JP2002098901A - Scanning laser microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a scanning laser microscope which is capable of correcting image deviation and makes it possible to obtain proper images. SOLUTION: The scanning laser microscope, which obtains image data by scanning a sample (112) with a spot beam and detecting the light from this sample, includes a specimen-loading member which is loaded with the specimen and is formed with an index as a reference for the specimen position, a first photoelectric conversion means (16a) which obtains the light from the specimen as the image data, a second photoelectric conversion means (16b) which obtains the light from the specimen loading member as the image data, a calculation means (1614) which calculates the position deviation quantity of the image of the specimen from the image data, obtained by the second photoelectric conversion means and a correction means (1611) which corrects the image data obtained by the first photoelectric conversion means with the position deviation quantity calculated by the calculation means.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a scanning type in which an acquired image is added to an original observation image, the image is corrected so that there is no displacement, and there is no image distortion, and storage and display are performed. Related to a laser microscope.

[0002]

2. Description of the Related Art (First Prior Art) An optical microscope is configured to magnify and observe a sample on a slide mounted on a stage with an objective lens. And a structure in which light from a light source such as a lamp is evenly applied to the entire observation region of the sample using a condenser lens.

Further, when it is desired to observe an image with good resolution and contrast, there is a problem such as flare as an illumination system in observation with an optical microscope, and it is very difficult to observe a low-contrast sample. There is a problem. In order to improve these problems, a scanning optical microscope, which is a point light projection type (spot light projection type) optical microscope, has been proposed.

[0004] This optical microscope irradiates an observation sample in a point-like manner with light from a point light source via an objective lens.
Light transmitted through the sample (transmitted light), reflected light from the sample, or fluorescent light generated from the sample is imaged again into a point-like image via the objective lens and the optical system, and the image is formed into a detector having a pinhole aperture. To obtain image density information.

However, with this configuration alone, only the density information of one point irradiated with the point light can be obtained.
X-Y scanning method for moving in the direction of the axis and mechanically moving in a two-dimensional plane, and C for synchronizing the optical path with scan scanning
An image display device such as an RT display displays a luminance corresponding to the signal of the density information according to the XY scanning so that the image can be observed.

The above are the principle components of the scanning optical microscope. Further, a device using laser light as a light source is called a scanning laser microscope, and it is widely known that the resolution of an image is improved. This scanning laser microscope saves and processes image data by converting transmitted light or reflected light of a sample being laser-scanned into electrical signals by photoelectric conversion using a photomultiplier tube or photodiode as a detector. , Display configuration.

FIG. 12 is a block diagram showing the basic structure of a conventional scanning laser microscope. In this configuration,
The laser light from the laser light source 12 provided in the optical microscope main body 11 causes the two-dimensional scanning mechanism 13 to perform XY scanning of the spot light on the surface of the sample 112 on the microscope stage 111.

FIG. 13 shows two galvanometer mirrors X and Y.
FIG. 3 shows an example of a schematic diagram of an XY scanning unit when the above-described correspondence is made. After the reflected light or the fluorescence from the sample resulting from the irradiation of the spot light passes through the pinhole plate 14 via the objective lens 113 and the two-dimensional scanning mechanism 13, the light detector 1
At 5, the light is received and photoelectrically converted into an electric signal. Pinhole plate 14
Indicates a pinhole having a predetermined diameter, and the light detection unit 15
Is located at the image forming position on the front surface of the sample, and only the information focused at the observation point on the sample surface can be detected by the light passing therethrough, and the confocal effect can be obtained. The two-dimensional scanning mechanism 1
3 is driven by generating a scanning control signal by the two-dimensional scanning drive control unit 169, and the signal processing unit 16 also performs data processing based on the signal from the two-dimensional scanning mechanism unit 13.

[0009] The light detected by the light detector 15 and converted into an electric signal is processed by the signal processor 16 and displayed on the display 17. First, a desired signal is amplified by the gain variable section 161, and then the desired signal is increased or decreased by the offset adjusting section 162. The set amount is determined by the CPU 16
6, the desired values are set in the D / A converters 167 and 168, respectively.

Next, the signal output from the offset adjusting section 162 is subjected to analog / digital conversion by the A / D converter 163 and then temporarily stored in the recording section 164 as image data. The stored image data is then processed, displayed,
Will be saved. For processing, desired image processing is performed by the CPU 166. The display can be performed by outputting image data from the storage unit 164 and performing the display on the display unit 17 via the D / A converter 165 to observe the image. When the depth direction, that is, three-dimensional information is required, the stage 111 is moved to a desired Z position by the Z scanning drive unit 1610, and the necessary images are sequentially constructed in the storage unit 164. This allows
It is also possible to display and observe a three-dimensional image. In addition, information can be obtained by performing measurement on a sample based on the image, that is, length, area, and volume measurement.

