WO2009151075A1 - Optical scanning microscope - Google Patents

Abstract

Provided is an optical scanning microscope wherein load to a sample is reduced without reducing a frame rate. A laser beam emitted from a light source (22) is permitted to perform scanning on a sample (12) by a horizontal scanner (25) and a vertical scanner (26).  A light blocking element (23) transmits or blocks the laser beam emitted from the light source (22) corresponding to an instruction given from a switching section (32).  The switching section (32) controls operation of the light blocking element (23) by monitoring image frame obtained by scanning the sample (12) with the laser beam, so that an irradiation pattern of the laser beam to the sample (12) changes.  This invention is applicable to a laser scanning microscope.

Description

Optical scanning microscope

The present invention relates to a light scanning microscope.

Conventionally, a laser scanning microscope is known in which a sample is observed by scanning laser light on a sample to be observed. For example, using a laser scanning microscope, the biological sample can be observed fluorescently.

By the way, when a biological sample into which a fluorescent dye is introduced is irradiated with intense light such as a laser beam many times, the amount of fluorescence generated from the fluorescent dye decreases accordingly, so a living body can be detected by a laser scanning microscope. When fluorescently observing a sample, it is important that the biological sample is not irradiated with unnecessary light.

Therefore, when the observer adjusts the position of the stage on which the sample is mounted so that an observation image of a desired portion of the sample can be obtained, for example, the scanning speed of the scanner itself is There has been proposed a laser scanning microscope which irradiates a sample with laser light at a constant time interval (decreases the frame rate) without any change and reduces the load on the sample (for example, see Patent Document 1).

JP, 2006-313355, A

However, in the above-described technique, since the sample is only intermittently irradiated with the laser light, the frame rate of the observation image is lowered, and there is a problem that the operability of the laser scanning microscope is deteriorated.

For example, when the frame rate of the displayed observation image is lowered, even if the observer adjusts the position of the stage while viewing the observation image, the operation of the observer is not immediately reflected in the displayed observation image. I will. Therefore, the observer moved the stage too much and took time to adjust the position of the stage, and it was difficult to prepare for observation quickly.
In addition, if a delay occurs before the operation of the observer is reflected in the observation image, the position of the stage may not be properly adjusted, and the expected observation image may not be obtained.

The present invention has been made in view of such circumstances, and an object thereof is to provide an optical scanning microscope capable of reducing the load on a sample without reducing the frame rate of an observation image. .

The optical scanning microscope according to the present invention comprises: a scanning means for scanning light emitted from a light source on a sample to be observed; and an optical path from the light source to the sample on the light source. Light shielding means for shielding the light, and a light shielding control means for controlling the light shielding means so that the light is irradiated to the sample, or the light is shielded and the sample is not irradiated. The light shielding control means is configured to emit the light to obtain the observation image of an arbitrary frame among a frame group consisting of several consecutive frames of an observation image obtained by scanning the light on the sample. The light shielding means is controlled such that the irradiation area of the sample is different from the irradiation area of the other frame of the frame group.

According to the present invention, the load on the sample can be reduced without reducing the frame rate of the observation image.

It is a figure which shows the structural example of one Embodiment of the optical scanning type | mold microscope to which this invention is applied. It is a figure which shows the example of a more detailed structure of a scanner control part. It is a figure which shows the example of a more detailed structure of a switching part. It is a figure which shows the irradiation pattern of the laser beam in each flame | frame.

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

FIG. 1 is a view showing a configuration example of an embodiment of an optical scanning microscope to which the present invention is applied.

The light scanning microscope 11 is, for example, a laser scanning microscope for fluorescence observation of a sample 12 such as a biological sample, and the sample 12 to be observed is placed on a stage 21 provided in the light scanning microscope 11. .

The light source 22 of the light scanning microscope 11 emits a laser beam to be applied to the sample 12, and the emitted laser beam passes through the light shielding element 23, the dichroic mirror 24, the horizontal scanner 25, and the vertical scanner 26. The sample 12 is irradiated. Here, the light blocking element 23 includes, for example, an acousto-optic modulator, an electro-optic modulator, a magneto-optic modulator, etc., and functions as a high-speed shutter that blocks the light from the light source 22.

