JP4963543B2 - Scanning laser microscope and control method thereof - Google Patents

Description

  The present invention relates to a scanning laser microscope for detecting transmitted light or reflected light from a sample when the surface of the sample is scanned with laser light, or fluorescence generated in the sample, and a control method therefor.

  A confocal scanning laser microscope irradiates a laser beam while scanning in the X-axis and Y-axis directions of the sample, detects transmitted light from the sample, reflected light, or fluorescence generated in the sample by a detector, and transmits transmitted light or It is a microscope for obtaining two-dimensional luminance information of reflected light or fluorescence. In addition, by displaying this luminance information as a two-dimensional distribution of luminance on a display or the like in correspondence with the XY scanning position, it is possible to observe a fluorescent image, a transmission image, or a reflection image of the sample.

  In addition, the confocal scanning laser microscope detects only the information on the focused surface by providing a stop with a diameter about the diffraction limit of the light to be measured at a position conjugate with the sample of the detection optical system. To do. Since this confocal scanning laser microscope can detect only information on the focal plane, an optical tomographic image, that is, three-dimensional information can be obtained without damaging the sample, and information on the non-focus plane is also obtained. By eliminating the above, a very clear image can be obtained.

When performing such an experiment of FRAP (Fluorescence Recovery After Photobleaching) using such a confocal scanning laser microscope, a sample image before execution of photobleaching is first acquired, and then photobleaching is performed. And an experiment is performed according to a procedure for acquiring a sample image during the photo bleaching and during the fluorescence recovery after the photo bleaching.
Conventionally, as a scanning laser microscope suitable for the FRAP experiment, an image scanning optical system for irradiating a sample with a first laser beam for acquiring a sample image, and photobleaching (in a specific part of the sample) There is known a scanning laser microscope provided with a scanning optical system for stimulation for irradiating a sample with a second laser beam (to give stimulation to the specimen).
In this scanning laser microscope, while irradiating the sample with the second laser, that is, while performing photobleaching, the sample is scanned with the first laser to irradiate the sample. A sample image at the return is acquired. As a result, it is possible to continuously observe the state of the sample at the return of fluorescence before the start of photobleaching.

  When performing the FRAP experiment as described above, it is very important to control the scanning timing of the first laser beam and the second laser beam. For example, when the first laser beam and the second laser beam irradiate the same position at the same time, the light intensity of the second laser beam is the first laser irradiated on the sample for image acquisition. Since the intensity of the light is much larger than the intensity of the light, there is a possibility that the second laser light is mixed into the first scanning optical system, and a scanning image in which the luminance value at the mixed point is saturated is obtained. is there.

  In response to these problems, Japanese Patent Application Laid-Open No. 2000-275529 (Patent Document 1) discloses a method of scanning from a scanning optical system for an image by scanning the scanning optical system for stimulation during a blanking period of the scanning optical system for an image. There has been proposed a method for avoiding the overlap of the first laser beam and the second laser beam from the stimulation scanning optical system.

JP 2000-275529 A

  However, in the technique disclosed in the above Japanese Patent Laid-Open No. 2000-275529, the second laser light is scanned by the stimulation scanning optical system during the blanking period of the first laser light by the image scanning optical system. Must be completed. Therefore, for example, when the first laser beam is scanned at high speed by the image scanning optical system, and the return period is extremely short, the stimulation scanning optics is used within the return period. Since it becomes impossible to complete the scanning of the second laser beam by the system, it is necessary to wait for the scanning of the first laser beam. For this reason, there is a problem that the first laser beam cannot be scanned at a constant speed, and the acquired image loses temporal quantification.

  The present invention has been made in view of such circumstances, and in a scanning laser microscope having a plurality of optical systems, the influence of each optical system on each other is eliminated and temporal quantification is maintained. Another object of the present invention is to provide a scanning laser microscope capable of acquiring a captured image and a control method thereof.

In order to solve the above problems, the present invention employs the following means.
The present invention includes an image scanning optical system that two-dimensionally scans a first laser beam for obtaining an image of a sample and irradiates the sample, and a return of the first laser beam by the image scanning optical system. A stimulation optical system that irradiates a specific part of the sample with a second laser beam for stimulation during a line period, and the irradiation of the second laser light by the stimulation optical system is completed during the blanking period Provided is a scanning laser microscope that can continuously perform the scanning of the first laser light by the image scanning optical system without stopping even if it cannot be performed .

According to the above configuration, since the second laser beam is irradiated to the specific part of the sample during the blanking period of the first laser beam by the image scanning optical system, the influence of each optical system on each other is eliminated. Is possible. As a result, for example, the problem of poor luminance of the acquired image caused by the second laser beam entering the image scanning optical system, or the first laser beam and the second laser beam irradiate the same position at the same time. It is possible to eliminate the problem of fluorescent discoloration caused by the occurrence of the problem.
Furthermore, since the first laser beam irradiation is prioritized over the second laser beam irradiation, for example, even if the second laser beam irradiation cannot be completed within one retrace period. The first laser beam can be continuously scanned without stopping. As a result, it is possible to acquire an image in which temporal quantification is maintained, and it is possible to obtain a highly accurate experimental result.