FIG. 14A shows an image in which no image shift has occurred, and FIG. 14B shows an acquired image in which the image shift has occurred. Although it is desired to finally obtain a good image with the scanning laser microscope as described above, an image may have a pixel shift due to the configuration conditions of the apparatus and the performance limit of each unit. Taking a raster scan image as an example of this pixel shift, as compared with an actual microscope image as shown in FIG. 14A, the acquired image as shown in FIG. 14B is slightly shifted for each line. Sometimes.

However, the shape and size of a biological specimen image are not always constant, and it is difficult to determine what is the original shape in an observed image. Depending on the type of image, it is easy to determine that a large image shift is a shift, but it is difficult to determine a slight image shift,
In fact, I sometimes don't know. In such a case, after all, the observation and recording are performed with the shifted image as it is.

The cause of such an image shift is a shift in the timing at which an image is obtained with respect to the sample position to which laser light is actually irradiated, so that the arrangement of each line data deviates from the original position. This is because a phenomenon occurs.

[0014] As described above, factors that cause the start of image capture to be inconsistent for each line include the effects of scanner drive errors and vibrations. In the scanner, the position is moved by the drive signal, but moves to a position having a slight shift error, not the position originally expected for the input signal. In this case, the faster the input signal, the less the mechanical response of the scanner will follow the signal change.

Therefore, the fluorescence signal from the designated scanning position should be acquired at a predetermined time. However, due to this error, information on the position is acquired at a different time. In the case of a raster scan image, this phenomenon is conspicuously observed in a driving portion where the speed is high, that is, in a lateral movement, and a shift occurs for each line. As a result, when the raster scan image is displayed, the image is shifted from the original image position between the upper and lower lines.

A change in position due to thermal deformation or the like can be ignored because it is a change that is slower than the scanning time. However, similar effects to this change include vibrations coming from the periphery of the device, specifically, the floor and the inside of the device. In addition, when vibration of a motorized part accompanied by movement at the same side portion or vibration by an observer is applied, the observation position on the sample is not always constant. Therefore,
In such a case, the above-described phenomenon also occurs. Further, when the frequency of the vibration includes a low frequency component, a vertical image shift also occurs in the raster scan image.

In order to prevent such an image shift from occurring, improvement measures have conventionally been considered. Regarding an error correction method for a scanning unit, a conventional example is disclosed in
No. 8 discloses an example in the case of a resonant galvanometer.
This is a control method in which even if the scanning frequency of the scanner changes, a sampling signal that accurately follows the scanning frequency at that time is generated, and an image without deviation is captured.

(Second Conventional Technique) FIG. 15A shows an image in which no image shift has occurred, and FIG. 15B shows an acquired image in which the image has been distorted. We want to finally obtain good images with a scanning laser microscope as described above, but due to the configuration conditions of the device and the performance limitations of each unit, the obtained image may be an image with distortion and uneven brightness. is there.
The distortion of the image increases as it goes to the periphery of the image. For example, when the original observation image is a circle as shown in FIG. 15A, the image acquired by the laser microscope becomes an elliptical image as shown in FIG. 15B.

In this example, a large image distortion makes it easy to judge. However, in general, the shape and size of a biological specimen image are not always constant, and it is necessary to judge what is the original shape in an observed image. Difficult things are included. Also,
Depending on the type of image, it is difficult to judge a slight image distortion or displacement, and there is a case where the user does not actually know. In such cases, the observer would not know
Observation and recording are performed with a distorted image. In addition, based on the image, measurement of the sample, that is, length, area,
If an attempt is made to know the volume, the value will include a measurement error in the peripheral image.

The cause of such image distortion largely depends on the performance of the system, such as the characteristics of the scanner and the control method, with respect to the irradiation position where laser light is to be actually irradiated. In a scanner, a position is moved by a drive signal, but if an input signal contains a high frequency component, it moves to a position having an error instead of a position originally expected. In this case, the higher the frequency of the input signal, the more difficult it is for the mechanical response of the scanner to follow the signal change.

Accordingly, although the fluorescence signal from the designated scanning position should have been acquired at a predetermined time, information of a position different from the original position is actually acquired due to this error. This phenomenon appears remarkably near the image. For example, when a grid-like image is obtained, images in which the grids are originally at regular intervals are not evenly spaced around the periphery. In the case of raster scan images, this phenomenon is noticeable in fast-moving lateral motion, resulting in distorted edges of the image.

