JP5052009B2 - Scanning microscope and specimen image acquisition method - Google Patents

Description

  The present invention relates to a scanning microscope and specimen image acquisition method used as a biological specimen research tool.

  Conventionally, as a scanning microscope used as a biological specimen research tool, for example, as disclosed in Japanese Patent Laid-Open No. 10-10436, laser light is converged to one point on the specimen, and this convergence point is set to 2 on the specimen. There is known a scanning laser microscope that scans in a two-dimensional direction to acquire two-dimensional luminance information of a specimen.

  A laser beam consisting of a single beam is used in a scanning laser microscope, but such a laser beam has good convergence, so that it is effective for obtaining optical information of one minute point on the XY plane of a specimen. Is. In addition, the detection light from the specimen is detected by a confocal optical system that detects it through a pinhole placed at a position optically conjugate with the convergence point on the specimen, so that light from an out-of-focus position is eliminated. Optical information can be obtained with higher accuracy.

  Therefore, according to the confocal laser microscope, one point of optical information in the three-dimensional space of the specimen can be acquired, and the laser beam is scanned along the XY plane, the XZ plane, and other two-dimensional planes. Since the information for each point described above can be arranged according to the scanned position, an optical slice image can also be formed. A galvanometer mirror is generally used for laser beam scanning. By moving two galvanometer mirrors that scan in the XY directions in combination, the imaging area is XY scanned line by line as in the raster scan of a television. In raster scanning, laser light is sequentially irradiated to a point adjacent in the X direction, and light (fluorescence, reflected light, etc.) from the specimen obtained at that time is detected by a detector. In this case, the time difference from the detection of fluorescence by irradiating a laser beam to a certain point (Xn, Yn) until moving to the next adjacent point (Xn + 1, Yn + 1) is on the order of microseconds.

By the way, the confocal laser microscope is effective for the Caged technique used for fluorescence observation. Here, the caged method is a method in which a caged reagent and a fluorescent indicator sensitive to calcium ion concentration are injected into a specimen, and a portion of the specimen is irradiated with a stimulating laser beam to cleave the caged group of the caged reagent. In this method, the substance contained therein is released, and the time-dependent change in the calcium ion concentration at that time is observed by irradiating the specimen with an observation excitation laser beam. According to this method, for example, when a Caged compound to which calcium ions are bound is introduced into a specimen, irradiation with a UV laser that releases the Caged compound at a point (Xn, Yn) causes cleavage of the Caged compound at that position. And the retained Ca ²⁺ ions are released. An observation specimen reacts with this calcium ion. By detecting the fluorescence generated by this reaction with a detector, the state of the sample's reaction to the calcium ion stimulation is observed.

  However, the influence of the calcium ions released from the point (Xn, Yn) may extend not only to the point (Xn, Yn) but also to the surroundings (for example, the next point (Xn + 1, Yn)). . Moreover, even if the released calcium ions remain only at the point (Xn, Yn), the reaction behavior of the observation specimen due to the stimulation may extend beyond the point (Xn, Yn). .

  This is because if the data acquisition of the next point (Xn + 1, Yn) immediately after the data acquisition of the point (Xn, Yn) is performed, the reaction behavior of the stimulus with respect to the previous point (Xn, Yn) is affected. There is a risk that the data will be combined. This is contrary to the researcher's request to acquire the reaction behavior data of each individual point (Xn, Yn) (Xn + 1, Yn) independently.

  In addition, when fluorescence observation is performed by simply exciting a fluorescent dye introduced into a specimen with a laser beam without stimulating it, if the lifetime of the fluorescence to be observed is short, it does not matter so much. When there is a lifetime, when the adjacent points are scanned in sequence, there is still a problem that the data at a certain point (Xn + 1, Yn) is affected by the previous point (Xn, Yn).

  An object of the present invention is to provide a scanning microscope that correctly detects the brightness of each point by eliminating the influence of the points of the sample from which data is acquired.