  The specific portion may be a single point (point) or a region having a predetermined area. Further, the stimulating optical system may have only a function of irradiating the second laser beam to one point on the sample, and the second laser beam is scanned two-dimensionally to obtain a predetermined on the sample. It may have a function of irradiating an area. Furthermore, a plurality of specific portions to be irradiated with the second laser light may be provided on the sample. Further, the stimulation optical system is not limited to one, and two or more stimulation optical systems may be provided.

The present invention relates to an image scanning optical system that two-dimensionally scans a first laser beam for obtaining an image of a sample and irradiates the sample, and a second laser beam for stimulation to a specific part of the sample. And the first laser beam is irradiated in the common area scanning period in which the first laser beam is irradiated to the scanning line in which the specific portion is set. Even when the irradiation of the second laser beam by the stimulation optical system cannot be completed during the blanking period, the first laser beam by the image scanning optical system is performed during the blanking period of the laser beam. Provided is a scanning laser microscope capable of continuously performing scanning without stopping .

According to the above configuration, in the common region scanning period in which the first laser beam is irradiated to the scanning line in which the specific part is set, the second laser beam is returned in the blanking period of the first laser beam by the image scanning optical system. Since the laser beam is irradiated, it is possible to eliminate the influence of each optical system on each other.
Furthermore, since the first laser beam scanning is prioritized over the second laser beam irradiation in the common region scanning period, for example, the second laser beam irradiation can be completed within one blanking period. Even if it is not, it can be continuously performed without stopping the scanning of the first laser beam. As a result, it is possible to acquire an image in which temporal quantification is maintained, and it is possible to obtain a highly accurate experimental result.
Further, in the period in which the first laser beam is irradiated to the scanning line in which the specific part is not set, the second laser beam is output regardless of whether the scanning state of the first laser beam is the blanking period. Irradiation efficiency of the second laser light can be increased by irradiating the specific portion with the laser light.

The stimulation optical system includes a specific part, and a period during which the first laser beam is irradiated to a scanning line in which an irradiation stop region that is a region smaller than the scanning region of the first laser beam is set. In this case, the irradiation of the second laser beam may be stopped.
As a result, the irradiation efficiency of the second laser beam can be increased as compared with the irradiation mode in which the second laser beam is irradiated during the blanking period of the first laser beam in all the scanning lines. Compared with the irradiation mode in which the second laser beam is irradiated during the blanking period of the first laser beam in the area scanning period, the influence of the optical systems on each other can be eliminated.

The present invention relates to an image scanning optical system that two-dimensionally scans a first laser beam for obtaining an image of a sample and irradiates the sample, and a second laser beam for stimulation to a specific part of the sample. and a stimulus optical system for irradiating, in a period in which the image scanning optical system for irradiating a first laser beam to the specific portion, wherein the stimulus optical system stops the irradiation of the second laser beam In the period in which the scanning optical system for image irradiates the first laser beam and the period in which the first laser beam is not irradiated to the specific part, the stimulation optical system has the second laser beam. Provided is a scanning laser microscope that irradiates the laser beam .

  According to the above configuration, in order that the irradiation of the second laser beam by the stimulation optical system is stopped during the period in which the specific laser beam is irradiated to the specific part by the image scanning optical system, the optical systems are mutually connected. It is possible to eliminate the influence on the. Furthermore, in the period when the specific part is not irradiated with the laser light by the image scanning optical system, the irradiation efficiency of the second laser light can be increased by irradiating the specific part with the second laser light.

According to the present invention, a first laser beam for obtaining an image of a sample is two-dimensionally scanned to irradiate the sample, and a second laser for stimulation is applied during a blanking period of the first laser beam. A method of controlling a scanning laser microscope that irradiates a specific part of the sample with light, and stops scanning of the first laser light even when irradiation of the second laser light cannot be completed in the blanking period. The present invention provides a control method for a scanning laser microscope that is continuously performed without any problems .

According to the above method, since the second laser beam is irradiated to the specific part of the sample during the blanking period of the first laser beam, it is possible to eliminate the influence of each laser beam on each other. Thereby, for example, the problem of poor luminance of the acquired image caused by mixing the second laser light into the image scanning optical system that scans the first laser light, or the first laser light and the second laser light. The problem of fluorescent fading caused by simultaneously irradiating the same position can be solved.
Furthermore, since the first laser light irradiation is prioritized over the second laser light irradiation, even if the second laser light irradiation cannot be completed within one retrace period, The scanning of one laser beam can be performed continuously without stopping. As a result, it is possible to acquire an image in which temporal quantification is maintained, and it is possible to obtain a highly accurate experimental result.

The present invention scans a first laser beam for obtaining an image of a sample two-dimensionally to irradiate the sample, and irradiates a specific part of the sample with a second laser beam for stimulation. A method for controlling a laser microscope, wherein the first laser beam is irradiated with the second laser beam in a common region scanning period in which the first laser beam is irradiated onto a scanning line in which the specific part is set. And a scanning laser microscope that continuously performs the scanning of the first laser light without stopping even when the irradiation of the second laser light cannot be completed during the blanking period. A control method is provided.

