JP2015025900A - Laser scanning microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To acquire images at a frame rate of several images per second, without deteriorating S/N, by a high-resolution technology employing a resonant scanner.SOLUTION: A laser scanning microscope 1 includes: a resonant scanner 3 for scanning a sample S with a laser beam; a drive circuit 5 which changes scan lines to be scanned by the resonant scanner 3, and causes the resonant scanner to repeatedly scan each scan line a plurality of times; a clock generator 11 which generates a sampling clock to be synchronized with scan speed of the resonant scanner 3; a phase control apparatus 15 which shifts a phase of the sampling clock by a predetermined amount, for every scanning of the scan lines; a detection apparatus 9 which detects light from the sample S in synchronization with the sampling clock, to obtain a light intensity signal corresponding to luminance; and an image forming apparatus 17 which generates an intermediate image for each scan line, on the basis of the light intensity signals for multiple scannings of the scan lines, and synthesizes the intermediate images to generate an image of the sample S.

Description

  The present invention relates to a laser scanning microscope.

  2. Description of the Related Art Conventionally, a scanning laser microscope (LSM) including a resonant scanner that scans a sample with laser light is known (see, for example, Patent Document 1). Since the resonant scanner drives the mirror with a sinusoidal motion, the scanning speed is fast at the center of the scanning range and slow at both ends. Therefore, in the scanning laser microscope described in Patent Document 1, the sampling clock corresponding to the inverse function of the scanning speed is set so that the center of one period is dense and both ends are sparse, and light from the sample is distributed at a constant distance interval. I try to detect it.

Japanese Patent Laid-Open No. 2003-344781

  However, in a conventional scanning laser microscope, a circuit that generates a sampling clock cannot freely set a high-resolution clock due to a limitation of a band or the like. Therefore, for example, in the case of an 8 KHz resonant scanner with a sampling number of 512 pixels, in order to increase the resolution without increasing the sampling clock generation circuit, ie, increase the number of samplings, the speed of the resonant scanner is reduced. Therefore, there is only a problem of 1024 pixels at 4 kHz, and there is a problem that the frame rate is lowered.

  In addition, even if the conventional scanning laser microscope can achieve high resolution without considering the scanning speed of the resonant scanner, the biological scanning laser microscope has a limit on the light from the specimen surface. At the same time, since the sampling period is narrowed, there is a problem that information per pixel is reduced and S / N is deteriorated.

  The present invention has been made in view of the above-described circumstances, and a laser capable of acquiring an image at a frame rate of a few frames without degrading S / N by using a high resolution technology employing a resonant scanner. The object is to provide a scanning microscope.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a resonant scanner that scans a sample with laser light emitted from a light source, a scanner control unit that switches scanning by the resonant scanner to a plurality of scanning lines, and repeatedly scans the scanning lines a plurality of times. A sampling clock generating unit that generates a sampling clock synchronized with a scanning speed by the resonant scanner, a phase control unit that shifts the phase of the sampling clock by a predetermined amount for each scanning in each scanning line, and laser light is scanned A detection unit that detects light from the sample in synchronization with the sampling clock and acquires a light intensity signal corresponding to the luminance of the detected light, and a plurality of scans of each of the scanning lines acquired by the detection unit For each scan line based on each light intensity signal for each Generated between images, to provide a laser scanning microscope and an image generation unit for generating a main image of the specimen by combining the intermediate image of each said scan line.

  According to the present invention, when the laser light emitted from the light source is scanned on the sample by the resonant scanner, the light from the sample is detected by the detection unit in synchronization with the sampling clock emitted by the sampling clock generation unit, An image is generated by the image generation unit based on the light intensity signal corresponding to the luminance.

  Here, since the resonant scanner is driven by a sinusoidal motion, the scanning speed is fast at the center of the scanning range and slows at both ends, but the sampling clock generator generates the sampling clock in synchronization with the scanning speed by the resonant scanner. The light intensity signal of the light from the sample can be acquired at a certain distance interval by the detection unit, and an image with reduced influence of distortion and deviation can be generated.

  In this case, the scanning by the resonant scanner is repeated a plurality of times for each scanning line by the scanner control unit to switch the scanning line, and the phase of the sampling clock is set to a predetermined amount for each scanning in each scanning line by the phase control unit. By shifting, the position where the light intensity signal is acquired by the detector with respect to the specimen can be shifted, and the light intensity signal between the pixels can be acquired.