The present invention is directed to a scanning microscope. The scanning microscope according to the present invention determines a data acquisition order in which adjacent data acquisition points are not continuous, light source means for emitting laser light, irradiation means for converging and irradiating the laser light to a data acquisition point on a specimen Data acquisition order determination means for performing scanning, scanning means for scanning the laser light in accordance with the data acquisition order, and a plurality of brightnesses of light from each data acquisition point by continuously performing sampling at each data acquisition point a plurality of times Detection means for detecting information, storage means for storing a plurality of pieces of luminance information of light from each data acquisition point detected by the detection means in association with position information of the data acquisition points, and the detection means among the plurality of luminance information of the light from each data obtaining point detected, each of the data detected luminance information with each other at the same rank in each data acquisition point While correlating with the location information of the acquired points generates two-dimensional image of a single, and an image forming means for forming a plurality of 2-dimensional images by performing this for each acquisition order.

The present invention is also directed to a specimen image acquisition method. The sample image acquisition method according to the present invention converges and irradiates a laser beam to a data acquisition point on a sample, scans the laser beam in an order in which the data acquisition points are not adjacent, and performs multiple times at each data acquisition point. By continuously sampling, a plurality of luminance information of light from each data acquisition point is detected, and a plurality of detected luminance information of light from each data acquisition point is associated with the position information of the data acquisition point. One piece of luminance information detected in the same order at each data acquisition point among the plurality of pieces of luminance information of light from each data acquisition point is associated with the position information of each data acquisition point. These two-dimensional images are generated and performed for each acquisition order to form a plurality of two-dimensional images.

  ADVANTAGE OF THE INVENTION According to this invention, the scanning microscope which eliminates the influence of the points of the sample which acquires data, and detects the brightness | luminance of each point correctly is provided.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

<First Embodiment>
FIG. 1 shows a schematic configuration of a confocal laser microscope according to the first embodiment of the present invention.

  As shown in FIG. 1, the confocal laser microscope has a laser light source unit 1 as light source means for emitting laser light (excitation light), a reflection mirror 7 that bends the optical path of the laser light, and two laser lights. Scanning optical unit 9 as a scanning means for dimensional scanning, objective lens 10 for converging laser light to a data acquisition point on specimen 11, dichroic mirror 8 for separating laser light and fluorescence, and fluorescence are detected. And a detector 12 as a detecting means.

  The laser light source unit 1 includes laser light sources 2 and 3, a reflection mirror 4, a dichroic mirror 5, and an acousto-optic variable filter (AOTF) 6. The laser light sources 2 and 3 emit laser beams having different wavelengths. The reflection mirror 4 is disposed on the optical path of the laser light from the laser light source 2. The dichroic mirror 5 is disposed on the optical path of the laser light from the laser light source 3 and at the intersection with the laser light reflected by the reflection mirror 4. The dichroic mirror 5 combines these two laser light paths. The dichroic mirror 5 transmits the laser light from the laser light source 3 and reflects the laser light reflected by the reflection mirror 4. The AOTF 6 is disposed on the optical path of the laser beam synthesized by the dichroic mirror 5. The AOTF 6 makes it possible to control the intensity of the laser light, the wavelength component of the laser light, on / off of the laser light irradiation, and the like.

  The reflection mirror 7 is disposed on the exit optical path of the AOTF 6. The dichroic mirror 8 is disposed on the reflection optical path of the reflection mirror 7. The dichroic mirror 8 transmits laser light (excitation light) reflected by the reflection mirror 7 and reflects detection light (fluorescence) emitted from a specimen 11 described later.

  The scanning optical unit 9 is disposed on the transmission optical path of the dichroic mirror 8. The scanning optical unit 9 includes a Y-direction scanner 9a and an X-direction scanner 9b for deflecting light in two orthogonal directions, and a laser beam converged on the sample 11 by the Y-direction scanner 9a and the X-direction scanner 9b. Light can be applied to any point on the two-dimensional plane.

  The objective lens 10 is disposed on the optical path of the laser light emitted from the scanning optical unit 9. The laser light emitted from the scanning optical unit 9 is converged to the data acquisition point on the specimen 11 by the objective lens 10 and irradiated. In other words, the objective lens 10 constitutes an optical system as irradiation means for converging and irradiating the laser beam to the data acquisition point on the specimen 11.

  The specimen 11 emits detection light (fluorescence) in response to irradiation with laser light (excitation light). Detection light (fluorescence) emitted from the specimen 11 travels backward along the optical path described above, and returns to the dichroic mirror 8 through the objective lens 10 and the scanning optical unit 9.