In order to respond to such a problem and to prevent the occurrence of image shift and distortion,
Conventionally, improvement measures have been considered. As described above, the error correction method of the scanning unit is disclosed in
Japanese Patent Application Publication No. 0-338338 discloses an example in the case of a resonant galvanometer. This is a control method that generates a sampling signal that accurately follows the scanning frequency at that time even if the scanning frequency of the scanner changes, and captures an image without distortion and without deviation.

[0023]

(Problems of the First Prior Art) Even if the error of the scanning section can be corrected by the control method disclosed in Japanese Patent Laid-Open No. Hei 11-338338, the scanner scanning section is only required. , And cannot be expected to prevent image shift caused by other factors. For example, when a pixel shift occurs due to the influence of vibration due to a floor or an electric part inside the apparatus, the pixel shift cannot be corrected as a whole of the apparatus by this control method alone. This control method is a correction method limited to the raster scan line direction, and cannot be corrected in the vertical direction.

This control method is a method limited to the resonance scanner, and cannot be applied to other scanning units. Further, even if a device for removing a plurality of vibrations, a conventional mechanism, and other measures are taken for all the factors, the control is complicated, the device is large, and the device is expensive. No correction can be expected.

(Problem of the second conventional technique) Even if an error in the scanning section can be corrected by the control method described in Japanese Patent Application Laid-Open No. H11-338338, the correction is limited to the scanner scanning section. When the combination of systems to be connected is changed, it is not possible to correct the entire laser microscope system including the error factors possessed by each connection unit. In addition, general compatibility cannot be maintained, and since the control method is limited to the resonance scanner, the method cannot be applied to other scanning units.

In addition, it is difficult to cope with a plurality of scanning conditions such as changing the observation conditions of a laser microscope, for example, changing the magnification of an objective lens and the scanning speed for image capture. Further, it cannot be expected to prevent image shift caused by other factors. For example, when a pixel shift occurs due to the influence of vibration due to a floor or an electric part inside the apparatus, the pixel shift cannot be corrected as a whole of the apparatus by this control method alone. Furthermore, since the correction of the image is incomplete in the end, it is not possible to correct the distance accurately, which results in the length, area,
Volume measurement will not be achievable.

A first object of the present invention is to provide a scanning laser microscope capable of correcting an image shift and obtaining a good image.

A second object of the present invention is to provide a scanning laser microscope capable of correcting image distortion and obtaining a good image.

[0029]

Means for Solving the Problems In order to solve the above problems and achieve the object, a scanning laser microscope of the present invention is configured as follows.

(1) In a scanning laser microscope according to the present invention, a specimen is placed on a scanning laser microscope which scans a specimen with spot light to detect light from the specimen and obtain image data. A sample mounting member on which an index serving as a reference for a sample position is formed; first photoelectric conversion means for obtaining light from the sample as image data; and a second photoelectric conversion means for obtaining light from the sample mounting member as image data. Photoelectric conversion means, calculating means for calculating the amount of displacement of the image of the sample from the image data obtained by the second photoelectric conversion means, and calculating the image data obtained by the first photoelectric conversion means Correction means for correcting based on the amount of positional deviation calculated in (1).

(2) A scanning laser microscope according to the present invention is the microscope described in (1) above, and has information on an absolute position at a reference position of the sample mounting member.

(3) In the scanning laser microscope of the present invention, a specimen is placed on a scanning laser microscope which scans a specimen with spot light to detect light from the specimen and obtain image data. A sample mounting member on which an index serving as a reference for a sample position is formed; first photoelectric conversion means for obtaining light from the sample as image data; and a second photoelectric conversion means for obtaining light from the sample mounting member as image data. Photoelectric conversion means, calculation means for calculating the amount of distortion of the image of the sample from the image data obtained by the second photoelectric conversion means, and image data obtained by the first photoelectric conversion means by the calculation means Correction means for correcting based on the calculated distortion amount.

(4) The scanning laser microscope of the present invention is the microscope according to the above (3), and further comprises a luminance correcting means for correcting the luminance of the image data obtained by the first photoelectric conversion means. .

As a result of taking the above-described measures, the following operations are obtained.

(1) According to the scanning laser microscope of the present invention, all the causes of the image shift can be corrected at once, and the correction can follow the display of the image in real time to provide a good image. become. In addition, no matter what scanning unit is used, or even if the scanning unit is not sufficiently controlled, image displacement can be corrected. The device can be realized.