According to the above method, in the common region scanning period in which the first laser beam is irradiated to the scanning line in which the specific part is set, the second laser beam is irradiated during the blanking period of the first laser beam. It becomes possible to eliminate the influence of each laser beam on each other.
Furthermore, since the first laser beam scanning is prioritized over the second laser beam irradiation in the common region scanning period, for example, the second laser beam irradiation can be completed within one blanking period. Even if it is not, it can be continuously performed without stopping the scanning of the first laser beam. As a result, it is possible to acquire an image in which temporal quantification is maintained, and it is possible to obtain a highly accurate experimental result.
Further, in the period in which the first laser beam is irradiated to the scanning line in which the specific part is not set, the second laser beam is output regardless of whether the scanning state of the first laser beam is the blanking period. Irradiation efficiency of the second laser light can be increased by irradiating the specific portion with the laser light.

The present invention scans a first laser beam for obtaining an image of a sample two-dimensionally to irradiate the sample, and irradiates a specific part of the sample with a second laser beam for stimulation. A method of controlling a laser microscope, wherein the irradiation of the second laser light is stopped and the first laser light is irradiated in the period of irradiating the specific part with the first laser light , In addition, the present invention provides a control method for a scanning laser microscope that irradiates the second laser beam during a period when the first laser beam is not irradiated to the specific part .

  According to the above method, since the irradiation of the second laser light is stopped during the period in which the first portion is irradiated with the specific portion, it is possible to eliminate the influence of the laser beams on each other. . Furthermore, in the period when the first laser beam is not irradiated to the specific part, the irradiation efficiency of the second laser light can be increased by irradiating the specific part with the second laser beam.

  According to the present invention, even when a plurality of optical systems are provided, it is possible to eliminate the influence of each optical system and to obtain an image with temporal quantification maintained. .

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[First Embodiment]
FIG. 1 is a block diagram showing a schematic configuration of a scanning laser microscope according to the first embodiment of the present invention.
The scanning laser microscope according to the present embodiment stimulates the sample 17 with the image scanning optical system 100 that irradiates the first laser beam for image acquisition on the focal plane of the sample 17 two-dimensionally. And a scanning optical system for stimulation 200 that irradiates a specific portion of the sample 17 with a second laser beam to be applied.
The image scanning optical system 100 includes a first light source 1, a dichroic mirror 2, a first scanning optical unit 3, a relay lens 4, and a mirror 5. A detection optical system 300 is arranged on the branch optical path of the dichroic mirror 2 of the image scanning optical system 100. The detection optical system 300 includes a photometric filter 6, a lens 7, a confocal pinhole 8, and a photoelectric conversion element 9.

  The stimulation scanning optical system 200 includes a second light source 10, a second scanning optical unit 11, a relay lens 12, and a dichroic mirror 13. The optical axis of the image scanning optical system 100 and the optical axis of the stimulation scanning optical system 200 are combined by the dichroic mirror 13 and guided to the imaging lens 14 and the objective lens 15. The relay lenses 4 and 12 are arranged so that the focal position coincides with the focal position of the imaging lens 14. The sample 17 is placed on the stage 18.

The dichroic mirror 13 has characteristics of transmitting light having a wavelength longer than that of the laser light from the image scanning optical system 100 and reflecting the wavelength of the laser light from the stimulation scanning optical system 200.
The first scanning optical unit 3 is controlled based on the scanning signal from the first scanning waveform generation circuit 19. The first scanning waveform generation circuit 19 generates the scanning signal based on the first clock supplied from the first clock generation circuit 20. The first clock generation circuit 20 generates a first clock based on various commands from the CPU 26, and supplies the first clock to the first data enable signal generation circuit 21.
The first data enable signal generation circuit 21 generates a first data enable signal based on the first clock from the first clock generation circuit 20 and the scanning waveform from the first scanning waveform generation circuit, and The generated first data enable signal is supplied to the second clock generation circuit 23, the A / D converter 25, and the first light source 1.

The second scanning optical unit 11 is controlled based on the scanning signal from the second scanning waveform generation circuit 22. The second scanning waveform generation circuit 22 generates the scanning signal based on the second clock supplied from the second clock generation circuit 23. The second clock generation circuit 23 generates a second clock based on the first data enable signal supplied from the first data enable signal generation circuit 24 and various commands supplied from the CPU 26, and the second clock generation circuit 23 generates the second clock. Are supplied to the second data enable signal generation circuit 24.
The second data enable signal generation circuit 24 generates a second data enable signal based on the second clock from the second clock generation circuit 23 and the scanning waveform from the second scanning waveform generation circuit 22. The generated second data enable signal is supplied to the second light source 10.

