JP2014224856A - Laser scanning microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To acquire an image of a sample reaction in a wide range including outside of a visual field range while reducing the influence of adjacent irradiation regions in a sample, without reducing resolution of the image.SOLUTION: There is provided a laser scanning microscope 100 comprising: an object lens 11; a scanning optical unit 9; a stage 1 capable of moving a sample S in a direction crossing an optical axis of the object lens 11; a photodetector 13 detecting a reaction of the sample S; a frame memory 43 storing therein brightness information on the sample S and scanning position information on laser light while making the brightness information and the scanning position information correspond to each other; an observation range designation unit setting an observation range on the sample S, and dividing the observation range into a plurality of sub-regions that can be scanned by the laser light in a state of fixing the stage 1; a scanning order determination unit determining an order of scanning irradiated regions of the continuously emitted laser light per sub-region so that the irradiated regions are not spatially adjacent to one another; and a control unit 49 sequentially moving the stage 1 for all ranges that can be scanned, and controlling the laser light to scan the sub-regions according to the determined scanning order.

Description

  The present invention relates to a laser scanning microscope.

  2. Description of the Related Art Conventionally, a laser scanning microscope is known in which laser light is scanned two-dimensionally on a specimen, and the reaction of the specimen caused by light irradiation is detected to obtain two-dimensional luminance information (see, for example, Patent Document 1). ). When the sample is irradiated with laser light, the reaction of the irradiated region may reach the surrounding region. Therefore, when the spatially adjacent regions on the specimen are sequentially scanned, there is a problem that the reaction of the immediately preceding irradiation region affects the current irradiation region and erroneously detects.

  The laser scanning microscope described in Patent Document 1 detects the reaction of each irradiation region by sequentially scanning spatially separated regions so as not to continuously scan spatially adjacent regions on the specimen. In addition, the influence of the reaction between the irradiation areas in the specimen is eliminated, and the brightness of each irradiation area is detected correctly.

JP 2006-227600 A

  However, in the case of observing the reaction of the light stimulus of the specimen, there is a demand for acquiring an image of a wide range of reaction in the specimen without reducing the resolution. In the laser scanning microscope described in Patent Document 1, There is an inconvenience that only the image of the region within the field of view of the microscope can be acquired.

  The present invention has been made in view of the above-described circumstances, and obtains an image of a specimen reaction in a wide range including the outside of the visual field range without reducing the resolution while reducing the influence between adjacent irradiation areas in the specimen. It is an object of the present invention to provide a laser scanning microscope that can be used.

In order to achieve the above object, the present invention provides the following means.
The present invention includes an objective lens that irradiates a specimen with laser light emitted from a laser light source, a scanning unit that scans the specimen with laser light emitted from the objective lens, and the specimen is placed thereon, A stage capable of moving the sample in a direction intersecting the optical axis of the lens, a detection unit for detecting a reaction of the sample caused by irradiation with laser light, and a detection unit configured to detect the sample detected by the detection unit. The brightness information corresponding to the reaction and the scanning position information relating to the position where the laser beam is scanned by the scanning section are stored in association with each other, the observation range to be observed on the specimen is set, and the stage is fixed An observation range dividing unit that divides the observation range into a plurality of scannable ranges in which laser light can be scanned by the scanning unit in a state; An order determining unit that determines the scanning order of the irradiation regions scanned by the scanning unit for each scannable range so that the irradiation regions of the light are not spatially adjacent to each other, and for all the scannable ranges There is provided a laser scanning microscope comprising: a control unit that sequentially moves the stage and causes the scanning unit to scan with laser light in accordance with the scanning order determined by the order determining unit.

  According to the present invention, the laser beam emitted from the laser light source is scanned by the scanning unit and irradiated on the sample by the objective lens, and the reaction of the sample generated thereby is detected by the detection unit. Then, the luminance information corresponding to the detected specimen reaction and the scanning position information of the laser beam by the scanning unit are associated with each other and stored in the storage unit. Therefore, a two-dimensional image of the sample can be acquired based on the luminance information and the scanning position information stored in the storage unit.