(2) According to the scanning laser microscope of the present invention, since the reference position is provided with the information on the absolute position, the image shift can be corrected more accurately, and the expandability of the apparatus is increased.

(3) According to the scanning laser microscope of the present invention, all the causes of image distortion can be corrected at once, and this correction can be made to follow the display of the image in real time, thereby eliminating distortion in peripheral parts and the like. It is possible to provide a perfect image.
In addition, since the absolute value of the image can be measured, accurate measurement of the length, area, and volume of the sample can be performed. Also, no matter what scanning unit is used, or even if the scanning unit is not sufficiently controlled, image distortion can be corrected. The device can be realized.

(4) According to the scanning laser microscope of the present invention, all the causes of luminance unevenness can be corrected at once, and this correction can be made to follow the display of the image in real time, and a good image without the luminance unevenness can be obtained. Can be provided. In addition, even if any scanning unit is used, and even if the control in the scanning unit is not sufficient, the brightness unevenness can be corrected. The device can be realized.

[0039]

DESCRIPTION OF THE PREFERRED EMBODIMENTS (First Embodiment) FIG. 1 is a block diagram showing the configuration of a scanning laser microscope according to a first embodiment of the present invention.

The optical microscope main body 11 guides the laser light from the laser light source 12 to the two-dimensional scanning mechanism 13 using a fiber or directly without using a fiber, and passes the sample 112 on the microscope stage 111 through the objective lens 113.
A spot light is irradiated on the surface. The laser beam applied to the sample 112 by the scanning control signal output from the two-dimensional scanning drive control unit 169 is transmitted to the two-dimensional scanning mechanism unit 1.
The XY scan is performed at 3. Here, scanning is performed in the X and Y directions using two galvanometer mirrors, and raster scanning is performed with laser light. The signals required for this XY scanning are X, Y
Alternatively, they are referred to as horizontal (H) and vertical (V) synchronization signals. As a result, as shown in FIG.
XY scanning is performed on the surface.

The sample 112 is, for example, a desired biological cell specimen stained with fluorescent light, and is placed on a glass plate. On the glass plate, a transparent material made of a fluorescent substance whose fluorescence wavelength is shifted from the wavelength of fluorescence of the biological specimen to be observed is embedded in a lattice shape so as to indicate a reference position. (Hereinafter referred to as a reference position sample.) FIG. 2 shows the above-described glass plate, for example, a reference position having a lattice shape in which vertical and horizontal intervals are constant.

The reference position sample is not limited with respect to the excitation wavelength, and can be similarly excited by laser light to the biological sample. The fluorescence wavelength to emit does not overlap with the wavelength of the fluorescence of the biological sample, and is optically the present state. Separated enough to allow separation by equipment. For example, as shown in FIG. 3, the excitation wavelength 51 of the reference position specimen is between the observed fluorescence wavelengths 52 in the range of visible to infrared fluorescence wavelengths of a commonly used fluorescent reagent, Uses the fluorescence wavelength 53 from the reference position sample which is separated and located on the long wavelength side.

As a result of the irradiation of the spot light, the reflected light or the fluorescent light from the sample 112 is detected by the optical path dividing unit 18 through the objective lens 113 and the two-dimensional scanning mechanism unit 13 and the fluorescent wavelength and position for biological specimen observation are detected. The optical path is divided into a fluorescent wavelength for use. Each detection light is transmitted to a pinhole plate 14,1.
After passing through No. 4, the photodetectors 151 and 152 photoelectrically convert the signals into electric signals. A photomultiplier tube (PMT) is often used for the photodetectors 151 and 152. Pinhole plates 14, 14
Indicates a pinhole having a predetermined diameter, and the light detection unit 15
1, 152 are arranged at the image forming position on the front surface, and only the luminance information at the focused position on the sample 112 can be detected by the light passing therethrough, and the confocal effect can be obtained.

The electric signals output from the light detectors 151 and 152 are processed by the signal processors 16a and 16b.
Since the configuration of the signal processing unit 16b is the same as that of the signal processing unit 16a, it is not shown.

The signal processor 16a performs signal processing on the fluorescence emitted from the target biological specimen. First, a desired signal is amplified by the gain variable section 161, and then the desired signal is increased or decreased by the offset adjusting section 162. The set amount of the offset adjustment unit 162 is determined by the CPU 166.
Thus, the desired values are set in the D / A converters 167 and 168, respectively.