Thus, when the sample 12 is irradiated with the laser light, fluorescence is expressed from the sample 12. The fluorescence from the sample 12 passes through the optical path of the laser light in the reverse direction to reach the dichroic mirror 24 and passes through the dichroic mirror 24. That is, the dichroic mirror 24 reflects light in the wavelength band of laser light (excitation light) and transmits light in the wavelength band of fluorescence.

Further, the fluorescence from the sample 12 is transmitted through the dichroic mirror 24, and then enters the light detection unit 28 through the barrier filter 27. The light detection unit 28 receives the incident fluorescence and performs photoelectric conversion to generate an electric signal indicating the intensity of the received fluorescence, and supplies the electric signal to the image processing unit 29.

Further, in the light scanning microscope 11, operations of a control unit 30 for controlling the overall operation of the light scanning microscope 11, a scanner control unit 31 for controlling the operations of the horizontal scanner 25 and the vertical scanner 26, and the light shielding element 23 A switching unit 32 is provided to control the

The control unit 30 instructs the scanner control unit 31 to start the fluorescence observation of the sample 12 according to the operation of the observer, and the scanner control unit 31 controls the horizontal scanner 25 and the vertical scanner according to the instruction from the control unit 30. Drive 26.

The horizontal scanner 25 is, for example, a resonant scanner and has a scan mirror that reflects laser light. The horizontal scanner 25 rotates the scan mirror with the straight line in the depth direction as a rotation axis in the drawing in accordance with the instruction of the scanner control unit 31 and scans the laser light on the sample 12 in the horizontal direction in the drawing.

The vertical scanner 26 is, for example, a galvano scanner and has a scan mirror that reflects laser light. The vertical scanner 26 rotates the scan mirror about a straight line in the upper right direction in the drawing and in a direction perpendicular to the rotation axis of the horizontal scanner 25 as a rotation axis, and the laser light is shown in FIG. Make it scan.

Hereinafter, the direction on the observation image of the sample 12 corresponding to the horizontal direction and the horizontal direction in the drawing is also referred to as the horizontal direction, and the direction on the observation image corresponding to the depth direction and the depth direction in the drawing is Also called vertical direction.

Further, the scanner control unit 31 supplies information indicating the scanning position of the laser beam by the horizontal scanner 25 and the vertical scanner 26 to the image processing unit 29 and the switching unit 32.

The image processing unit 29 generates an image signal of an observation image which is an image of the sample 12 based on the electric signal supplied from the light detection unit 28 according to the information supplied from the scanner control unit 31. For example, as the image signal of the observation image, the image processing unit 29 is an image of an image of one frame including 512 pixels in the horizontal direction and 512 pixels in the vertical direction and having a frame rate of 10 to 40 fps (frame per second). Generate an image signal. The image processing unit 29 supplies the generated observation image to the display unit 33 connected to the light scanning microscope 11. Thereby, the observation image of the sample 12 is displayed on the display unit 33.

In the light scanning microscope 11, when the light detection unit 38 detects fluorescence, the barrier filter 27 transmits only light in the wavelength band of fluorescence, and unnecessary components from light incident from the dichroic mirror 24, for example, the wavelength band of laser light A clearer observation image can be obtained by removing the component of.

Further, the switching unit 32 controls the operation of the light shielding element 23 based on the information supplied from the scanner control unit 31. That is, in the switching unit 32, the laser beam from the light source 22 is transmitted through the light shielding element 23 and the sample 12 is irradiated, or the laser light from the light source 22 is blocked by the light shielding element 23 and irradiated to the sample 12 The light blocking element 23 is controlled so as to be in either one of the light blocking state where it disappears.

Next, FIG. 2 is a diagram showing a more detailed configuration example of the scanner control unit 31 of FIG. In FIG. 2, parts corresponding to the case in FIG. 1 are given the same reference numerals, and descriptions thereof will be omitted.