  The detector 12 is disposed on the optical path of the detection light that is selectively reflected by the dichroic mirror 8. Although not limited to this, the detector 12 is configured by, for example, a photomultiplier. The detector 12 outputs an analog electric signal reflecting the luminance of the detection light from the sample 11.

  The confocal laser microscope further includes an A / D converter 13 as signal processing means, a personal computer (PC) 14 as control means, and scanner drive as drive means for driving the Y-direction scanner 9a and the X-direction scanner 9b. Unit 21, an input device 22 as information input means, and a monitor 23 as display means.

  Although the input device 22 is not limited to this, for example, it is configured by a keyboard. Alternatively, the input device 22 may be configured by a pointing device such as a mouse and a GUI (graphical user interface). The monitor 23 is configured by, for example, a CRT, although not limited thereto.

  A personal computer (PC) 14 is connected to the detector 12 via an A / D converter 13. The A / D converter 13 converts the analog electrical signal from the detector 12 into a digital signal and outputs it to the PC 14.

  The PC 14 includes a control program unit 15, a laser output control unit 16, a frame memory 17, an X direction scanner drive waveform memory 18, a Y direction scanner drive waveform memory 19, and a clock generation unit 20.

  The frame memory 17 associates luminance data detected by the detector 12 and converted into a digital signal via the A / D converter 13 with each point coordinate (position information of the data acquisition point) in the imaging region. save. The frame memory 17 constitutes storage means for storing the luminance information of the detected light in association with the position information of the data acquisition point.

  As illustrated in FIG. 2, the control program unit 15 includes a scanning condition input unit 151, a scanning point array generation unit 152, and an XY and XYT image generation unit 153. The scanning condition input unit 151 receives scanning conditions such as an XY scan size, a sampling speed, and a data acquisition time per point in the imaging area from the input device 22. The scanning point array generation unit 152 creates a random scanning point array that does not continuously acquire data from two adjacent points in the imaging region. The scanning point array generation unit 152 constitutes a data acquisition order determination unit that determines a data acquisition order in which adjacent data acquisition points are not consecutive. For example, a random number is used to create this random scan point array. Then, it is checked whether there is an adjacent point in the generated array, and if there is an adjacent point, processing such as replacing with another point is performed. The XY, XYT image generation unit 153 generates image data based on the association between the luminance data written in the frame memory 17 and each point coordinate (position information of the data acquisition point) in the imaging region. The XY and XYT image generation unit 153 constitutes an image forming unit that forms a two-dimensional image based on the correspondence between the luminance information of the detection light and the position information of the data acquisition point.

  The laser output control unit 16 controls the output of the laser light from the laser light source unit 1 according to the data acquisition time per point set in the scanning condition input unit 151.

  The X-direction scanner drive waveform memory 18 and the Y-direction scanner drive waveform memory 19 scan the X-direction scanner 9b and the Y-direction scanner 9a that are converted from the random scan point array generated by the scan point array generation unit 152. Drive waveform data (waveform DAC data applied to the D / A converter of the scanner drive unit 21) is stored.

  The scanner drive unit 21 is connected to the X direction scanner drive waveform memory 18 and the Y direction scanner drive waveform memory 19. The scanner drive unit 21 is connected to the Y-direction scanner 9a according to the waveform data (waveform DAC data) from the X-direction scanner drive waveform memory 18 and the Y-direction scanner drive waveform memory 19 read in synchronization with the clock pulse of the clock generation unit 20. The X direction scanner 9b is driven.

  The clock generation unit 20 generates clock pulses that determine the operation timing of the control program unit 15, the laser output control unit 16, the frame memory 17, the X direction scanner drive waveform memory 18, and the Y direction scanner drive waveform memory 19.

  Next, the operation of the first embodiment will be described.

  First, scanning conditions are input from the input device 22 to the scanning condition input unit 151 of the PC 14. The input scanning conditions are an XY scan size for determining an imaging region (for example, 512 × 512 points), a sampling speed for determining a sampling interval of detection signals in the A / D converter 13, and 1 in the imaging region. Data acquisition time per point. Then, the scan point array generation unit 152 creates a random scan point array that does not continuously acquire data from two adjacent points in the imaging region, and determines a data acquisition order in which the data acquisition points are not adjacent. Also, the number of samplings per point is calculated based on the sampling speed and the data acquisition time per point. In the first embodiment, the number of samplings per point is set to one.