Next, the signal output from the offset adjusting section 162 is subjected to analog / digital conversion by the A / D converter 163 and then stored in the first storage section 164. In normal observation without any image shift correction, the data is directly passed through the third storage unit 1615, digital-to-analog converted by the D / A converter 165, and displayed on the display unit 17.

On the other hand, the signal for which the reference position has been detected is subjected to the same analog / digital processing as described above in the signal processing section 16b. However, since the contents of the data to be handled are different, the variable gain value and the offset adjustment value are also different. The digitally converted data after the appropriate processes of the variable gain and the offset adjustment are performed are temporarily stored in the second recording unit 1612. Based on the recorded data, an error calculator 1
At 614, the shift amount of each line is calculated. The actual acquired image is an image slightly larger in size than the display image. As a result, information on an image whose periphery is shifted is also recorded as correction data, and correction can be performed.

For example, as shown in FIG. 4, the reference line for adjusting the image shift is the first line. In this case, if the boundary between white and black forming the stripe-shaped luminance on the second line is shifted with a shift compared to the luminance information on the first line, the shifted pixel L2 corresponds to the shift. Similarly, after the third line, compared to the first line,
The shift amounts L3, L4,... Ln are calculated. The calculated shift amount is passed to the third storage control unit 1611 and the third storage unit 1
615 is used as an initial value of write control. That is, the shift amount Ln of each line is added as an offset of the write start address when writing data from the first storage unit 164 to the third storage unit 1615, and the image data subjected to the shift correction is written to the third storage unit 1615. .

In this case, since the image shift is a shift in the horizontal direction, if the reference position sample is a reference position sample consisting of only vertical lines, the processing can be advanced by the above-described method. When a grid-like reference position sample is used, there are specific lines that are covered by the grid-like data, and a stripe that serves as a reference for a lateral shift for comparison cannot be seen, and there are places where determination cannot be made. In this case, since the line does not protrude greatly and shifts as compared to the upper and lower lines, the average of the positions of the upper and lower lines is interpolated between two points, and the value at that time is used to calculate the shift amount. . As a result, the value of the pixel shift of the biological specimen image can be obtained from the shift of each line, and the image can be corrected so that there is no shift. The corrected image is converted to an analog signal by the D / A converter 165 and displayed on the display unit 17.

When an image shift occurs in the vertical direction due to vibration or the like, the error calculating unit 1614 similarly detects a vertical position shift error. In the processing in the vertical direction, since the vibration direction is the vertical direction of the image, the shift amount is not the pixel shift but the number of lines. Therefore, how many lines are shifted in the vertical direction is calculated, and the data of the shift amount is stored in the third storage controller 161.
1 and shifts the required shift amount in the vertical data, and rewrites the image data in the third storage unit 1615.

In the first embodiment, a case where both the fluorescence observation sample and the reference position sample are excited at the same excitation wavelength is considered.
The same laser light is shared for excitation. In this case, since the fluorescence wavelengths are different, the detection unit side is divided. However, since the laser light source 12 is generally determined by the excitation wavelength of the observation sample and the photodetector, there may be a plurality of laser light sources instead of one. .

In addition, in the case of a multi-stained sample, a plurality of laser light sources having excitation wavelengths suitable for the dye may be prepared, and a plurality of light detection units may be prepared, so that multi-channel processing may be performed. That is, in the case of triple staining, three lasers are used, and four detection units including those for the reference position specimen are provided. Further, a laser light source for exciting the reference position sample may be separately provided.

(Second Embodiment) In the second embodiment, a material having an emission wavelength characteristic shifted from the fluorescence wavelength of the observation sample is used as the reference position sample. Then, a laser beam having a wavelength not used in the fluorescence observation and in the infrared region that does not affect the biological specimen is used for detecting the reference position specimen. The reference position sample uses a substance that reflects infrared light and transmits a shorter wavelength of light, and corrects pixel shift.

In the second embodiment, basically, the first
A laser light source for a reference position sample is added to the configuration of the first embodiment. The infrared laser light for position detection does not overlap with the excitation laser light and the fluorescence wavelength of the biological specimen. Then, in actual image observation, a plurality of laser beams are simultaneously irradiated to capture an image. Subsequent processing is the same as in the first embodiment.

As a result, the reference position sample can be easily prepared as compared with the first embodiment, and the optical separation of the detection light becomes easier. Further, since the reflected light of the laser light is used, the amount of detected light is more sufficient than in the case of the fluorescent light, and adjustment is easy.