  In this case, for each scannable range in which the observation range is divided by the observation range dividing unit, the control unit scans the laser beam with the scanning unit according to the scanning order determined by the order determining unit, so that spatially adjacent irradiation is performed. The region is not irradiated with laser light continuously in time. Therefore, even when the reaction of the irradiation region irradiated with the laser beam reaches the surrounding irradiation region adjacent spatially, the irradiation region irradiated with the laser beam immediately before the reaction of the irradiation region irradiated with the current laser beam It is possible to prevent the reaction of.

In addition, by moving the stage and scanning the laser beam for all the scannable ranges within the observation range to detect the reaction of the sample, the specimens outside the scanable range, that is, outside the visual field range, A two-dimensional image can be acquired.
Therefore, it is possible to acquire an image of the reaction of the specimen in a wide range including the outside of the visual field range without reducing the resolution, while reducing the influence between spatially adjacent irradiation regions in the specimen.

  The present invention includes an objective lens that irradiates a specimen with laser light emitted from a laser light source, a scanning unit that scans the specimen with laser light emitted from the objective lens, and the specimen is placed thereon, A stage capable of moving the sample in a direction intersecting the optical axis of the lens, a detection unit for detecting a reaction of the sample caused by irradiation with laser light, and a detection unit configured to detect the sample detected by the detection unit. The brightness information corresponding to the reaction and the scanning position information relating to the position where the laser beam is scanned by the scanning section are stored in association with each other, the observation range to be observed on the specimen is set, and the stage is fixed An observation range dividing unit that divides the observation range into a plurality of scannable ranges in which laser light can be scanned by the scanning unit in a state; An order determining unit that determines the main scanning order of the irradiation regions to be scanned by the scanning unit for each scannable range and the order determining unit so that the irradiation regions of the light are not spatially adjacent to each other Prior to the main detection in which the laser beam is scanned in accordance with the main scanning order, the stage is sequentially moved with respect to all the scannable ranges, so that each of the scannable ranges is substantially completed in a shorter time than the scan in the main detection. Performing simple detection by irradiating light, and moving the stage again for the scannable range in which the reaction of the sample detected by the detection unit in the simple detection exceeds a predetermined threshold at least in part, Provided is a laser scanning microscope including a control unit that scans laser light with the scanning unit in accordance with the main scanning order.

  According to the present invention, when laser light is irradiated onto irradiation regions that are not spatially adjacent to each other by this detection (this is referred to as mapping scan), between spatially adjacent irradiation regions in the sample. However, it takes time to irradiate the entire scannable range with the laser beam as much as the distance between the irradiation regions irradiated with the laser beam continuously in time is increased.

  Therefore, with simple detection, light is irradiated over substantially the entire scanable range in a shorter time than the scan in the main detection, and the scanable range where the sample response is large is selected in a short time in advance, and the selected scanable range By performing the mapping scan only by the main detection, it is possible to efficiently observe the scanable range in which the reaction in the specimen is large in a short time while reducing the influence between spatially adjacent irradiation regions.

  The selection of the scannable range in which the specimen response is large is based on whether the response in any one irradiation region within the scannable range exceeds a predetermined threshold or the average of the responses in a plurality of irradiation regions within the scannable range. It may be determined whether or not exceeds a predetermined threshold.

  In the above invention, the control unit may perform the simple detection by causing the scanning unit to sequentially scan the irradiation areas spatially adjacent to each other with laser light.

  When laser light is sequentially irradiated to the irradiation areas that are spatially adjacent to each other on the specimen by scanning with laser light (this is referred to as raster scanning), irradiation that is spatially adjacent compared to the mapping scan. Although affected by the regions, the entire scanable range can be irradiated with the laser light in a short time as the distance between the irradiation regions irradiated with the laser beam continuously in time is close.

  In the above-described invention, a wide-area illumination light source capable of irradiating illumination light at a time to substantially the entire scannable range is provided, and the control unit causes the scannable range to be irradiated with illumination light by the wide-range illumination light source. Simple detection may be performed.

  When illuminating illumination light at a time over substantially the entire scanable range with a wide range illumination light source, the illumination light is affected by spatially adjacent illumination areas compared to mapping scanning on the specimen. The illumination light can be irradiated in a shorter time than the mapping scan or the raster scan by the amount that is not scanned.