  Next, the random scanning point array (coordinate data) generated by the scanning point array generation unit 152 is converted into drive waveform data (waveform DAC data) for scanning the X-direction scanner 9b and the Y-direction scanner 9a. It is stored in the direction scanner drive waveform memory 18 and the Y direction scanner drive waveform memory 19.

  For example, as shown in FIG. 3A, the random scan point array includes the X position data array 301 of (10), (250), (120)..., And (20), (40), (200),. It is given as coordinate data of 512 × 512 points in the Y position data array 302. As shown in FIG. 3B, these coordinate data are converted into an XDAC data array 401 (2500), (1000)... And a YDAC data array 402 (1000), (500). The DAC data in the XDAC data array 401 and the YDAC data array 402 are stored in the X direction scanner drive waveform memory 18 and the Y direction scanner drive waveform memory 19, respectively.

  The drive waveform data of the X direction scanner drive waveform memory 18 and the Y direction scanner drive waveform memory 19 are read out to the scanner drive unit 21 in synchronization with the clock pulse of the clock generation unit 20, and the scanner drive unit 21 is connected to the Y direction scanner 9a. The X direction scanner 9b is driven. At the same time, the laser light source unit 1 emits laser light according to an instruction from the laser output controller 16. The laser light is deflected by the Y-direction scanner 9a and the X-direction scanner 9b, and is sequentially applied to each data acquisition point on the sample 11 via the objective lens 10.

  In response to the laser light irradiation, detection light (fluorescence) is emitted from each data acquisition point on the specimen 11. The detection light (fluorescence) enters the dichroic mirror 8 through the objective lens 10 and the scanning optical unit 9, is reflected by the dichroic mirror 8, and is detected by the detector 12. The detector 12 outputs an analog signal reflecting the luminance of the detection light. The analog signal output from the detector 12 is converted into a digital signal by the A / D converter 13 and written in the frame memory 17. The luminance data of each point in the imaging region acquired by the detector 12 is generated by random scanning points generated by the scanning point array generation unit 152 (X position data array 301 and Y position data array 302 shown in FIG. 3A). Are written in the frame memory 17 in association with.

  Then, the XY, XYT image generation unit 153 generates XY image data based on the association between the luminance data written in the frame memory 17 and each point coordinate in the imaging region. An image corresponding to the XY image data is displayed on the monitor 23.

  In fluorescence observation by conventional raster scanning, data of the next adjacent point (Xn + 1, Yn) is acquired immediately after data of a certain point (Xn, Yn) is acquired. For example, in the observation by the caged method, the influence of calcium ions released from the point (Xn, Yn) is not limited to the point (Xn, Yn), but around it (for example, the next point (Xn + 1, Yn)). There is a possibility to extend to. Therefore, the data at the point (Xn + 1, Yn) may be affected by the response behavior of the stimulus to the previous point (Xn, Yn). Even in fluorescence observation without stimulation, if the fluorescence has a long lifetime, for example, on the order of microseconds, the data of a certain point (Xn + 1, Yn) is also affected by the previous point (Xn, Yn). Problems arise.

On the other hand, in the present embodiment, for an imaging region (for example, 512 × 512 points), a random scanning point array that does not continuously acquire data from two adjacent points is created to determine the data acquisition order. In accordance with the determined acquisition order, drive signals for the Y-direction scanner 9a and the X-direction scanner 9b are generated, point scanning is performed with the drive signals, and detection light from each data acquisition point is detected to acquire luminance data. The acquired luminance data of each point is stored in the frame memory 17 in association with a random scanning point array, and finally the luminance data of all points is acquired to complete a two-dimensional image. For this reason, the acquired data from each point does not include data obtained by successively acquiring two adjacent points, and is not affected by light from other points. That is, the influence between points when data is acquired is surely eliminated. Therefore, the brightness of each point is correctly detected without being affected by other points. In the above-described embodiment, the laser beam continues to be emitted while moving the data acquisition point. The AOTF 6 may be controlled in synchronization with the operations of the scanner 9a and the X-direction scanner 9b, and laser light emission may be stopped while the data acquisition point is moved to the next data acquisition point. In this way, all the effects of the laser beam when moving the data acquisition point are eliminated. Further, the imaging region is not limited to a fixed rectangle, and may be limited to a region where a specimen exists.