Scanner control unit 31 includes vertical synchronization signal generation circuit 61, vertical scanner drive circuit 62, horizontal synchronization signal generation circuit 63, horizontal scanner drive circuit 64, pixel clock generation circuit 65, horizontal line count circuit 66, and frame count circuit 67. Configured

When the vertical synchronization signal generation circuit 61 receives an instruction to start fluorescence observation from the control unit 30, the vertical synchronization signal VD indicating the timing of starting scanning of the laser light in the vertical direction is output to the switching unit 32 and the vertical scanner drive circuit 62. , The horizontal synchronization signal generation circuit 63, and the frame count circuit 67.

In the light scanning microscope 11, scanning of the laser light in the vertical direction is performed by the vertical scanner 26, and at the same time, scanning of the laser light in the horizontal direction is performed by the horizontal scanner 25. Is irradiated with laser light. At this time, for example, during a period in which one scan is performed in the vertical direction, a plurality of predetermined scans are performed in the horizontal direction. Also, in order to obtain an observation image of one frame, one scan in the vertical direction is performed.

Assuming that the entire area in which the laser light is scanned by the horizontal scanner 25 and the vertical scanner 26 is referred to as a scanning area, the vertical synchronization signal VD is a laser light which is generated from one end to the other end in the vertical direction of the scanning area. Is a signal that indicates the timing at which the vertical scanning of H. is started.

The vertical scanner drive circuit 62 generates a drive signal VDD for driving the vertical scanner 26 based on the vertical synchronization signal VD supplied from the vertical synchronization signal generation circuit 61 and supplies the drive signal VDD to the vertical scanner 26 for vertical Make a direction scan.

The horizontal synchronization signal generation circuit 63 generates the horizontal synchronization signal HD based on the vertical synchronization signal VD supplied from the vertical synchronization signal generation circuit 61, and the horizontal scanner drive circuit 64, the pixel clock generation circuit 65, and the horizontal line count circuit 66. , And the frame count circuit 67 and the switching unit 32. The horizontal synchronization signal HD is a signal indicating the timing at which the horizontal scanning of the laser light is started, which is performed from one end of the scanning region in the horizontal direction to the other end.

The horizontal scanner drive circuit 64 generates a drive signal HDD for driving the horizontal scanner 25 based on the horizontal synchronization signal HD supplied from the horizontal synchronization signal generation circuit 63, and supplies the drive signal HDD to the horizontal scanner 25 to perform horizontal operation. Make a direction scan.

The pixel clock generation circuit 65 generates a pixel clock PC indicating the sampling timing of the electrical signal generated by the light detection unit 28 based on the horizontal synchronization signal HD supplied from the horizontal synchronization signal generation circuit 63. Then, the pixel clock generation circuit 65 supplies the generated pixel clock PC to the horizontal line count circuit 66, the image processing unit 29, and the switching unit 32.

The pixel clock PC is a signal indicating the timing at which the image processing unit 29 should sample (acquire) an electrical signal from the light detection unit 28. Data obtained from the electrical signal sampled by the image processing unit 29 is an observation image Image data for one pixel in the image.

Horizontal line count circuit 66 starts counting pixel clocks PC from pixel clock generation circuit 65 when horizontal synchronization signal HD is supplied from horizontal synchronization signal generation circuit 63, and the number of pixel clocks PC counted is When the predetermined value is reached, the trigger signal TR1 is generated. That is, by counting the number of pixel clocks PC, the horizontal line count circuit 66 detects the timing to start the scan of the next horizontal line, and generates a trigger signal TR1 indicating the timing.

Here, assuming that a region irradiated with laser light to obtain an image displayed on a predetermined pixel of an observation image in a scanning region is referred to as a partial scanning region, the horizontal line means the horizontal direction in the scanning region. A horizontally long area consisting of partial scan areas arranged in parallel. In the light scanning microscope 11, when the area of the sample 12 corresponding to the partial scanning area is irradiated with the laser light, the fluorescence from the area is detected and the area irradiated with the laser light (partial scanning area) The image of is to be displayed at the pixel on the observation image corresponding to the area.