  In the said invention, it is good also as providing the irradiation area | region restriction | limiting part which restrict | limits the illumination light irradiated to the said scannable range by the said wide range illumination light source to the said other scannable range spatially adjacent. .

  With this configuration, the illumination area controller emits illumination light emitted from the wide range illumination light source only to the scanable range that is the original irradiation target. Accordingly, it is possible to prevent erroneous detection of a reaction caused by leakage of illumination light in another scannable range as being a part of the reaction in the scannable range that is the original irradiation target. As a result, it is possible to more accurately select a scannable range where the response in the specimen is large.

  In the above invention, the scanable range in which the order determining unit irradiates illumination light with the wide range illumination light source so that illumination light is not continuously irradiated onto the scannable ranges spatially adjacent to each other in time. The irradiation order may be determined, and the control unit may perform the simple detection by moving the stage according to the irradiation order determined by the order determination unit.

  With this configuration, the illumination light emitted from the wide range illumination light source is not continuously irradiated in time to the spatially adjacent scannable range. Therefore, even when the response of the scannable range irradiated with the illumination light from the wide range illumination light source extends to the spatially adjacent scannable range, immediately before the response of the scannable range irradiated with the current illumination light. It is possible to prevent the influence of the reaction in the scannable range irradiated with the illumination light. As a result, it is possible to more accurately select a scannable range having a large response in the sample while reducing the influence between spatially adjacent scannable ranges in the sample.

In the above invention, an image construction unit that constructs an image of the specimen is provided, and the storage unit stores coordinate information related to the position coordinates of the stage in association with the luminance information and the scanning position information, and the image construction A unit arranges the luminance information stored in the storage unit based on the scanning position information to construct a two-dimensional image of the scannable range, and arranges the image of the scannable range according to the coordinate information Then, a two-dimensional image of the observation range may be constructed.
With this configuration, the image construction unit can acquire an entire observation range two-dimensional image in which two-dimensional images of all scannable ranges are arranged in the coordinate order of the stage.

  The present invention includes a specimen stage on which a specimen is placed, an objective lens that irradiates the specimen with laser light emitted from a laser light source, and a scanning unit that scans the specimen with laser light emitted by the objective lens. The microscope body comprising: a microscope stage that movably supports the microscope body in a direction intersecting the optical axis of the objective lens with respect to the specimen stage; and the reaction of the specimen caused by laser light irradiation A storage unit that detects brightness information corresponding to the reaction of the specimen detected by the detection unit, and scanning position information related to a position where the scanning unit scans the laser beam, An observation range to be observed on the specimen is set, and laser light can be scanned by the scanning unit with the microscope body fixed to the stage. An observation range dividing unit that divides the observation range into a number of scannable ranges and a laser beam irradiation region that is continuously irradiated in time on the specimen are not spatially adjacent to each other by the scanning unit. An order determining unit that determines the scan order of the irradiation areas to be scanned for each scannable range, and the microscope body is sequentially moved by the microscope stage with respect to all the scannable ranges, and is determined by the order determination unit. And a control unit that scans the laser beam with the scanning unit in accordance with the order of the irradiation regions.

  According to the present invention, for each scannable range obtained by dividing the observation range by the observation range dividing unit, the control unit scans the laser beam with the scanning unit according to the scanning order determined by the order determining unit, so that the laser beam is irradiated. Even if the reaction of the irradiated region reaches the surrounding irradiation region that is spatially adjacent, the reaction of the irradiation region irradiated with the laser beam immediately before that affects the reaction of the irradiation region irradiated with the current laser beam. Can be prevented.

In addition, the control unit moves the microscope main body with the microscope stage and scans the laser beam for all the scannable ranges within the observation range to detect the reaction of the specimen, so that it is out of the scanable range, that is, outside the field of view. A two-dimensional image of the specimen in the entire observation range including it can be acquired.
Therefore, it is possible to acquire an image of the reaction of the specimen in a wide range including the outside of the visual field range without reducing the resolution, while reducing the influence between spatially adjacent irradiation regions in the specimen.