<Second Embodiment>
The schematic configuration of the confocal laser microscope according to the second embodiment is the same as that shown in FIG.

  In the first embodiment, one XY image on the specimen 11 is acquired by setting the data acquisition time per point to one sampling, but in this second embodiment, one point is acquired. By performing sampling a plurality of times during the pertinent data acquisition time, an XYT image that is a stack of a plurality of XY images that are temporally continuous is acquired.

  It is assumed that the number of samplings per point calculated by the sampling speed and the data acquisition time per point is set to a plurality, for example, 50 samplings. In addition, 50 frames are prepared corresponding to the number of samplings per point.

  As for the random scanning point array generated by the scanning point array generation unit 152, for example, the X position data array 301 and the Y position data array 302 shown in FIG. 3A described above are given as the coordinate data of the imaging region. The drive waveform data (waveform DAC data) converted from the X position data array 301 and the Y position data array 302 is 50 DAC data (2500) for the first X position data (10) shown in FIG. 3A. Similarly, YDAC data (1000) for 50 data is also generated for the first Y position data (20). Thereafter, similarly, 50 DACs of XDAC data and YDAC data are generated for the XY position data of each data acquisition point. 4A and 4B show the XDAC data array 501 and the YDAC data array 502 generated in this way, respectively.

  The XDAC data array 501 and the YDAC data array 502 are stored in the X direction scanner drive waveform memory 18 and the Y direction scanner drive waveform memory 19, respectively.

  The drive waveform data of the X direction scanner drive waveform memory 18 and the Y direction scanner drive waveform memory 19 are read out to the scanner drive unit 21 in synchronization with the clock pulse of the clock generation unit 20, and the scanner drive unit 21 reads the Y direction scanner 9a. And the X-direction scanner 9b is driven.

  In this state, the laser light emitted from the laser light source unit 1 is deflected by the Y-direction scanner 9 a and the X-direction scanner 9 b and is irradiated to each data acquisition point on the sample 11 through the objective lens 10. In response to the laser light irradiation, detection light (fluorescence) is emitted from each data acquisition point on the specimen 11. Detection light (fluorescence) is detected by the detector 12. The detector 12 outputs an analog signal reflecting the luminance of the detection light. The analog signal output from the detector 12 is converted into a digital signal by the A / D converter 13 and written in the frame memory 17.

  In this case, the luminance data corresponding to the first XDAC data in the XDAC data array 501 and the first YDAC data in the YDAC data array 502 are random scan points generated by the scan point array generation unit 152 (shown in FIG. 3A). Are written in the # 1 frame memory 17 associated with the X position data array 301 and the Y position data array 302). The luminance data corresponding to the second XDAC data in the XDAC data array 501 and the second YDAC data in the YDAC data array 502 are associated with random scanning points generated by the scanning point array generation unit 152. 2 is written in the second frame memory 17. Similarly, the luminance data corresponding to the 3rd to 50th XDAC data in the XDAC data array 501 and the 3rd to 50th YDAC data in the YDAC data array 502 are also in the same order, from # 3 to # 50. Are written in the frame memory 17. This operation is repeated for all the luminance data acquired corresponding to the XDAC data of the XDAC data array 501 corresponding to the random scanning points and the YDAC data of the YDAC data array 502, and from # 1 as shown in FIG. It is written in the frame memory 17 of # 50.

  Then, the XY, XYT image generation unit 153 generates XYT image data based on the correspondence between the luminance data written in the frame memories 17 of # 1 to # 50 and the respective data acquisition point coordinates in the imaging region. Images corresponding to the XY image data are individually displayed on the monitor 23.

  In the present embodiment, data acquisition (sampling) is repeated a plurality of times at predetermined time intervals for each of randomly set data acquisition points, and a plurality of these acquired points (t = T1). , T2,..., Tn) (n = 50) detected in the same order are individually written in the frame memory 17 of # 1 to # 50, so that the detected data of each point is influenced by other points. In addition, based on the luminance data written in each frame memory 17, it is possible to observe the dynamic change in the response behavior to the stimulus of the specimen 11 by the plurality of XYT images generated by the XY and XYT image generation unit 153.

<Third Embodiment>
The schematic configuration of the confocal laser microscope according to the third embodiment is the same as that shown in FIG.