For example, if one horizontal line consists of 512 partial scan areas and the count of the pixel clock PC is started at the timing when the horizontal synchronization signal HD is input, the number of counted pixel clocks PC becomes 512. At timing, the next horizontal synchronization signal HD should be generated.

Therefore, when the number of pixel clocks PC counted is 512, the horizontal line count circuit 66 generates a trigger signal TR1 indicating the timing of generation of the horizontal synchronization signal HD and supplies the trigger signal TR1 to the horizontal synchronization signal generation circuit 63. Also, when the trigger signal TR1 is supplied from the horizontal line count circuit 66, the horizontal synchronization signal generation circuit 63 newly generates a horizontal synchronization signal HD. The horizontal line count circuit 66 newly starts counting of the pixel clock PC when a new horizontal synchronization signal HD is supplied from the horizontal synchronization signal generation circuit 63.

When the vertical synchronization signal VD is supplied from the vertical synchronization signal generation circuit 61, the frame count circuit 67 starts counting the horizontal synchronization signal HD from the horizontal synchronization signal generation circuit 63, and the number of horizontal synchronization signals HD counted is counted. Generates a trigger signal TR2 when it reaches a predetermined value.

That is, the frame count circuit 67 detects the timing to start the next vertical scanning by counting the number of horizontal synchronization signals HD, and generates a trigger signal TR2 indicating the timing. Then, the frame count circuit 67 supplies the generated trigger signal TR2 to the vertical synchronization signal generation circuit 61 to generate the next vertical synchronization signal VD.

For example, when the scan area consists of 512 horizontal lines, the trigger signal TR2 is generated at the timing when the count number of the horizontal synchronization signal HD becomes 512.

As described above, the scanner control unit 31 generates the vertical synchronization signal VD and the horizontal synchronization signal HD indicating the timing of scanning start in the vertical direction and the horizontal direction, and controls the operation of the vertical scanner 26 and the horizontal scanner 25.

Next, FIG. 3 is a diagram showing a more detailed configuration example of the switching unit 32 of FIG. In FIG. 3, parts corresponding to the case in FIG. 1 are given the same reference numerals, and descriptions thereof will be omitted.

The switching unit 32 includes an effective horizontal synchronization signal count circuit 91, an effective pixel clock count circuit 92, and a drive circuit 93.

When the vertical synchronization signal VD is supplied from the vertical synchronization signal generation circuit 61, the effective horizontal synchronization signal count circuit 91 starts counting the horizontal synchronization signal HD from the horizontal synchronization signal generation circuit 63, and reaches a predetermined number. Count. Then, the effective horizontal synchronization signal count circuit 91 outputs the enable signal HDE at H (High) level while counting the horizontal synchronization signal HD, and after stopping the counting of the horizontal synchronization signal HD, the next vertical is generated. The enable signal HDE of L (Low) level is output until the synchronization signal VD is supplied. The enable signal HDE output from the valid horizontal synchronization signal count circuit 91 is supplied to the drive circuit 93.

When laser light is scanned over the scanning area to obtain an observation image, the wobbling of the scanning mirrors of the horizontal scanner 25 and the vertical scanner 26 distorts the image in the vicinity of the boundary of the scanning area. An image of the area of the part is taken as an observation image. Here, if an area displayed as an observation image in the scanning area is referred to as an effective scanning area, when the horizontal synchronization signal HD with respect to the horizontal line including a part of the effective scanning area is counted, An H level enable signal HDE indicating that the effective scanning area is included is output.

That is, when the horizontal synchronization signal HD specified by the count number of the horizontal synchronization signal HD is the horizontal synchronization signal HD indicating the timing to start the scanning of the horizontal line including a part of the effective scanning area, the H level is enabled. A signal HDE is output. Conversely, when the horizontal synchronization signal HD specified by the count number is the horizontal synchronization signal HD indicating the timing of the start of scanning of a horizontal line not including a part of the effective scanning area, the enable signal HDE at L level is Is output.