(Third Embodiment) In the third embodiment, a light detecting unit 151 for observation and a transmitted image light detecting unit 19 for transmission are provided, and similar processing is performed. When detecting the transmitted image, the laser light is transmitted through the reference position sample 111 and then detected by the transmitted image light detector 19 via the desired optical filter 18. As a result, the signal from the transmitted image light detection unit 19 is input to the processing unit 16b, and is processed in the same manner as in the first embodiment. Thus, an image of the reference position signal is obtained in the same manner.

In the case of a lattice-like reference position sample, if the positional deviation within the lattice, that is, the image deviation, cannot be accommodated, it can be dealt with by increasing the lattice interval.

If the reference position is described in such a way that the absolute position can be understood, the above case can be dealt with more. In this case, a scalable use is possible by attaching a numerical value indicating the absolute coordinate, a mark of the reference position, or the like, or by gradually changing the line width so that the absolute position can be determined. Further, these alignment processes can be realized by either hardware or software.

These processes are not limited to images by raster scan, but can be applied to various laser scans, and there are no limitations on the scan speed, scan size, and the like. Also, the light detection unit 15
1, 152 is a photodiode (PD), CCD, C
It is not limited to PMT as long as photoelectric conversion can be performed efficiently, such as MD. Further, the two-dimensional scanning mechanism unit 13 may be a galvanomirror, a resonance galvanomirror, a polygon mirror, an AOD, or the like, as long as the XY scanning can be controlled.

(Fourth Embodiment) FIG. 5 is a block diagram showing a configuration of a scanning laser microscope according to a fourth embodiment of the present invention. In the configuration of FIG. 5, a correction value calculation unit 16141 is provided instead of the error calculation unit 1614 of FIG.

In the fourth embodiment, when the optical system is generally determined, the observation viewing angle is determined.
It is owned and managed by U166. Since the grid interval of the reference position sample used as the reference is already known, it can be associated with the viewing angle. Since the reference grid interval changes depending on the observation magnification, the observation magnification of the objective lens can be automatically recognized. Based on this information, the CPU 16
6 calculates and determines the reference grid interval. Alternatively, these parameters may be manually input conditions each time.

In the fourth embodiment, the second storage unit 16
12 based on the data stored in the
In step 1, the amount of distortion is calculated. Thereby, it is possible to understand how the acquired images change from the regular intervals and the degree of distortion of the images.
The actual acquired image acquires an image slightly larger in size than the display image. As a result, the correction data including the information on the image outside the visual field is recorded in the third storage unit 1615, and sufficient correction is performed in the periphery.

The case where the periphery of the image of the lattice pattern thus obtained is distorted will be described with reference to FIGS. 6 (a) and 6 (b). In this case, of course, the biological specimen image is also distorted, so that processing for distortion correction is performed.
Assuming that the interval between the equally-spaced grid-like patterns shown in FIG. 6A is L1 shown in FIG. 7, the space between the grid-like patterns near the center of the acquired image shown in FIG. 6B is shown in FIG. L2. When the lattice pattern of the acquired image has spread near the center (FIG. 6B), thinning is performed based on the number of pixels obtained from the distance ratio between L1 and L2, and the distance approaches the reference lattice pattern. Thus, the correction pixel data is obtained. For example, when L2 = 2 × L1, the obtained image can be corrected by thinning out one pixel.

On the other hand, when the lattice-like pattern of the acquired image becomes narrower (horizontal direction near the left and right ends of the image shown in FIG. 6B), pixel interpolation obtained from the interval ratio between L1 and L3 is performed. Pixel data is interpolated so as to approach the interval of the reference grid pattern. For example, when L3 = L1 / 2, two pixels are successively interpolated as the same data.

Each of the above processes is a process performed based on one grid-like pattern. This is performed using a plurality of grid-like patterns in the entire field of view to perform image correction. Further, the average value of the data of the peripheral pixels may be used instead of the same data for the interpolation pixels.

Actually, the calculated correction value is passed to the third storage control unit 1611, and the final correction image is stored in the third storage unit 1615 by controlling the address of the write control unit of the third storage unit 1615. It is memorized. For example, when compressing data and using image data of every other pixel as correction data,
As shown in FIG. 8, the third storage control unit 1611 transfers and stores data to the third storage unit 1615 at an even or odd read address. Alternatively, all read image data is stored in the third storage unit 1615 at an even or odd write address. In the case of every 2, 3,... M pixels, the image data of the pixels is 2n, 3n,.
It is stored at intervals of n (n and m are natural numbers).

In the case of interpolation, the second storage control unit 161
The reading of data No. 3 or the writing of data by the third storage control unit 1611 is repeated with the same address value to read or write data. For example, as shown in FIG. 9, when interpolating one pixel at a time, the address value is set to 1, 1, 2, 2, 3,
Control as 3,.