  In the third embodiment, sampling is performed a plurality of times during the data acquisition time per point, and further, the laser light intensity, the wavelength component of the laser light, and the laser light corresponding to the plurality of samplings per point. Set laser conditions such as irradiation on / off.

  Assume that the number of samplings per point calculated by the sampling speed and the data acquisition time per point is set to 10 samplings, for example.

  As for the random scanning point array generated by the scanning point array generation unit 152, for example, the X position data array 301 and the Y position data array 302 shown in FIG. 3A described above are given as the coordinate data of the imaging region. By the same procedure as described in the second embodiment, driving waveform data (X (Y) DAC data) A1 for 10 data with respect to the first position data in the X position data array 301 and the Y position data array 302, A2,... A10 are generated, and similarly, X (Y) DAC data B1, B2,..., C1, C2,. The upper part of FIG. 6 shows the X (Y) DAC data array 601 generated in this way.

  Further, the laser conditions are written as shown in the lower part of FIG. 6 in correspondence with the X (Y) DAC data A1, A2,..., B1, B2,..., C1, C2,. A laser setting data array 701 is generated. In the laser setting data, laser data irradiation on data ON is set only for X (Y) DAC data A3, B3, C3,..., And laser light irradiation off data is set for other X (Y) DAC data. OFF is set.

  The laser setting data array 701 is stored in the memory 16 a of the laser output control unit 16. The laser output controller 16 reads the laser setting data array 701 stored in the memory 16a in synchronization with the sampling timing at the time of data acquisition, and controls on / off of the laser beam irradiation by the AOTF6.

  According to this configuration, the X (Y) DAC data A1, A2,..., B1, B2,..., C1, C2,. Accordingly, the data acquisition point on the specimen 11 is set. At the same time, on / off of laser light irradiation is controlled based on the laser setting data in the laser setting data array 701. In this case, in the laser setting data array 701, on-data ON of laser light irradiation is applied only to A3, B3, C3,... Among 10 (X) (Y) DAC data A, B, C,. Therefore, AOTF 6 is turned ON only at these timings, and the sample is irradiated with laser light.

  In the present embodiment, light from each data acquisition point is detected a plurality of times at a predetermined time interval, and different laser conditions can be set in accordance with the detection timings of the plurality of detections. Here, for a plurality of (t = T1, T2,... Tn) (n = 10) luminance data acquired by a plurality of times of data acquisition (sampling) at a data acquisition point, t = T3 ( The laser beam irradiation is turned on only when A3, B3, C3,..., And the laser beam irradiation is turned off at the other t = T1, T2, T4,. In addition to being observable, it is possible to observe the dynamic behavior of the specimen 11 when the stimulus is applied and after the stimulus is stopped.

  The data condition to be set at the time of data acquisition at a data acquisition point is not limited to on / off of laser beam irradiation, and can be set by changing one or both of laser beam intensity and wavelength component. . For example, in observing multiple stained fluorescent specimens, laser light having an excitation wavelength corresponding to each fluorescent dye may be sequentially irradiated.

<Fourth embodiment>
FIG. 7 shows a schematic configuration of a confocal laser microscope according to the fourth embodiment of the present invention. In FIG. 7, members indicated by the same reference numerals as those shown in FIG. 1 are similar members, and detailed description thereof is omitted.

  As shown in FIG. 7, the confocal laser microscope of the present embodiment further includes a current detector 24 as a current detecting means for detecting a current value from the specimen 11 in addition to the apparatus configuration of FIG. is doing. The current detector 24 outputs an analog electrical signal that reflects the detected current value.

  The analog signal of the current value output from the current detector 24 is input to the A / D converter 13 and converted into a digital signal, similarly to the luminance of the detection light from the detector 12. The current value data is written in the frame memory 17 as luminance information. That is, the current value is acquired at the same timing as the fluorescence detection sampling timing. The current value data is written in the frame memory 17 in association with the random scanning points (X position data array 301 and Y position data array 302 shown in FIG. 3A) generated by the scanning point array generation unit 152.

  Then, the XY and XYT image generation unit 153 generates XY image data based on the association between the current data written in the frame memory 17 and each point coordinate in the imaging region. An image corresponding to the XY image data is displayed on the monitor 23.

  In the present embodiment, the current value can be measured without the influence of the last laser irradiation. Therefore, the electrical reaction of the sample 11 when each data acquisition point of the sample 11 is irradiated with laser light can be accurately visualized. This embodiment may be implemented in combination with the first to third embodiments.