  Therefore, the image generation unit obtains the light intensity signal acquisition position that is an intermediate image of each scanning line based on each light intensity signal for each scanning over each scanning line, that is, between pixels of the original image. By synthesizing images that are shifted little by little to generate a main sample image, it is possible to achieve higher resolution than before without greatly reducing the frame rate. Further, it is not necessary to change the period per sampling of each scanning line, and it is possible to prevent the S / N from being lowered due to insufficient light quantity from the sample. Therefore, an image can be acquired at a frame rate of several sheets per second without degrading S / N by a high resolution technology employing a resonant scanner. Also, when scanning a high resolution image with a galvano scanner, the time is 0. Since the frame rate is several frames, it is possible to take an image at a sufficiently high speed even if the scanning is performed several times to improve the resolution.

  The present invention provides a resonant scanner that scans a sample with laser light emitted from a light source, and switches the scanning by the resonant scanner to a plurality of scanning lines, and scans a plurality of times for each frame composed of the plurality of scanning lines. A scanner control unit, a sampling clock generation unit that generates a sampling clock synchronized with a scanning speed by the resonant scanner, and a phase control unit that shifts the phase of the sampling clock of each scanning line by a predetermined amount for each scanning in each frame Detecting the light from the sample scanned with the laser light in synchronization with the sampling clock, and outputting a light intensity signal corresponding to the luminance of the detected light, and each output from the detection unit For each light intensity signal for each scan over the scan line Zui generates an intermediate image of each of the scanning lines, to provide a laser scanning microscope and an image generating unit that generates the image of the specimen the intermediate image synthesizing and of each said scan line.

  According to the present invention, the scanning by the resonant scanner is switched to a plurality of scanning lines by the scanner control unit, and is repeated a plurality of times for each frame, and the sampling clock for each scanning line for each scanning in each frame by the phase control unit. Is shifted by a predetermined amount, the position where the detector acquires the light intensity signal with respect to the sample can be shifted, and the light intensity signal between the pixels can be acquired. Therefore, it is possible to achieve higher resolution than before without greatly reducing the frame rate, and to acquire an image at a frame rate of several seconds without deteriorating S / N due to insufficient light quantity from the sample.

  In the above invention, the image generation unit may correct the light intensity signal based on positional deviation information of the acquisition position of the light intensity signal by the detection unit with respect to the scanning position of the laser beam by the resonant scanner. Good.

  If the phase of the sampling clock is shifted by a predetermined amount according to the pixel in the center of the image, the error between the resonant scanner speed and the sampling clock generation timing cannot be ignored as it moves toward the edge of the image, and it deviates from the desired pixel shift amount. There is a tendency to end up. Therefore, by configuring in this way, the light intensity signal is corrected to a value obtained at the acquisition position at a fixed distance interval, and the influence of the error between the speed of the resonant scanner and the sampling clock generation timing at the edge of the image is reduced. Thus, it is possible to suppress image displacement (image distortion).

In the above invention, the image generation unit may correct the light intensity signal by linear interpolation.
With this configuration, it is possible to easily estimate a value obtained at an acquisition position at a constant distance interval and to quickly correct the light intensity signal.

In the above invention, the image generation unit may correct the light intensity signal after scanning of the entire scanning range by the resonant scanner is completed.
With this configuration, the light intensity signals can be collectively corrected.

In the above invention, the image generation unit may correct the light intensity signal for each scanning line or for each frame including a plurality of scanning lines.
With this configuration, an intermediate image can be generated from the corrected light intensity signal, and the sample can be observed in real time using the main image without positional deviation (distortion).

In the said invention, it is good also as adjusting the acquisition period of each light intensity signal so that the said detection part may not overlap the acquisition period which acquires the light intensity signal between adjacent pixels.
When all the light intensity signals of the light from the sample generated between the sampling clocks are integrated, information on the position of each pixel includes information on the position of other pixels, resulting in blurring of the image. With this configuration, it is possible to prevent information on the position of each pixel from overlapping, and to reduce image blur.

  According to the present invention, there is an effect that an image can be acquired at a frame rate of several sheets without degrading S / N by a high resolution technique employing a resonant scanner.