The effective pixel clock count circuit 92 holds irradiation information indicating the irradiation pattern of the laser light with respect to the scanning area of each frame of the observation image. One piece of irradiation information includes information indicating whether or not laser light is to be irradiated for each partial scan area of the scan area for one frame. When the horizontal synchronization signal HD is supplied from the horizontal synchronization signal generation circuit 63, the effective pixel clock count circuit 92 starts counting the pixel clock PC from the pixel clock generation circuit 65, refers to the illumination information, and enables the enable signal PCE. Are supplied to the drive circuit 93.

That is, when the pixel clock PC specified by the count number of the pixel clock PC is the pixel clock PC corresponding to the partial scan region to be irradiated with the laser light indicated by the irradiation information, the H level enable signal PCE is output. Ru. Further, when the pixel clock PC specified by the count number is the pixel clock PC corresponding to the partial scan area to which the laser light indicated by the irradiation information should not be irradiated, the L level enable signal PCE is output.

The pixel clock PC corresponding to the partial scanning area refers to the pixel clock PC indicating the timing of sampling of the electrical signal obtained by detecting the fluorescence from the partial scanning area.

When the enable signal HDE of H level is supplied from the effective horizontal synchronization signal count circuit 91 and the enable signal PCE of H level is supplied from the effective pixel clock count circuit 92, the drive circuit 93 transmits the optical path of the laser light through A control signal to the effect that the light shielding element 23 is supplied is supplied to the light shielding element 23. In addition, drive circuit 93 sets the light shielding state when at least one of enable signal HDE from valid horizontal synchronization signal count circuit 91 and enable signal PCE from valid pixel clock count circuit 92 is at L level. A control signal is supplied to the light shielding element 23.

By the way, at the preparation stage of the fluorescence observation of the sample 12, the observer adjusts the position of the stage 21 so that the desired part of the sample 12 can be observed on the observation image, or adjusts the intensity of the laser light and various gains. It is necessary to select an appropriate barrier filter 27 or the like.
At the time of preparation for these fluorescence observations, the observer operates the light scanning microscope 11 while looking at the observation image displayed on the display unit 33, so that the laser light is irradiated to the sample 12 also at the preparation stage. Become.

Therefore, in the preparation step of the fluorescence observation of the sample 12, the light scanning microscope 11 changes the irradiation pattern of the laser light to the sample 12 for each frame of the observation image, for example, as shown in FIG. Reduce the load on 12

In each of A to D in FIG. 4, the horizontal direction in the drawing indicates the horizontal direction, that is, the direction in which the laser beam is scanned by the horizontal scanner 25, and the vertical direction in the drawing indicates the vertical direction, That is, the direction in which the laser beam is scanned by the vertical scanner 26 is shown. Also, one square indicates one partial scan area. Further, the shaded partial scanning area in the scanning area represents the area irradiated with the laser light.

A of FIG. 4 shows a pattern of laser light emitted to the scanning region R11 when obtaining an observation image of the first frame. In the example of FIG. 4A, the scan area R11 is divided into five in the horizontal direction and five in the vertical direction, and the scan area R11 includes a total of 25 (= 5 × 5) partial scan areas. .

Hereinafter, in the drawing of the scanning region R11, it is i-th (1 ≦ i ≦ 5) in the horizontal direction (rightward) from the upper left, and j-th (1 ≦ j ≦ 5) in the vertical direction (downward) The partial scan area located is referred to as a partial scan area BR (i, j).

For example, when the optical path of the laser beam is continuously made to be in the transmission state and the laser beam is scanned on the scanning region R11 to obtain an observation image for one frame, one from the horizontal line at the top in the figure. The scanning is performed so that the laser light is sequentially irradiated downward to the bottom horizontal line. At this time, laser beams are sequentially emitted in the right direction from the left end to the right end in the figure, and the even lines from the top are in the figure, Laser light is sequentially emitted in the left direction from the right end to the left end.