Application Example of Fourth Embodiment As shown in FIG. 8, the region 26 including the spine 25a of the nerve cell 25 is irradiated with stimulation light by a random scan method, and the response of the nerve cell 25 to this stimulation is applied to the region 25b. Measurement is performed by the inserted current detector 24.

  The current value may be measured for a plurality of parts. In that case, the current detector 24 is inserted into each measurement site.

<Fifth embodiment>
In the first embodiment, the data acquisition order in which adjacent data acquisition points are not consecutive is determined at random. In contrast, in the present embodiment, a data acquisition order in which adjacent data acquisition points are not consecutive with a regularity is determined.

  The schematic configuration of the confocal laser microscope according to the fifth embodiment is the same as that shown in FIG.

  In the present embodiment, the scanning point array generation unit 152 generates a scanning point array in the irradiation order as shown in FIGS. 9 and 10, for example. The XY and XYT image generation unit 153 generates image data based on the correspondence between the acquired luminance data and each scanning point, as in the first embodiment.

  In the example of FIG. 9, one scanning area is divided into eight areas A to H having the same size. Then, the upper left point of each divided region is irradiated in the order of ACEGBDFH. After one round, the next point in the X-line direction is irradiated in the same order as ACEGBD. When one line is completed, the second line of each region is irradiated in the same way. That is, the regions A to H are irradiated with laser light according to the same rule in a certain order. FIG. 9 shows the irradiation order in a scanning region of 64 × 64 pixels. The numbers in each square indicate the irradiation order. In this example, since the point next to the first irradiated point is irradiated ninth, the laser irradiation time interval between the adjacent points is the data acquisition time for eight pixels.

  In the example of FIG. 10, one scanning area is divided into four areas A to D having the same size. Then, each divided region is irradiated in the same order as described above in the order of ABCD. In this example, the point next to the first irradiated point is irradiated fifth, so the time interval of laser irradiation of the adjacent points is an acquisition time for four pixels. Further, when irradiation is performed in the order of ABCD, the movement distances of B → C and D → A are larger than others, but in this embodiment, irradiation is performed in the order of ABCC, so that the movement distances between regions can be made almost uniform. Therefore, it is advantageous to increase the stability of scanning in a non-continuous scanning in units of pixels.

  In addition to FIGS. 9 and 10, the irradiation time interval between adjacent irradiation points may be adjusted by appropriately setting the divided regions in the X direction and the Y direction. Further, the irradiation order may be changed as appropriate, for example, irradiation is sequentially performed in the Y direction.

  Such an irradiation order (point arrangement) may be stored in a memory in advance and recalled at the time of use, or may be determined each time using a calculation formula for determining the arrangement. Such a scanning method may be used in combination with the above-described second to fourth embodiments.

  In addition, this invention is not limited to the said embodiment, In the implementation stage, you may change variously in the range which does not change the summary.

  For example, first, an image of one frame is acquired (either normal XY scanning or random scanning according to the present invention), the position where the sample exists is detected or specified on the monitor, and only the position where the sample exists Alternatively, laser scanning may be performed by determining a data acquisition order in which adjacent data acquisition points are not continuous. In this way, the position where the sample does not exist is not scanned, so that the image acquisition time is greatly shortened. Further, the plane on which image acquisition is performed may be an XZ plane or a plane inclined with respect to the laser optical axis instead of the XY plane. In this case, in order to move the convergence point in the Z direction at high speed, an optical element such as a deformable mirror may be provided in the optical path.

  Furthermore, the above embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent elements. For example, if some of the configuration requirements are deleted from all the configuration requirements shown in the embodiment and the advantages described in the embodiment are obtained, the configuration in which this configuration requirement is deleted It can be extracted as an invention.