The A / D converter 25 converts the analog electric signal from the photoelectric conversion element 9 into a digital electric signal and supplies it to the CPU 26.
The CPU (central processing unit) 26 outputs the first scan generation command, the scan stop command, and the like to the first clock generation circuit 20 and the second clock generation circuit 23 at an appropriate timing, thereby performing the first scan. Indirectly control each part of the microscope, including the optical unit 3 and the second scanning optical unit 11, and create image data based on the digital signal supplied from the A / D converter 25, By storing this in the frame memory 29, the experimental results and the like are displayed on the display device 30.
The CPU 26 is connected to a main storage device 27 such as a RAM (Random Access Memory) and an auxiliary storage device 28 such as an HD (Hard Disk) ROM (Read Only Memory).
For example, various control application programs are stored in the auxiliary storage device 28, and the CPU 26 reads out the control application program from the auxiliary storage device 28 to the main storage device 27 and executes it, whereby various types of FRAP and the like as will be described later. Make the experiment happen. A general personal computer may be used as the CPU 26, the main storage device 27, and the auxiliary storage device 28. Further, an input device such as a keyboard or a mouse may be further connected to the CPU 26.

  In the scanning laser microscope having such a configuration, the laser light from the first light source 1 is guided to the first scanning optical unit 3 that is scan-controlled by the first scanning waveform generation circuit 19 and is in an arbitrary direction. Are deflected and scanned. This laser light is further condensed on the cross section 16 of the sample 17 via the relay lens 4, the mirror 5, the dichroic mirror 13, the imaging lens 14, and the objective lens 15, and is scanned two-dimensionally within the cross section 16. .

  A fluorescent indicator that is excited by the wavelength of the first light source 1 is introduced into the sample 17, and when the laser light is scanned two-dimensionally within the cross section 16, the fluorescent indicator is excited to generate fluorescence. Fluorescence captured by the objective lens 15 travels in the opposite direction along the same optical path as the laser light, passes through the objective lens 15, the imaging lens 14, and the dichroic mirror 13, and is reflected by the mirror 5, the relay lens 4, and the first scanning optics. The light is guided to the dichroic mirror 2 through the unit 3. The dichroic mirror 2 has a characteristic of reflecting light having a wavelength longer than that of the laser light from the first light source 1, whereby the fluorescence is reflected by the dichroic mirror 2 and introduced into the detection optical system 300. .

  In the detection optical system 300, the fluorescence is selectively transmitted through the photometric filter 6, and only the light from the cross section 16 is selected by the lens 7 and the confocal pinhole 8, and enters the photoelectric conversion element 9. And converted into an electrical signal. The output signal of the photoelectric conversion element 9 is guided to the A / D converter 25, converted into a digital signal in synchronization with the signal generated from the first data enable signal generation circuit 20, and supplied to the CPU 26. In the CPU 26, image data is created based on the digital signal from the A / D converter 25, and this image data is stored in the frame memory 29 and displayed on the display provided in the display device 30. As a result, a fluorescent image (two-dimensional distribution of fluorescent luminance) at the cross section 16 is displayed on the display of the display device 30.

  On the other hand, the laser light from the second light source 10 is combined with the optical axis from the image scanning optical system 100 via the second scanning optical unit 11, the relay lens 12, and the dichroic mirror 13. Then, the light passes through the imaging lens 14 and the objective lens 15 and is irradiated onto the cross section 16 of the sample 17. The irradiation position in the cross section 16 at this time is controlled by the second clock generation circuit 23 and the second scanning waveform generation circuit 22 to control the second scanning optical unit 11, thereby scanning the image scanning optical system 100. Any position that does not depend on the position can be selected.

Subsequently, the control of the image scanning optical system 100 and the stimulation scanning optical system 200 described above will be described in detail. Here, as shown in FIG. 2, a specific part having a predetermined area is provided in a part of the scanning region of the first laser light, and the second part is provided in this specific part (second laser light scanning region). A case where scanning is performed with laser light will be described as an example.
First, as shown in FIG. 1, the first scanning optical unit 3 includes a horizontal scanner 3a for scanning the laser beam from the light source 1 in the horizontal direction on the sample surface, and also in the vertical direction on the sample surface. A vertical scanner 3b for scanning is provided.

As shown in FIG. 3, the first scanning waveform generating circuit 19 is configured to horizontally swing the horizontal scanner 3 a around the axis so as to scan the first laser beam in the horizontal direction on an arbitrary cross section of the sample 17. In order to scan the direction waveform and the first laser beam in the vertical direction, the vertical direction waveform that swings the vertical scanner 3b about the axis is generated.
Here, the horizontal waveform for swinging the horizontal scanner 3a about the axis is an X blanking period A in which the horizontal scanner 3a is moved from the center position O to the swing angle G at the start of luminance data sampling, and A luminance data sampling period B for sampling the luminance data and an X blanking period C for returning the horizontal scanner 3a from the swing angle H at the end of the luminance data sampling to the center position O are constituted. In this horizontal waveform, the X blanking period A and the X blanking period C correspond to the X blanking period of the first laser beam.

  Similarly, a vertical waveform for swinging the vertical scanner 3b about the axis is a Y blanking period D for moving the vertical scanner 3b from the center position O to the swing angle I at the start of luminance data sampling, and A luminance data sampling period E for sampling the luminance data and a Y blanking period F for returning the vertical scanner 3b from the swing angle J at the end of the luminance data sampling to the center position O are configured. In this vertical waveform, the Y blanking period D and the Y blanking period F correspond to the Y blanking period of the first laser beam.