It is a block diagram which shows the laser scanning microscope which concerns on one Embodiment of this invention. It is a figure which shows the relationship between the time interval of a sampling clock, and the distance interval of the sampling position of a light intensity signal. It is a figure which shows the sampling timing and the positional relationship of a sample. It is a figure which shows the relationship between the speed of the resonant scanner in the laser scanning microscope which concerns on the 1st modification of one Embodiment of this invention, and the generation timing of a sampling clock. (A) is a figure which shows an example of the linear interpolation in the laser scanning microscope which concerns on the 1st modification of one Embodiment of this invention, (b) is a figure which shows an example of the linear interpolation different from (a). (C) is a figure which shows an example of the linear interpolation different from (a) and (b). It is a figure which shows the comparison before and after change of the step amount of the drive signal of the galvano scanner in the laser scanning microscope which concerns on the 4th modification of one Embodiment of this invention. It is a figure which shows the comparison before and after the change of the distance between the scanning lines corresponding to the step amount of the drive signal of FIG.

A laser scanning microscope according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the laser scanning microscope 1 according to the present embodiment includes a resonant scanner 3 and a galvano scanner (not shown) that scan laser light emitted from a light source (not shown) on a specimen S, and a resonant. A drive circuit (scanner controller) 5 for driving the scanner 3 and the galvano scanner, a dichroic mirror 7 for branching the fluorescence generated in the sample S by scanning the laser light from the optical path of the laser light, and a branch by the dichroic mirror 7 And a detection device (detection unit) 9 for detecting the emitted fluorescence.

  Further, the laser scanning microscope 1 includes a clock generator (sampling clock generator) 11 that generates a sampling clock that specifies the timing at which fluorescence is detected by the detection device 9, and synchronization based on the sampling clock generated from the clock generator 11. A synchronization signal generation circuit 13 that generates a signal, a phase control device (phase control unit) 15 that controls the phase of a sampling clock generated from the clock generation device 11, and an image processing device (image generation unit) that generates an image of the sample S ) 17.

  The resonant scanner 3 includes a mirror unit 4A that deflects laser light and a main body unit 4B that drives the mirror unit 4A. The resonant scanner 3 is driven by the drive circuit 5 and can vibrate the mirror part 4A with a sinusoidal motion by the main body part 4B, thereby allowing the laser light to scan on the specimen S in the X-axis direction. ing. The resonant scanner 3 sends scanning position information regarding the scanning position of the laser beam to the clock generator 11.

  The galvano scanner includes a galvano mirror (not shown) that can swing around a predetermined swing axis that intersects the optical axis of the laser beam. This galvano scanner can scan the laser beam on the specimen S in the Y-axis direction by controlling the swing angle of the galvanometer mirror.

  The driving circuit 5 scans the laser beam along a scanning line in the X-axis direction (hereinafter simply referred to as “scanning line”) by the resonant scanner 3 and moves it in the Y-axis direction by the galvano scanner. The scanning lines are switched. The drive circuit 5 repeats scanning by the resonant scanner 3 n times continuously for each scanning line.

  The dichroic mirror 7 is disposed between the light source and the resonant scanner 3 and the galvano scanner. The dichroic mirror 7 transmits the laser light from the light source, while reflecting the fluorescence returning from the sample S through the resonant scanner 3 toward the optical path of the laser light toward the detection device 9 and branches off from the optical path of the laser light. It is supposed to let you.

  The detection device 9 has an integral photoelectric conversion circuit. The detection device 9 detects the fluorescence from the sample S in synchronization with the sampling clock emitted from the clock generation device 11, and photoelectrically converts the detected fluorescence to sample (acquire) a light intensity signal corresponding to the luminance of the fluorescence. It is supposed to be. The detection device 9 sends the sampled light intensity signal to the image processing device 17.

  The clock generator 11 generates a sampling clock synchronized with the scanning speed of the resonant scanner 3 based on the scanning position information output from the resonant scanner 3. In other words, since the resonant scanner 3 driven by a sinusoidal motion has a high scanning speed at the center of the scanning range and is slow at both ends, the clock generator 11 is dense at the center of one period and sparse at both ends as shown in FIG. An unequal interval sampling clock is generated. In FIG. 2, the horizontal axis indicates time, and the vertical axis indicates a position where the light intensity signal is sampled.

  Further, the clock generator 11 shifts the phase of the sampling clock by a predetermined amount for each scan of each scanning line by the resonant scanner 3 under the control of the phase controller 15.

  The synchronization signal generation circuit 13 counts the sampling clock generated from the clock generation device 11 and generates a horizontal synchronization signal every time the sampling clock for one scanning line is counted. The synchronization signal generation circuit 13 generates a vertical synchronization signal when the generated horizontal synchronization signal reaches one frame. The synchronization signal generation circuit 13 sends a horizontal synchronization signal in units of one scanning line and a vertical synchronization signal in units of one frame to the phase control device 15 and the image processing device 17.