1 shows a schematic configuration of a confocal laser microscope according to a first embodiment of the present invention. The schematic structure of the control program part used for 1st Embodiment is shown. The X position data arrangement | sequence and Y position data arrangement | sequence of 1st Embodiment are shown. FIG. 3B shows an XDAC data array and a YDAC data array corresponding to the X position data array and the Y position data array in FIG. 3A, respectively. The XDAC data arrangement | sequence of the 2nd Embodiment of this invention is shown. The YDAC data arrangement | sequence of the 2nd Embodiment of this invention is shown. 3 shows a schematic configuration of a frame memory used in the second embodiment. 10 shows a laser setting data array according to a third embodiment of the present invention. 10 shows a schematic configuration of a confocal laser microscope according to a fourth embodiment of the present invention. The application example of the current measurement by the confocal laser microscope of 4th Embodiment is shown. An example of the data acquisition order in the confocal laser microscope concerning the 4th Embodiment of this invention is shown. The other example of the data acquisition order in the confocal laser scanning microscope concerning the 4th Embodiment of this invention is shown.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Laser light source unit, 2 ... Laser light source, 3 ... Laser light source, 4 ... Reflection mirror, 5 ... Dichroic mirror, 6 ... Acousto-optic variable filter, 7 ... Reflection mirror, 8 ... Dichroic mirror, 9 ... Scanning optical unit, 9a ... Y direction scanner, 9b ... X direction scanner, 10 ... objective lens, 11 ... sample, 12 ... detector, 13 ... A / D converter, 14 ... personal computer, 15 ... control program section, 16 ... laser output control section 16a ... memory, 17 ... frame memory, 18 ... X direction scanner drive waveform memory, 19 ... Y direction scanner drive waveform memory, 20 ... clock generation unit, 21 ... scanner drive unit, 22 ... input device, 23 ... monitor, 24 DESCRIPTION OF SYMBOLS ... Current detector, 25 ... Nerve cell, 25a ... Spine, 25b ... Region, 26 ... Region, 151 ... Scanning condition input unit, 152 Scanning point array generation unit, 153... XYT image generation unit, 301... X position data array, 302... Y position data array, 401... XDAC data array, 402 ... YDAC data array, 501. 601 ... XDAC data array, 701 ... laser setting data array.

Claims (8)

Light source means for emitting laser light;
Irradiating means for converging and irradiating the laser beam to a data acquisition point on the specimen;
Data acquisition order determination means for determining a data acquisition order in which adjacent data acquisition points are not continuous;
Scanning means for scanning the laser beam according to the data acquisition sequence;
Detecting means for detecting a plurality of luminance information of light from each data acquisition point by continuously performing sampling a plurality of times at each data acquisition point;
Storage means for storing a plurality of pieces of luminance information of light from each data acquisition point detected by the detection means in association with position information of the data acquisition point;
Among the plurality of luminance information of light from each data acquisition point detected by the detection means, the luminance information detected in the same order at each data acquisition point is associated with the position information of each data acquisition point. A scanning microscope comprising image forming means for generating a plurality of two-dimensional images by generating one two-dimensional image for each acquisition order .   The detection means detects a reaction of the sample as a current value in synchronization with detection of light from the data acquisition point when the data acquisition point is irradiated with laser light. Scanning microscope.   The apparatus further comprises laser light control means for controlling at least one of the wavelength and intensity of the laser light emitted from the light source means, and the laser light control means corresponds to each timing of the plurality of consecutive samplings at each data acquisition point. The scanning microscope according to claim 1, wherein irradiation change of the laser beam is set.   The scanning microscope according to claim 3, wherein the irradiation condition of the laser beam is at least one of on / off of laser beam irradiation, intensity of the laser beam, and wavelength component.   The scanning microscope according to any one of claims 1 to 4, wherein the data acquisition order determination unit randomly determines the data acquisition order.   The scanning microscope according to any one of claims 1 to 4, wherein the data acquisition order determination unit determines the data acquisition order with regularity.   The scanning microscope according to claim 6, wherein the data acquisition order determining unit divides the scanning region into a plurality of regions, and determines the order in which the regions are irradiated with laser light according to the same rule in a certain order. Converging and irradiating laser light to the data acquisition point on the specimen,
Scanning the laser light according to an order in which the data acquisition points are not adjacent,
Detecting multiple pieces of luminance information of light from each data acquisition point by continuously performing multiple samplings at each data acquisition point,
A plurality of luminance information of light from each detected data acquisition point is stored in association with the position information of the data acquisition point,
Among a plurality of pieces of luminance information of light from each data acquisition point, luminance information detected in the same order at each data acquisition point is associated with the position information of each data acquisition point, and one two-dimensional image A sample image acquisition method for generating a plurality of two-dimensional images by generating the two-dimensional images by performing the above- described process for each acquisition order .

Images