Further, as shown in FIG. 4, the first data enable signal generation circuit 21 includes a horizontal direction data enable signal generation circuit 211, a vertical direction data enable signal generation circuit 212, and a data enable signal calculation circuit 213. ing.
As shown in FIG. 3, the horizontal data enable signal generation circuit 211 is turned off in the X blanking period A, turned on in the X luminance data sampling period B, and turned off in the X blanking period C. Is generated.
Similarly, as shown in FIG. 3, the vertical data enable signal generation circuit 212 is vertical data that is OFF in the Y blanking period D, ON in the Y luminance data sampling period E, and OFF in the Y blanking period F. Generate an enable signal.

  Here, the horizontal direction data enable signal is generated in a plurality of cycles in one cycle of the vertical waveform. For example, when scanning with a resolution of 512 pixels × 512 pixels, the horizontal data enable signal is generated 512 times in one cycle of the vertical waveform.

  As shown in FIG. 5, the data enable signal operation circuit 213 shown in FIG. 4 performs a logical product operation of the horizontal direction data enable signal and the vertical direction data enable signal, and performs the first operation as shown in FIG. 1 data enable signal is generated. As a result, the first data enable signal is turned on during the period when the irradiation position of the first laser beam is on the scanning region of the first laser beam, in other words, the first laser beam is applied to the sample 17. This is a period for irradiating the upper section 16.

  Since the A / D converter 25 and the first light source 1 are controlled based on the first data enable signal, the first laser is used except when the first laser beam is scanning the sample 17. Is not performed, and the A / D conversion of the luminance data is not performed. Further, the data enable signal generation circuit 21 is configured not to operate unless the clock from the first clock generation circuit 20 is input.

  Similarly, as shown in FIG. 1, the second scanning optical unit 11 includes a horizontal scanner 11a for scanning the laser light from the second light source 10 in the horizontal direction on the sample surface, and similarly on the sample surface. And a vertical scanner 11b for scanning in the vertical direction.

As shown in FIG. 7, the second scanning waveform generation circuit 22 has a horizontal waveform for causing the horizontal scanner 11a to swing about the axis in order to scan the second laser beam on the sample 17 in the horizontal direction, and In order to scan the second laser beam in the vertical direction, a vertical waveform is generated that swings the vertical scanner 11b about the axis.
Here, the horizontal waveform for swinging the horizontal scanner 11a about the axis is an X blanking period A in which the horizontal scanner 11a is rotated from the center position O to the swing angle G ′ at the start of stimulation light irradiation. ′, A stimulation light irradiation period B ′ for irradiating the stimulation light, and an X blanking period C ′ for returning the horizontal scanner 11a from the swing angle H ′ at the end of the stimulation light irradiation to the center position O. . In this horizontal waveform, the X blanking period A ′ and the X blanking period C ′ correspond to the X blanking period of the second laser beam.

  Similarly, the vertical waveform for swinging the vertical scanner 11b about the axis is a Y blanking period D in which the vertical scanner 11b is rotated from the center position O to the swing angle I ′ at the start of stimulation light irradiation. ′, A stimulation light irradiation period E ′ for irradiating the stimulation light, and a Y blanking period F ′ for returning the vertical scanner 11b from the swing angle J ′ at the end of the stimulation light irradiation to the center position O. . In this vertical waveform, the Y blanking period D ′ and the Y blanking period F ′ correspond to the Y blanking period of the second laser beam.

As shown in FIG. 8, the second data enable signal generation circuit 24 includes a horizontal direction data enable signal generation circuit 241, a vertical direction data enable signal generation circuit 242, and a data enable signal calculation circuit 243. ing.
As shown in FIG. 7, the horizontal data enable signal generation circuit 241 is horizontal data that is OFF in the X blanking period A ′, ON in the stimulation light irradiation period E ′, and OFF in the X blanking period C ′. Generate an enable signal.
Similarly, as shown in FIG. 7, the vertical data enable signal generation circuit 242 is vertical so that it is OFF in the Y blanking period D ′, ON in the stimulation light irradiation period E ′, and OFF in the Y blanking period F ′. A direction data enable signal is generated.

The data enable signal operation circuit 243 illustrated in FIG. 8 performs a logical product operation on the horizontal direction data enable signal and the vertical direction data enable signal as illustrated in FIG. 7 to generate a second data enable signal. As a result, the second data enable signal is turned on during a period in which the second laser beam is applied to a specific portion set in the cross section 16 on the sample 17.
Further, the data enable signal calculation circuit 243 is configured to operate only during a period in which the second clock is input from the second clock generation circuit 23 shown in FIG. Here, the second clock generation circuit 23 generates the second clock based on the scan start command and the scan stop command from the CPU 26, and the second clock is generated only during the period when the first data enable signal is OFF. Is generated. In other words, as shown in FIG. 6B, the second clock generation circuit 23 is a period from when the scan start command is output by the CPU 26 to when the operation stop command is output, The second clock is generated only during the period when the data enable signal is off.
Thus, the second data enable signal output from the above-described data enable signal calculation circuit 243 is a period during which the second clock is input, and the second laser beam is on the sample 17. Only in a period during which the specific part set in the cross-section 16 is irradiated is in the ON state.