  The phase controller 15 shifts the phase of the sampling clock generated from the clock generator 11 by a predetermined amount each time the horizontal synchronizing signal sent from the synchronizing signal generator 13 is input. For example, the phase control device 15 records the time corresponding to 1 / n pixel in terms of the center pixel, and this time is set as the phase shift amount of the sampling clock.

  As shown in FIG. 3, when the number of times of scanning repeated in one scan line is n = 4, the clock generator 11 causes the sampling clock phase to be 1 for each scan of each scan line by the resonant scan 3. Each pixel is shifted by / 4 pixel, and when it is repeated four times, it returns to the initial phase. FIG. 3 shows the positional relationship between the sampling timing and the sample S, and Phases 1, 2, 3, and 4 show the first, second, third, and fourth scans in one scan line.

  In addition, the phase control device 15 sends the amount of phase shift of the sampling clock to the image processing device 17 every time a horizontal synchronization signal is sent from the synchronization signal generation circuit 13.

  The image processing device 17 performs sampling based on each light intensity signal for each scanning of each scanning line by the resonant scanner 3, ie, an intermediate image for each scanning line, that is, between pixels of the original image. An image with the position shifted little by little is generated. Specifically, the image processing device 17 is transmitted from the light intensity signal transmitted from the detection device 9, the horizontal and vertical synchronization signals transmitted from the synchronization signal generation circuit 13, and the phase control device 15. Based on the amount of phase shift coming, n scan line data obtained by n scans for each scan line are arranged for each scan line in accordance with the position on the sample S as shown in FIG. By changing, an intermediate image for each scanning line is generated.

  The image processing device 17 combines the intermediate images of the respective scanning lines and combines the intermediate images for one frame to generate the main image of the sample S.

The operation of the laser scanning microscope 1 configured as described above will be described.
In this embodiment, the number n = 4 of repeating scanning in one scanning line will be described as an example.
In order to observe the specimen S with the laser scanning microscope 1 according to the present embodiment, the resonant scanner 3 and the galvanometer mirror are driven by the drive circuit 5, and laser light is generated from the light source.

  The laser light emitted from the light source passes through the dichroic mirror 7, is deflected by the resonant scanner 3, and is scanned along the scanning line in the X-axis direction on the sample S. Then, after the scanning by the resonant scanner 3 is continuously repeated four times per scanning line by the driving circuit 5, the scanning line of the resonant scanner 3 is switched by moving by one scanning line in the Y-axis direction by the galvano scanner. .

  When fluorescence is generated in the specimen S by scanning the laser light, the fluorescence returns through the optical path of the laser light via the resonant scanner 3 and the galvanometer mirror, is reflected by the dichroic mirror 7 and enters the detection device 9.

  In the detection device 9, the fluorescence from the sample S is detected in synchronization with the sampling clock generated by the clock generator 11, the light intensity signal corresponding to the luminance is sampled, and the sampled light intensity signal is converted into the image processing device 17. Sent to. Thereby, the image processing device 17 generates an image of the sample S based on the input light intensity signal.

  Here, since the resonant scanner 3 is driven by a sine wave motion, the scanning speed is high at the center of the scanning range and slows at both ends, but the clock generator 11 generates the sampling clock in synchronization with the scanning speed of the resonant scanner 3. Thus, the light intensity signal of the fluorescence from the specimen S can be sampled at a constant distance interval by the detection device 9. Thereby, it is possible to generate an image in which the influence of distortion and shift is reduced.

  In this case, the phase of the sampling clock of the clock generator 11 is set to ¼ pixel in accordance with the horizontal synchronizing signal sent from the synchronizing signal generating circuit 13 for each scan in the scanning line by the phase controller 15. Shifted. Thereby, the position where the detection device 9 acquires the light intensity signal with respect to the sample S can be shifted, and the light intensity signal between the pixels can be acquired.

  Further, the image processing device 17 rearranges the data of the four scanning lines obtained by the four scans for each scanning line for each scanning line, and generates an intermediate image for each scanning line. Then, the intermediate image of each scanning line is synthesized by the image processing device 17 to generate the main image of the sample S.