However, in practice, to reduce the load on the sample 12, the scanning is performed while the optical path of the laser light is switched to either the transmission state or the light shielding state, and only some partial scan areas included in the scan area R11 Is irradiated with laser light.

Specifically, in the first frame of the observation image, of the scan area R11, the partial scan area BR (1, 1), the partial scan area BR (5, 1), the partial scan area BR (2, 2), the partial The laser beam is irradiated to the scan area BR (3, 3), the partial scan area BR (4, 4), the partial scan area BR (1, 5), and the partial scan area BR (5, 5). Therefore, the observation image of the first frame is an image in which each of the portions of the sample 12 located in each of these seven partial scan areas is displayed. That is, in the observation image, an image is displayed on each of the pixels of the observation image corresponding to each of the partial scanning regions to which the laser light is irradiated, and the image of the sample 12 is not displayed on the other pixels.

Further, when obtaining an observation image of the second frame, at the time of scanning of the laser light, as shown in B of FIG. 4, the partial scan area BR (3, 1) and the partial scan area BR (4 , 2), partial scan area BR (1, 3), partial scan area BR (5, 3), partial scan area BR (2, 4), and partial scan area BR (3, 5) are irradiated with laser light. Ru.

Furthermore, when obtaining the observation image of the third frame, at the time of scanning of the laser light, as shown in FIG. 4C, the partial scanning region BR (2, 1) and the partial scanning region BR (3 , 2), partial scan area BR (4, 3), partial scan area BR (1, 4), partial scan area BR (5, 4), and partial scan area BR (2, 5) are irradiated with laser light. Ru.

Furthermore, when obtaining the observation image of the fourth frame, at the time of scanning of the laser light, as shown in D of FIG. 4, the partial scan area BR (4, 1) and the partial scan area BR of the scan area R11 Laser light is transmitted to (1, 2), partial scan area BR (5, 2), partial scan area BR (2, 3), partial scan area BR (3, 4), and partial scan area BR (4, 4) It is irradiated.

As described above, when the scanning for obtaining the observation image of the fourth frame after the scanning of the laser light is started, that is, when the fourth scanning in the vertical direction is completed, the entire scanning region R11 is irradiated with the laser light. It will be. Here, in the partial scan area to which the laser light is applied in the scan for obtaining an observation image of an arbitrary frame among the 1 to 4 frames, the laser for a scan for obtaining an observation image of another frame No light is emitted.

In other words, laser light is applied to any partial scan area of the scan area R11 in any one of the first to fourth scans in the first to fourth scans in the vertical direction, The laser beam is not irradiated at the time of other scans.

Therefore, even in the fifth and subsequent frames, if scanning is repeatedly performed with the same irradiation pattern as in the first to fourth frames, each partial scan area is irradiated with laser light only once for four frames. It can be done. That is, for example, the irradiation pattern in each of the fifth to eighth frame scans is the same as the irradiation pattern in each of the first to fourth frame scans.

Further, in this case, each of the irradiation information J1 to the irradiation information J4 indicating each of the irradiation patterns of the first to fourth frames is held in the effective pixel clock counting circuit 92. Then, the effective pixel clock count circuit 92 supplies the enable signal PCE to the drive circuit 93 while repeatedly referring to the irradiation information J1 to the irradiation information J4 in order. Thereby, the irradiation pattern of the laser beam to the scanning region R11 is repeatedly changed in the order of the irradiation patterns shown in A to D of FIG.

In this manner, a predetermined number of temporally consecutive frames (four frames in the example of FIG. 4) are regarded as one frame group, and an irradiation pattern different from other frames is applied to each frame of the frame group. By performing the scanning of the laser light at this time, the irradiation amount of the laser light to the sample 12 can be reduced.

That is, the laser beam to the sample 12 is applied by causing the laser beam to be emitted only once by the four-degree scan where the laser beam is always irradiated by the one-time scan normally in the partial scan region. The amount of light irradiation can be reduced to one fourth of the normal, and the load on the sample 12 can be reduced. Moreover, in this case, it is not necessary to reduce the frame rate of the observation image in order to reduce the irradiation amount of the laser beam.