  Such a second data enable signal is supplied to the second light source 10, and the second light source 10 is controlled based on the second data enable signal. As a result, during the blanking period of the first laser beam by the image scanning optical system 100, the second laser beam is irradiated to a specific portion set in the cross section 16 on the sample 17.

Next, a case where an FRAP experiment is performed in the scanning laser microscope having the above configuration will be described.
First, a scan start command from the CPU 26 is supplied to the first clock generation circuit 20, and the first clock generation circuit 20 generates a first clock signal at regular intervals. The first clock signal is supplied to the first scanning waveform generation circuit 19 and the first data enable signal generation circuit 21. The first scanning waveform generation circuit 19 generates the horizontal waveform and the vertical waveform shown in FIG. 3 during the period in which the first clock signal is generated. The generated horizontal waveform and vertical waveform are supplied to the first scanning optical unit 3 in synchronization with the first clock signal input from the first clock generation circuit 20, and are incorporated in the horizontal scanner. 3a, the vertical scanner 3b is driven.

On the other hand, the first data enable signal generation circuit 21 generates a horizontal direction data enable signal and a vertical direction data enable signal as shown in FIG. 5, and the first data enable signal as shown in FIG. A data enable signal is generated.
The generated first data enable signal is supplied to the light source 1 and the A / D converter 25. When the first data enable signal is in the ON state, the first laser light is irradiated and the A / D The D converter 25 is activated. As a result, signal conversion is performed by the A / D converter 25 during the period in which the first laser beam scanning region shown in FIG. 2 is irradiated with the first laser beam, and this digital signal is supplied to the CPU 26. As a result, the result of fluorescence observation is displayed on the display of the display device 30.

  On the other hand, the second clock generation circuit 23 starts driving in response to a scanning start command from the CPU 26, and the first data enable signal supplied from the first data enable signal generation circuit 21 is OFF. Only a second clock signal having a constant frequency is generated and supplied to the second scanning waveform generation circuit 22 and the second data enable signal generation circuit 24.

  The second scanning waveform generation circuit 22 generates a horizontal waveform and a vertical waveform as shown in FIG. The generated horizontal waveform and vertical waveform are supplied to the second optical scanning unit 11 in synchronization with the second clock signal supplied from the second clock generation circuit 23, and are incorporated in the horizontal scanner. 11a, the vertical scanner 11b is driven.

On the other hand, the second data enable signal generation circuit 24 generates a horizontal data enable signal and a vertical data enable signal as shown in FIG. 7, and the second data enable signal is generated based on these signals and the second clock. A signal is generated.
The generated second data enable signal is supplied to the second light source 10, and irradiation of the second laser light is performed when the second data enable signal is in the ON state.
Thereby, in the blanking period of the first laser light, photobleaching is executed by irradiating the specific part set on the cross section 16 of the sample 17 with the second laser light. . Then, at the end of the scanning of the second laser beam for the specific part set on the cross section 16 of the sample 17, the CPU 26 supplies a scanning stop command to the second clock generation circuit 23, and the second clock generation circuit 23 stops driving. Thereby, the operation of the second scanning optical system 100 is stopped and the photo bleaching is finished.

As described above, according to the scanning laser microscope according to the present embodiment, as shown in FIGS. 6A and 6B, the second data only when the first data enable signal is OFF. A second clock is supplied from the clock generation circuit 23 to the second scanning waveform generation circuit 22 and the second data enable signal generation circuit 24. As a result, even if the second scanning optical system 200 performs scanning of the second laser light during the scanning period of the first laser light by the first scanning optical system 100, the second scanning optical system 200 Photo bleaching can be executed during the blanking period of the first laser beam by the image scanning optical system 100. As a result, the influence of each optical system on each other can be eliminated. For example, the problem of poor luminance of an acquired image caused by the second laser light entering the image scanning optical system 100, or the first laser light and the second laser light irradiate the same position at the same time. The problem of fluorescent fading caused by this can be solved.
Furthermore, since the image scanning optical system 100 can scan the first laser beam at a constant speed, it is possible to acquire an image with temporal quantification maintained. Thereby, a highly accurate experimental result can be obtained.

[Second Embodiment]
Next, a scanning laser microscope according to the second embodiment of the present invention will be described.
FIG. 8 is a block diagram showing a schematic configuration of the scanning laser microscope according to the present embodiment.
As shown in FIG. 9, the scanning laser microscope according to the present embodiment has a configuration in which a data enable signal conversion circuit 31 is further added to the configuration of the scanning laser microscope according to the first embodiment shown in FIG. Take. The data enable signal conversion circuit 31 is provided between the first data enable signal generation circuit 21 and the second clock generation circuit 23.
In the data enable signal conversion circuit 31, the start line number and the end line number of the second laser beam scanning area (specific part) of the stimulation scanning optical system 200 are registered in advance. For example, as shown in FIG. 10, the number of scanning lines in the scanning region of the first laser beam by the image scanning optical system 100 is “516”, and the scanning line numbers “208” to “308” in the scanning region. When the second laser light scanning region by the stimulation scanning optical system 200 is set, the data enable signal conversion circuit 31 has “208” as the start line number of the second laser light scanning region. ",""308" is registered as the end line number.