  Thereby, according to the laser scanning microscope 1 which concerns on this embodiment, the high-resolution image of 512 pixels or more can be produced | generated, without reducing a frame rate significantly. Further, it is not necessary to change the period per sampling of each scanning line from the case of 512 pixels, and it is possible to prevent the S / N from being lowered due to insufficient light quantity from the sample S. Therefore, an image can be acquired at a frame rate of several sheets without degrading the S / N by the high resolution technology employing the resonant scanner 3.

The present invention can be modified as follows.
As a first modification, the image processing device 17 may correct the light intensity signal based on positional deviation information of the sampling position of the light intensity signal by the detection device 9 with respect to the scanning position of the laser light by the resonant scanner 3. Good.

  The image processing device 17 reads the scanning position information of the scanning position of the laser light from the resonant scanner 3, and also reads the sampling position information of the sampling position of the light intensity signal from the detection device 9, and calculates the light intensity from the difference between these position information. The signal is corrected to a value obtained from sampling positions at regular distance intervals.

  For example, when the phase of the sampling clock is shifted by ½ pixel in accordance with the pixel at the center of the image, as shown in FIG. 4, the speed of the resonant scanner 3 and the generation timing of the sampling clock are increased toward the end of the image. The error cannot be ignored and tends to deviate from the desired pixel shift amount. In FIG. 4, the horizontal axis indicates the number of pixels (Pixcel), and the vertical axis indicates the error amount (%).

  As shown in FIG. 5A, when the sampling timing of Phase 2 that should sample the intermediate position of the first pixel and the second pixel of Phase 1 is shifted as shown in FIG. 5A, the sampling values of Phase 1 and Phase 2 Is extended to a desired sampling position, and the light intensity signal of Phase 2 is corrected to the value of the desired sampling position. The image processing device 17 generates an intermediate image using the corrected light intensity signal. In FIG. 5A, ◯ indicates the actual sampling position value, and □ indicates the desired sampling position value. The same applies to FIGS. 5B and 5C.

  In this way, according to this modification, it is possible to reduce an error between the speed of the resonant scanner at the edge of the image and the generation timing of the sampling clock, and to suppress image displacement (distortion).

  In the present modification, the locus of the sampling values of Phase 1 and Phase 2 is extended and corrected to the value of the desired sampling position. Instead, for example, as shown in FIG. 5B, Phase 1 When the sampling timing of Phase 2 that should sample the intermediate position of the first pixel and the second pixel is shifted, linear interpolation is performed from the sampling values of two adjacent sampling timings of Phase 1 and is estimated by the average value thereof. The light intensity signal may be corrected to the intermediate position value. Further, as shown in FIG. 5C, a desired intermediate position value may be estimated using an interpolation formula such as spline interpolation.

  In the present modification, the image processing device 17 may correct the light intensity signal after the scanning of the entire scanning range by the resonant scanner 3 is completed. In this way, each light intensity signal can be corrected together.

  Further, the image processing device 17 may correct the light intensity signal for each scanning line or for each frame including the scanning lines. In this way, an intermediate image can be generated from the corrected light intensity signal, and the specimen S can be observed in real time from the main image without positional deviation (distortion).

  As a second modification, when the duty ratio of the sampling clock from the clock generator 11 is always 50%, the phase controller 15 uses the reverse edge of the sampling clock signal to double the resolution. It is good to do. In this way, the same effect as when the phase of the sampling clock is shifted by 180 ° can be easily obtained.

  As a third modification, the detection device 9 may adjust the sampling period of each light intensity signal so that the sampling periods for acquiring the light intensity signals between adjacent pixels do not overlap.

  If all the fluorescence from the sample S generated between the sampling clocks is integrated in the detection device 9, information on the position of each pixel also includes information on the position of other pixels, resulting in blurring of the image. For example, in FIG. 2, the information on the positions of the second to fourth pixels is included in the data of the fifth pixel. In such a case, according to the present modification, by shortening the sampling period so as not to overlap each other, it is possible to prevent the information on the position of each pixel from overlapping and to reduce the blur of the image.

  In this modification, the sampling period is adjusted. Instead, the original image is estimated by using a deconvolution operation from a degradation function corresponding to the blur (blur) and the sampling position (motion) of the image. A common super-resolution technique may be used to eliminate crosstalk between pixels. Even in this case, the sharpness of the image can be improved.

  As a fourth modified example, as shown in FIG. 6, the drive circuit 5 increases the step amount of the drive signal for driving the galvano scanner by 1 / n times (in the example of the present embodiment, 1/4 times). It is good. In FIG. 6, the horizontal axis indicates time (t), and the vertical axis indicates the step amount (Pos) of the drive signal.