Although the observation image of each frame does not necessarily display the portion of the sample 12 at all pixels, the display of the observation image of each frame is switched at a high speed, so the observation image is actually viewed. An observer perceives an image obtained by averaging observation images of several consecutive frames as an observation image. For example, assuming that an image obtained by averaging the observation images of the first frame to the fourth frame is perceived by the observer as the observation image, the observer recognizes each part of the sample 12 in all pixels on the observation image. It looks like it is displayed.

That is, it appears to the observer that an observation image with a somewhat deteriorated image quality is displayed as compared with the observation image when the optical path of the laser light is always in the transmission state.

Further, it has been described that when the laser beam is scanned, the laser beam is sequentially irradiated downward from the top horizontal line to the bottom horizontal line in A of FIG. Any scanning may be performed as long as the scanning is performed so that the entire laser light is irradiated.

For example, in the scanning of the odd-numbered frame, the scanning is performed sequentially from the uppermost horizontal line to the lowermost horizontal line downward in FIG. 4A, and in the scanning of the even-numbered frame, the odd-numbered frames. The scanning may be performed in the opposite direction to the time. In such a case, in the scanning of the even-numbered frame, laser light is sequentially emitted upward from the lowest horizontal line to the first horizontal line in FIG. 4A. At this time, in FIG. 4A, the horizontal lines from the lowermost side to the odd-numbered ones are sequentially irradiated with the laser light from the right end to the left end in the figure and the horizontals from the lowermost side to the even-numbered ones. The lines are sequentially irradiated with laser light in the right direction from the left end to the right end in the drawing.

Next, the operation of the light scanning microscope 11 in the preparation stage of the fluorescence observation of the sample 12 will be described.

First, when the observer instructs to display the observation image, the scanner control unit 31 generates the vertical synchronization signal VD, the horizontal synchronization signal HD, and the pixel clock PC according to the instruction of the control unit 30, and The three signals are supplied to the switching unit 32, and the pixel clock PC is supplied to the image processing unit 29. Further, the scanner control unit 31 generates a drive signal VDD and a drive signal HDD from the generated vertical synchronization signal VD and horizontal synchronization signal HD, and supplies them to the vertical scanner 26 and the horizontal scanner 25.

Then, the horizontal scanner 25 rotates the scan mirror based on the drive signal HDD from the scanner control unit 31, and the vertical scanner 26 also rotates the scan mirror based on the drive signal VDD from the scanner control unit 31.

Further, the switching unit 32 refers to the laser information for each frame while referring to the irradiation information held in advance based on the vertical synchronization signal VD, the horizontal synchronization signal HD, and the pixel clock PC supplied from the scanner control unit 31. The control signal is generated so as to change the irradiation pattern of The irradiation pattern of the laser light here is, for example, the irradiation pattern described with reference to A of FIG. 4 to D of FIG. 4.

More specifically, effective horizontal synchronization signal count circuit 91 supplies enable signal HDE to drive circuit 93 based on vertical synchronization signal VD and horizontal synchronization signal HD from vertical synchronization signal generation circuit 61 and horizontal synchronization signal generation circuit 63. . Further, the effective pixel clock count circuit 92 refers to the horizontal synchronization signal HD and the pixel clock PC from the horizontal synchronization signal generation circuit 63 and the pixel clock generation circuit 65 while referring to the held irradiation information J1 to the irradiation information J4. Based on this, the enable signal PCE is supplied to the drive circuit 93.

Then, drive circuit 93 generates a control signal based on enable signal HDE and enable signal PCE from valid horizontal synchronization signal count circuit 91 and valid pixel clock count circuit 92. The drive circuit 93 supplies the generated control signal to the light shielding element 23 to control the operation of the light shielding element 23.