Hereinafter, the scanning laser microscope according to the present embodiment will be described with respect to the differences from the first embodiment described above.
First, as in the first embodiment described above, when a scan start command is supplied from the CPU 26 to the first clock generation circuit 21, the first clock signal is sent from the first clock generation circuit 20 to the first clock. The data enable signal generation circuit 21 is supplied. As a result, the first data enable signal generation circuit 21 generates a first data enable signal and supplies it to the data enable signal conversion circuit 31.
The data enable signal conversion circuit 31 counts the falling edge or rising edge of the first data enable signal as shown in FIG. 11, so that which one is currently in the scanning region of the first laser light as shown in FIG. 10. The scanning line number is monitored whether the first laser beam is being scanned, and based on the monitoring result, the first data enable signal supplied from the first data enable signal generation circuit 21 is converted, This is supplied to the second clock generation circuit 23.

Specifically, as shown in FIG. 10, when “208” is registered as the start line number and “308” as the end line number of the scanning region of the second laser beam by the stimulation scanning optical system 200, Then, it is determined whether or not the scanning line number of the first laser beam based on the count number satisfies the condition of the starting line number “208” or more and “308” or less. As a result, when the above condition is not satisfied, that is, when the current scanning line number of the first laser beam is “any one of 1 to 207” or “any one of 309 to 516”. As shown in FIG. 11, the data enable signal conversion circuit 31 converts all the first data enable signals in this period to the OFF state and outputs them to the second clock generation circuit 23. On the other hand, when the above condition is satisfied, that is, when the scanning line number of the first laser beam is “any one of 208 to 308 (common area scanning period)”, the data enable signal conversion circuit 31 As shown in FIG. 11B, the first data enable signal in this period is output as it is to the second clock generation circuit without being converted.
As a result, the second clock generated by the second clock generation circuit 23 is generated when the first data enable signal is OFF, as shown in FIG. 11C.

  Then, by controlling the stimulation scanning optical system 200 based on the first data enable signal subjected to such conversion processing, the scanning region of the first laser beam and the stimulation by the image scanning optical system 100 are controlled. Since the scanning by the stimulating scanning optical system 200 can always be performed in a region other than the common region where the scanning region of the second laser light by the scanning optical system 200 overlaps, the irradiation efficiency of the second laser light is increased. It becomes possible.

As described above, according to the scanning laser microscope according to the present embodiment, the second laser beam by the stimulation scanning optical system 200 is used during the blanking period of the first laser beam by the image scanning optical system 100. Therefore, it is possible to eliminate the influence of each optical system on each other. As a result, for example, the problem of poor brightness of the acquired image caused by the second laser light entering the image scanning optical system 100, or the first laser light and the second laser light irradiate the same position at the same time. This makes it possible to eliminate the problem of fluorescent fading caused by the failure.
Furthermore, since the image scanning optical system 100 can scan the first laser beam at a constant speed, it is possible to acquire an image with temporal quantification maintained. Thereby, a highly accurate experimental result can be obtained.

  Further, in a region other than the overlapping region between the scanning range of the first laser beam by the scanning optical system 100 for image and the scanning range of the second laser beam by the scanning optical system 200 for stimulation, the scanning optical system 200 for the stimulation uses the second scanning region. Since the second laser beam can always be scanned, the sample can be more efficiently stimulated with light.

  In the above embodiment, a case where the start line number and the end line number of the second laser beam scanning area by the stimulation scanning optical system 200 are registered in the data enable signal conversion circuit 31 in advance. As described above, the information registered in the data enable signal conversion circuit 31 is not limited to the above information. For example, the number of scanning lines in the scanning region of the second laser beam (for example, “308−208 = 100” in the example of FIG. 10) and the scanning line number that is the center of the scanning region (for example, FIG. 10). In the example, if “208+ (308−208) / 2 = 258”) is registered, it is possible to calculate the start line number and the end line number of the scanning area of the second laser beam. . Thus, the present invention is not limited to the above example, and the data enable signal conversion circuit 31 specifies a common area between the scanning area scanned with the first laser beam and the scanning area scanned with the second laser beam. It is sufficient if the information for registration is registered.

In the above embodiment, the irradiation timing of the second laser light is set to the first laser in the period in which the first laser light is irradiated to the scanning line in which the scanning region of the second laser light is set. Although the light blanking period is used, not only the common region but also a predetermined region including the common region (for example, a region obtained by adding two lines above and below the common region, that is, in the above embodiment, “scanning line number 206 Thru | or the range of the investigation line number 310 "), it is good also considering the irradiation timing of the said 2nd laser beam as a blanking period of the 1st laser beam.
Further, the irradiation with the second laser beam may be stopped during the period in which the first laser beam irradiates the scanning region of the second laser beam.

  According to such a configuration, the second laser beam is applied to the specific part in a period in which the specific laser beam (scanning region of the second laser beam) is not irradiated by the image scanning optical system 100. Therefore, it is possible to further increase the irradiation efficiency of the second laser beam.

  As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the specific structure is not restricted to this embodiment, The design change etc. of the range which does not deviate from the summary of this invention are included.