  Since the galvano scanner operates following the input drive signal, the step amount of the drive signal is increased by 1 / n times (1/4), thereby reducing the distance between the scan lines to 1 as shown in FIG. / N (1/4), and the resolution can be improved.

  Therefore, according to this modification, high resolution is achieved by both scanning in the X-axis direction by the resonant scanner 3 and scanning in the Y-axis direction by the galvano scanner, and an image having n times the resolution is S / N and frame rate. Can be realized without significant sacrifice.

  In the present embodiment, the driving circuit 5 causes the resonant scanner 3 to repeatedly scan a plurality of scanning lines continuously for each scanning line, and the phase control device 15 shifts the phase of the sampling clock by a predetermined amount for each scanning in each scanning line. However, as a fifth modification, the drive circuit 5 repeatedly scans the frame multiple times for each frame by the resonant scanner 3, and the phase control device 15 performs the phase of the sampling clock of each scan line for each scan in each frame. May be shifted by a predetermined amount.

  In this case, every time each scanning line is scanned once by the resonant scanner 3 by the drive circuit 5, the scanning line is switched by the galvano scanner, and after scanning for one frame, the scanning line returns to the first scanning line and scanning is performed in the same procedure. You can repeat it.

  Even in this case, the light intensity signal between the pixels can be acquired by shifting the position at which the light intensity signal is sampled by the detection device 9 with respect to the sample S. Accordingly, it is possible to achieve higher resolution than before without greatly reducing the frame rate, and to acquire an image at a frame rate of several seconds without deteriorating S / N due to insufficient light quantity from the sample S.

DESCRIPTION OF SYMBOLS 1 Laser scanning microscope 3 Resonant scanner 5 Drive circuit (scanner control part)
9 Detector (Detector)
11 Clock generator (sampling clock generator)
15 Phase control device (phase control unit)
17 Image processing device (image generation unit)
S specimen

Claims (7)

A resonant scanner that scans the specimen with laser light emitted from a light source;
A scanner controller that switches the scanning by the resonant scanner to a plurality of scanning lines, and repeatedly scans the scanning lines a plurality of times;
A sampling clock generator for generating a sampling clock synchronized with the scanning speed by the resonant scanner;
A phase controller that shifts the phase of the sampling clock by a predetermined amount for each scan in each scan line;
A detection unit that detects light from the sample scanned with laser light in synchronization with the sampling clock, and obtains a light intensity signal corresponding to the luminance of the detected light;
An intermediate image for each scan line is generated based on each light intensity signal for each scan of each of the scan lines acquired by the detection unit, and the intermediate image for each scan line is synthesized to produce the sample. A laser scanning microscope comprising: an image generation unit that generates the main image. A resonant scanner that scans the specimen with laser light emitted from a light source;
A scanner control unit that switches the scanning by the resonant scanner to a plurality of scanning lines and scans a plurality of times for each frame composed of the plurality of scanning lines;
A sampling clock generator for generating a sampling clock synchronized with the scanning speed by the resonant scanner;
A phase control unit that shifts the phase of the sampling clock of each scanning line by a predetermined amount for each scanning in each frame;
A detector that detects light from the sample scanned with laser light in synchronization with the sampling clock and outputs a light intensity signal corresponding to the luminance of the detected light;
An intermediate image for each scanning line is generated based on each light intensity signal for each scanning over a plurality of times of each scanning line output from the detection unit, and the intermediate image for each scanning line is synthesized to produce the sample A laser scanning microscope comprising: an image generation unit that generates the main image.   The said image generation part correct | amends the said light intensity signal based on the positional offset information of the acquisition position of the said light intensity signal by the said detection part with respect to the scanning position of the laser beam by the said resonant scanner. The laser scanning microscope described.   The laser scanning microscope according to claim 3, wherein the image generation unit corrects the light intensity signal by linear interpolation.   5. The laser scanning microscope according to claim 3, wherein the image generation unit corrects the light intensity signal after scanning of the entire scanning range by the resonant scanner is completed.   The laser scanning microscope according to claim 3 or 4, wherein the image generation unit corrects the light intensity signal for each scanning line or for each frame including a plurality of scanning lines. The laser scanning microscope according to any one of claims 1 to 6, wherein the detection unit adjusts an acquisition period of each light intensity signal so that acquisition periods of acquiring light intensity signals between adjacent pixels do not overlap. .

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