Furthermore, the light source 22 emits laser light as excitation light and causes the light to enter the light shielding element 23. The light shielding element 23 transmits the laser light from the light source 22 with the optical path of the laser light in a transmission state or shields the laser light from the light source 22 in a light shielding state according to the control signal from the switching unit 32. The laser light transmitted through the light shielding element 23 is reflected by the dichroic mirror 24, and is irradiated to the sample 12 through the horizontal scanner 25 and the vertical scanner 26.

At this time, the horizontal scanner 25 rotates the scan mirror to scan the laser light horizontally on the sample 12, and the vertical scanner 26 rotates the scan mirror to vertically move the laser light on the sample 12. Let it scan.

Further, when the sample 12 is irradiated with the laser light, fluorescence is expressed from the sample 12. The fluorescence from the sample 12 passes through the vertical scanner 26 and the horizontal scanner 25 and further through the dichroic mirror 24. Then, the fluorescence transmitted through the dichroic mirror 24 is transmitted through the barrier filter 27 and received by the light detection unit 28 to be photoelectrically converted.

The image processing unit 29 acquires (samples) an electrical signal obtained by photoelectric conversion of fluorescence from the light detection unit 28 based on the pixel clock PC from the scanner control unit 31. Then, the image processing unit 29 generates an observation image based on the electrical signal acquired from the light detection unit 28, and supplies the generated observation image of each frame to the display unit 33 for display.

As a result, the observation image is displayed on the display unit 33, and the observer can adjust the position of the stage 21 while checking the portion of the sample 12 on the observation image.

As described above, by changing the irradiation pattern of the laser light for each frame, the load of the sample 12 due to the irradiation of the laser light can be reduced. Moreover, since the frame rate of the observation image does not decrease, the operation of the observer on the light scanning microscope 11 is immediately reflected on the observation image displayed on the display unit 33. Therefore, the operability of the light scanning microscope 11 does not decrease either.

In the above description, the light shielding element 23 is described as being disposed between the light source 22 and the dichroic mirror 24. However, the light shielding element 23 may be disposed anywhere on the light path of the laser light, that is, between the light source 22 and the sample 12 It may be done.

Further, in the example shown in A to D of FIG. 4, while the scanning of the first frame to the fourth frame is performed, the laser beam is irradiated only once to one partial scanning region. Although it has been described, the partial scanning area may be irradiated with the laser beam a plurality of times. In addition, there may be a partial scanning area where the laser beam is not irradiated even once.

That is, in a period in which scanning of a frame group including the first frame to the fourth frame is performed, the laser beam is irradiated to the scanning range of the laser beam by the horizontal scanner 25 and the vertical scanner 26, that is, substantially the entire scanning region R11. As long as the laser beam is scanned, the laser beam may be scanned.

Furthermore, the irradiation intensity of the laser beam of each flame | frame which comprises a flame | frame group may be all the same intensity, and may be mutually different intensity | strength. When the irradiation intensity of the laser beam of each frame is different from each other, it is preferable that the average irradiation intensity of each frame group is substantially equal.

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

Reference Signs List 11 light scanning microscope, 12 samples, 21 stages, 22 light sources, 23 light blocking elements, 24 dichroic mirrors, 25 horizontal scanners, 26 vertical scanners, 28 light detection units, 29 image processing units, 31 scanner control units, 32 switching units

Claims (2)

Scanning means for scanning light emitted from a light source on a sample to be observed;
A light shielding unit disposed on an optical path from the light source of the light to the sample to shield the light from the light source;
A light blocking control means for controlling the light blocking means to be in a light transmitting state in which the light is irradiated to the sample or in a light blocking state in which the light is blocked and not irradiated to the sample;
The light shielding control means is configured to emit the light to obtain the observation image of an arbitrary frame among a frame group consisting of several consecutive frames of an observation image obtained by scanning the light on the sample. The light shielding means is controlled such that the irradiation area of the sample is different from the irradiation area of another frame of the frame group. The light shielding control means is configured to irradiate the light over substantially the entire scanning range of the light by the scanning means in a period in which the light is scanned to obtain the observation images of all the frames of the frame group. The optical scanning microscope according to claim 1, wherein the light shielding means is controlled.

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