First, in the first and second embodiments, the specific part may be a single point.
Second, the stimulation scanning optical system 200 may have only a function of irradiating one point on the sample 17 with the second laser light.
Thirdly, a plurality of the specific parts may be set.
Fourth, the stimulation scanning optical system 200 is not limited to one, and two or more may be provided.

1 is a block diagram showing a schematic configuration of a scanning laser microscope according to a first embodiment of the present invention. It is the figure which showed an example of the scanning area | region of the 1st laser beam, and the scanning area | region of the 2nd laser beam. It is a figure which shows an example of the horizontal direction and a vertical direction waveform produced | generated by the 1st scanning waveform generation circuit, and the horizontal method data enable signal and the vertical method data enable signal which were produced | generated by the 1st data enable signal generation circuit. . FIG. 2 is a diagram illustrating an example of an internal structure of a first data enable signal generation circuit in FIG. 1. FIG. 5 is a diagram for explaining a first enable signal output from the data enable signal arithmetic circuit of FIG. 4. It is a figure which shows the relationship between a 1st enable signal and a 2nd clock. FIG. 6 is a diagram illustrating an example of a horizontal waveform and a vertical waveform generated by a second scanning waveform generation circuit, and a horizontal method data enable signal and a vertical method data enable signal generated by a second data enable signal generation circuit. is there. FIG. 3 is a diagram showing an example of an internal structure of a second data enable signal generation circuit shown in FIG. 1. It is a block diagram which shows schematic structure of the scanning laser microscope which concerns on the 2nd Embodiment of this invention. It is the figure which showed an example of the relationship between the scanning area | region of a 1st laser beam, and the scanning area | region of a 2nd laser beam. In the scanning laser microscope according to the second embodiment of the present invention, (A) a data enable signal output from the first data enable signal generation circuit, (B) a post-conversion signal output from the data enable signal conversion circuit It is a figure which shows an example of a data enable signal and (C) 2nd clock.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 1st light source 3 1st scanning optical unit 10 2nd light source 11 2nd scanning optical unit 17 Sample 19 1st scanning waveform generation circuit 20 1st clock generation circuit 21 1st data enable signal generation circuit 22 Second scanning waveform generation circuit 23 Second clock generation circuit 24 Second data enable signal generation circuit 100 Image scanning optical system 200 Stimulus scanning optical system

Claims (6)

An image scanning optical system that scans two-dimensionally with a first laser beam for obtaining an image of the sample and irradiates the sample;
A stimulation optical system for irradiating a specific part of the sample with a second laser beam for stimulation during a blanking period of the first laser beam by the scanning optical system for images,
Even if the irradiation of the second laser light by the stimulation optical system cannot be completed during the blanking period, the scanning is continuously performed without stopping the scanning of the first laser light by the image scanning optical system. Laser microscope. An image scanning optical system that scans two-dimensionally with a first laser beam for obtaining an image of the sample and irradiates the sample;
A stimulation optical system for irradiating a specific part of the sample with a second laser beam for stimulation,
Irradiating the second laser light in a blanking period of the first laser light in a common region scanning period in which the first laser light is irradiated to a scanning line in which the specific part is set; and Even when the irradiation of the second laser light by the stimulation optical system cannot be completed in the blanking period, the scanning of the first laser light by the image scanning optical system is continuously performed without stopping. Scanning laser microscope. An image scanning optical system that scans two-dimensionally with a first laser beam for obtaining an image of the sample and irradiates the sample;
A stimulation optical system for irradiating a specific part of the sample with a second laser beam for stimulation,
During the period in which the image scanning optical system irradiates the first laser beam on the specific part, the stimulation optical system stops the irradiation of the second laser beam, and the image scanning optical system Scanning laser in which the stimulation optical system irradiates the second laser light during a period in which the laser beam is irradiated and the specific laser beam is not irradiated on the specific part. microscope. A first laser beam for obtaining an image of the sample is two-dimensionally scanned to irradiate the sample, and a second laser beam for stimulation is applied to the sample during a blanking period of the first laser beam. A method for controlling a scanning laser microscope that irradiates a specific part of
A control method for a scanning laser microscope, in which, even when the irradiation of the second laser light cannot be completed during the blanking period, the scanning of the first laser light is continuously performed without stopping . Control of a scanning laser microscope that two-dimensionally scans a first laser beam for obtaining an image of a sample to irradiate the sample and irradiates a specific portion of the sample with a second laser beam for stimulation A method,
Irradiating the second laser light in a blanking period of the first laser light in a common region scanning period in which the first laser light is irradiated to a scanning line in which the specific part is set; and A control method for a scanning laser microscope, in which irradiation of the first laser light is continuously performed without stopping even when irradiation of the second laser light cannot be completed in the blanking period . Control of a scanning laser microscope that two-dimensionally scans a first laser beam for obtaining an image of a sample to irradiate the sample and irradiates a specific portion of the sample with a second laser beam for stimulation A method,
In the period of irradiating the specific part with the first laser beam, the irradiation of the second laser beam is stopped and the first part is irradiated with the first laser beam, and the first part is applied to the first part. A method for controlling a scanning laser microscope , wherein the second laser beam is irradiated during a period in which the laser beam is not irradiated .

Images