position information for the second scanning system 16-2, and scanning for driving the second scanning system 16-2 based on the scanning position information is performed. The signal is set.

  In step S15, the laser light source 21-2 emits stimulation laser light, and the second scanning system 16-2 is driven according to the scanning signal set in step S14 to scan the laser light. Light stimulation is performed on the spot.

  In step S16, similarly to step S11, the laser beam for observation is rescanned by the first scanning system 16-1, an image after the light stimulation is performed is acquired, and the process ends. At this time, as shown in FIG. 15, a spot faded by light stimulation is detected at coordinates (Xt, Yt) that coincide with the coordinates specified in step S12.

  As described above, in the laser scanning microscope 11, the correction coordinates in the second scanning system 16-2 are obtained from the coordinates based on the image obtained by the first scanning system 16-1 by the correction calculation using the correction coefficient. Therefore, it is possible to reliably irradiate the intended place on the image obtained by the first scanning system 16-1 with the laser light via the second scanning system 16-2.

  That is, in the conventional laser scanning microscope, if the coordinates obtained in the first scanning system are set as they are in the second scanning system, they are caused by an error between the coordinates of the first scanning system and the coordinates of the second scanning system. The laser beam via the second scanning system was irradiated to a position different from the intended location on the image obtained by the first scanning system. On the other hand, in the laser scanning microscope 11, such an error between coordinates can be eliminated, so that the target location and the location where the laser beam is actually irradiated can be matched.

  In the present embodiment, correction is performed on the primary imaging plane 34 so that the coordinates of the first scanning system and the coordinates of the second scanning system coincide with each other. In the correction, since the error due to the objective lens 33 of the microscope unit 14 is not corrected, it is actually necessary to correct the distortion of the objective lens 33 and the like. However, the amount of deviation due to the error caused by the objective lens 33 is slight compared to the case where the correction for the error in the scanning unit 13 (the correction in the present embodiment) is not performed, and the same objective lens. Since the same correction can be applied to 33, it is not necessary to acquire a correction value every time observation is performed. Thereby, the convenience of the laser scanning microscope 11 can be remarkably improved.

  The embodiment of the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the gist of the present invention.

  DESCRIPTION OF SYMBOLS 11 Laser scanning microscope, 12 Observation surface, 13 Scan part, 14 Microscope part, 15 Coupling part, 16-1 1st scanning system, 16-2 2nd scanning system, 21-1 and 21-2 Laser light source, 22 -1 and 22-2 condenser lenses, 23-1 and 23-2 X-axis scanning means, 24-1 and 24-2 X-axis scanning mirrors, 25-1 and 25-2 Y-axis scanning means, 26-1 and 26-2 Y-axis scanning mirror, 31 dichroic mirror, 32 condenser lens, 33 objective lens, 34 primary imaging surface, 34a sample fixing adapter, 34b fluorescent sample, 35 dichroic mirror, 36 imaging lens, 37 photodetector

Claims (5)

In a scanning laser microscope that scans a laser beam on a sample and acquires an image of the sample using light from the sample,
A plurality of scanning means for scanning the laser beam;
The optical paths of the laser beams emitted from the plurality of scanning units are arranged on a common optical path synthesized, and the laser beams are condensed and primary coupled with an objective lens arranged on the sample side. A light collecting means for forming an image plane,
Irradiation spot of the laser beam irradiated to the fluorescent sample arranged on the primary imaging plane through another scanning unit based on the coordinate system of the specific scanning unit among the plurality of scanning units Set the coordinates of
Irradiating the irradiated portion with the laser light via the other scanning means;
Scanning the fluorescent material with the laser light through the specific scanning means to acquire an image of a scanning region of the laser light,
In the image, the coordinates of the location where the laser beam is actually irradiated through the other scanning means are detected,
Based on the set coordinates and the detected coordinates, a correction coefficient for performing correction for matching the coordinate system of the other scanning unit with the coordinate system of the specific scanning unit is obtained. Scanning laser microscope. A positioning means for arranging the fluorescent sample on the primary imaging plane;
The scanning laser microscope according to claim 1, wherein the fluorescent sample is attached to the positioning unit. The laser light irradiation spot irradiated through the other scanning means is set to be a predetermined position in the field of view for each predetermined magnification at the time of acquiring the image. The scanning laser microscope according to claim 1 or 2. The size per irradiation spot where the fluorescent sample is irradiated with the laser light via the other scanning means is set to be larger than the range on the fluorescent sample corresponding to one pixel of the image. The scanning laser microscope according to any one of claims 1 to 3. In a control method of a scanning laser microscope that scans a laser beam on a sample and acquires an image of the sample using light from the sample,
The scanning laser microscope is disposed on a common optical path in which a plurality of scanning means for scanning the laser light and the optical paths of the laser light emitted from the plurality of scanning means are combined, and collects the laser light. A light condensing means for forming a primary imaging plane with the objective lens arranged on the sample side,
Irradiation spot of the laser beam irradiated to the fluorescent sample arranged on the primary imaging plane through another scanning unit based on the coordinate system of the specific scanning unit among the plurality of scanning units Set the coordinates of
Irradiating the irradiated portion with the laser light via the other scanning means;
Scanning the fluorescent material with the laser light through the specific scanning means to acquire an image of a scanning region of the laser light,
In the image, the coordinates of the location where the laser beam is actually irradiated through the other scanning means are detected,
Obtaining a correction coefficient for performing correction for matching the coordinate system of the other scanning unit with the coordinate system of the specific scanning unit based on the set coordinates and the detected coordinate. A control method characterized by the above.

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