The finally corrected image is converted into an analog signal by the D / A converter 165 and displayed on the display unit 17. In the displayed image, the reference of the absolute position is known because the interval between the captured lattice pattern and the pixel can be associated with each other. That is, the CPU 166 calculates the interval between pixels. Therefore, since the corrected image has been corrected based on this, in image measurement, a highly reliable length, area, and volume can be calculated and calculated from the pixel interval of the corrected image.

Furthermore, since the scanning speed may increase in the Y direction, that is, in the vertical direction, the same processing can be performed. Therefore, if distortion correction is performed simultaneously in the X and Y directions, a more reliable image can be obtained. Further, when performing more precise interpolation, it can be realized by setting a grid-like pattern more finely.

(Fifth Embodiment) In addition to the case where the reference position sample is a lattice pattern, the distortion can be similarly corrected using, for example, an oblique line. For example, when a straight line along the diagonal line of the field of view is used as a reference signal, if a line of an actually acquired image has a bend, the portion is similarly distorted. For example, in the case of raster scanning, distortion in the X direction is conspicuous.

As shown in FIG. 10A, when there is a distortion in the corner of the image, the image in the vicinity thereof is shifted laterally by the amount of the distortion and corrected so as to approach a normal image position without distortion. As shown in FIG. 10B, when focusing on the pixel P (x1, y1) in the area where the distortion occurs, the deviation from the reference diagonal line 10a on the M line is defined as Lm. The pixel of interest p (x1, y1) is shifted laterally along the M line by Lm as shown in FIG.
The distortion is corrected as a new image at the position (x2, y1). This correction is performed for each pixel of each line in the area with distortion. Further, the reference line does not need to be particularly diagonal, and may be anything as long as it belongs to the observation visual field and can acquire an image.

Such processing can be applied in raster scan, particularly when the distortion in the Y direction is negligible compared to the distortion in the X direction.

(Sixth Embodiment) FIG. 11 is a block diagram showing a configuration of a scanning laser microscope according to a sixth embodiment of the present invention. In the configuration of FIG. 11, an image calculation unit 1617 is provided in addition to the configuration of FIG.

In the sixth embodiment, it is possible to use a reference position sample, monitor the amount of light therefrom, and correct the uneven brightness. If there is no luminance unevenness, the detection signal at each position from the reference position sample has a uniform value,
If luminance unevenness occurs in a certain portion, the detected signal may have high or low luminance. In the sixth embodiment, the acquired image is corrected based on the characteristics of the uneven brightness of these images.

The luminance unevenness is uniform at the center of the image, but mainly occurs at the periphery. This similarly affects the observed biological specimen. For example, there is a decrease in brightness at the periphery of the image, and the image becomes dark, resulting in unevenness.
Therefore, using the luminance of the central portion of the image as a reference value, the correction value calculating section 16142 calculates the degree of the peripheral luminance unevenness, and the image calculating section 1617 adds or subtracts the degree to the actual luminance of the image.

The luminance at the central portion serving as a reference is I (x1, y
In the case of 1), if the luminance of the peripheral part is I (x2, y2), the difference ΔI is ΔI (x1, x2, y1, y2) = | I (x1, y1)
−I (x2, y2) | Accordingly, the corrected luminance of the peripheral portion is I '(x2, y2) = I (x2, y2) + [Delta] I.
Is processed as new image data in the third storage unit 161.
5 is stored. In addition, since luminance information in the lattice of the lattice pattern cannot be obtained, a luminance difference is obtained by performing linear interpolation of the luminance during that time.

In these processes, first, image distortion correction, that is, position correction according to the fourth and fifth embodiments is performed, and then luminance correction at each position is performed. As a result, distortion due to position can be corrected, and unevenness due to luminance can be corrected.
Further, these processes can be realized by either hardware or software.

These processes are not limited to raster scan images, and can be applied to various laser scans, and there are no limitations on the scan speed, scan size, and the like. Also, the light detection unit 15
1, 152 is a photodiode (PD), CCD, C
It is not limited to PMT as long as photoelectric conversion can be performed efficiently, such as MD. Further, the two-dimensional scanning mechanism unit 13 may be a galvanomirror, a resonance galvanomirror, a polygon mirror, an AOD, or the like, as long as the XY scanning can be controlled.

Further, after the image shift correction described in the first to third embodiments is performed, the distortion correction and the unevenness correction described in the fourth to sixth embodiments are performed, whereby the reliability is improved. Image can be obtained.

The present invention is not limited to the above embodiments, but can be implemented with appropriate modifications without departing from the scope of the invention. For example, the image may be corrected each time by always obtaining an image using a glass plate on which a lattice-like pattern is formed, but is not limited thereto.
When using a scanning laser microscope, data is acquired and stored once using a glass plate on which a lattice-like pattern is formed, and in subsequent processing, image correction is performed based on the stored data. Is also good.

Further, the above embodiments may be used in combination. In the above embodiment, the XY scanning is performed with the objective lens fixed.
It may be scanning.

[0082]

According to the present invention, it is possible to provide a scanning laser microscope capable of correcting an image shift and obtaining a good image.

Further, according to the present invention, it is possible to provide a scanning laser microscope capable of correcting image distortion and obtaining a good image.

[Brief description of the drawings]

FIG. 1 is a block diagram showing a configuration of a scanning laser microscope according to a first embodiment of the present invention.

FIG. 2 is a diagram showing a reference position sample according to the first embodiment of the present invention.

FIG. 3 is a diagram showing excitation light and fluorescence wavelength characteristics according to the first embodiment of the present invention.

FIG. 4 is a view showing an image shift between lines according to the first embodiment of the present invention.

FIG. 5 is a block diagram showing a configuration of a scanning laser microscope according to a fourth embodiment of the present invention.

FIG. 6 is a diagram illustrating distortion of an image of a lattice pattern according to a fourth embodiment of the present invention.

FIG. 7 is a diagram showing distortion of a grid interval of an image according to a fourth embodiment of the present invention.

FIG. 8 is a diagram showing pixel thinning correction according to a fourth embodiment of the present invention.

FIG. 9 is a diagram showing pixel interpolation correction according to a fourth embodiment of the present invention.

FIG. 10 is a diagram showing diagonal distortion correction according to a fifth embodiment of the present invention.

FIG. 11 is a block diagram showing a configuration of a scanning laser microscope according to a sixth embodiment of the present invention.

FIG. 12 is a block diagram showing a basic configuration of a scanning laser microscope according to a conventional example.

FIG. 13 is a schematic diagram of XY scanning according to the embodiment of the present invention and a conventional example.

FIG. 14 is a diagram showing an observation image according to a conventional example.

FIG. 15 is a view showing an observation image according to a conventional example.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 11 ... Optical microscope main body 12 ... Laser light source 13 ... Two-dimensional scanning mechanism part 14 ... Pinhole plate 151, 152 ... Light detection part 16 ... Signal processing part 16a, 16b ... Signal processing part 161 ... Gain variable part 162 ... Offset adjustment part 163: A / D converter 164: first storage unit 165: D / A converter 166: CPU 167, 168: D / A converter 169: two-dimensional scanning drive control unit 1610: Z drive unit 1611: third storage Control unit 1612 second recording unit 1613 second storage control unit 1614 error calculator 16141 correction value calculation unit 16142 correction value calculation unit 1615 third storage control unit 1616 first storage control unit 1617 image calculation Unit 17: Display unit 18: Optical path division unit 19: Transmitted image light detection unit

Claims (4)

[Claims] 1. A scanning laser microscope that scans a sample with spot light to detect light from the sample and obtains image data, wherein the sample is placed and an index is formed as a reference for the position of the sample. A sample mounting member, a first photoelectric conversion unit that obtains light from the sample as image data, a second photoelectric conversion unit that obtains light from the sample mounting unit as image data, Calculating means for calculating a displacement amount of the image of the sample from the image data obtained by the photoelectric conversion means; and image data obtained by the first photoelectric conversion means based on the displacement amount calculated by the calculation means. A scanning laser microscope, comprising: a correction unit configured to perform correction. 2. The scanning laser microscope according to claim 1, wherein information on an absolute position is provided at a reference position of said sample mounting member. 3. A scanning laser microscope which scans a sample with spot light to detect light from the sample and obtains image data, wherein the sample is placed and an index is formed as a reference for the position of the sample. A sample mounting member, a first photoelectric conversion unit that obtains light from the sample as image data, and a second photoelectric conversion unit that obtains light from the sample mounting member as image data
Photoelectric conversion means, calculation means for calculating the amount of distortion of the image of the sample from the image data obtained by the second photoelectric conversion means, and image data obtained by the first photoelectric conversion means by the calculation means A correction means for correcting based on the calculated amount of distortion, and a scanning laser microscope. 4. The scanning laser microscope according to claim 3, further comprising a luminance correcting means for correcting the luminance of the image data obtained by said first photoelectric conversion means.