  The present invention includes a specimen stage on which a specimen is placed, an objective lens that irradiates the specimen with laser light emitted from a laser light source, and a scanning unit that scans the specimen with laser light emitted by the objective lens. A microscope body that includes a microscope body, a microscope stage that movably supports the microscope body in a direction that intersects the optical axis of the objective lens, and the laser beam is emitted to the specimen on the specimen stage. A detection unit that detects the reaction of the sample, and a memory that stores the luminance information corresponding to the reaction of the sample detected by the detection unit and the scanning position information related to the position where the scanning unit scans the laser beam in association with each other And an observation range to be observed on the specimen, and a laser beam is run by the scanning unit with the microscope body fixed to the stage. The scanning is performed so that an observation range dividing unit that divides the observation range into a plurality of scannable ranges and a laser light irradiation region that is continuously irradiated onto the specimen in time are not spatially adjacent to each other. Prior to the main detection in which the laser beam is scanned in accordance with the main scanning order determined by the order determination unit, the order determination unit that determines the main scanning order of the irradiation region scanned by the unit for each scannable range. The microscope light main body is sequentially moved by the microscope stage with respect to the scannable range, and simple detection is performed in which light is applied to substantially the entire scannable range in a shorter time than the scan in the main detection. In the detection, the microscope body is moved again for the scannable range in which the reaction of the sample detected by the detection unit exceeds at least a predetermined threshold. Serial to provide a laser scanning microscope and a control unit for scanning the laser beam by the scanning unit in accordance with the present scan order.

  According to the present invention, by performing the main detection after the simple detection, it is possible to efficiently observe the scanable range in which the reaction in the specimen is large in a shorter time while reducing the influence between spatially adjacent irradiation regions. Can do.

  In the above invention, the control unit may perform the simple detection by causing the scanning unit to sequentially scan the irradiation areas spatially adjacent to each other with laser light.

  In the above-described invention, a wide-area illumination light source capable of irradiating illumination light at a time to substantially the entire scannable range is provided, and the control unit causes the scannable range to be irradiated with illumination light by the wide-range illumination light source. Simple detection may be performed.

  In the said invention, it is good also as providing the irradiation area | region restriction | limiting part which restrict | limits the illumination light irradiated to the said scannable range by the said wide range illumination light source to the said other scannable range spatially adjacent. .

  In the above invention, the scanable range in which the order determining unit irradiates illumination light with the wide range illumination light source so that illumination light is not continuously irradiated onto the scannable ranges spatially adjacent to each other in time. The irradiation order may be determined, and the control unit may perform the simple detection by moving the microscope main body by the microscope stage according to the irradiation order determined by the order determination unit.

In the above-described invention, an image constructing unit that constructs an image of the specimen is provided, and the storage unit stores coordinate information related to the position coordinates of the microscope body in association with the luminance information and the scanning position information, and the image The construction unit arranges the luminance information stored in the storage unit based on the scanning position information to construct a two-dimensional image of the scannable range, and the image of the scannable range according to the coordinate information A two-dimensional image of the observation range may be constructed by arranging the images.
With this configuration, the image construction unit can acquire a two-dimensional image of the entire observation range in which two-dimensional images of all scannable ranges are aligned in the coordinate order of the microscope body.

  According to the present invention, it is possible to observe the reaction of the specimen in a wide range including the outside of the visual field range while reducing the influence between adjacent irradiation areas in the specimen.

1 is a schematic configuration diagram illustrating a laser scanning microscope according to a first embodiment of the present invention. It is a block diagram which shows the control program part of FIG. It is a figure which shows the observable area | region and ROI creation tool which are displayed on a monitor by the observation range designation | designated part. It is a figure which shows a mode that the observation range currently displayed on the observable area | region of FIG. 3 was divided | segmented into the several sub area | region. It is a figure which shows the scanning order in the sub area | region of 64x64 pixel. It is a figure which shows a mode that the 1st sub area | region has been arrange | positioned in the visual field range. It is a figure which shows a mode that the mapping scan is carried out to the sub area | region of FIG. It is a figure which shows a mode that the 2nd sub area | region has been arrange | positioned in the visual field range. It is a figure which shows a mode that the mapping scan is carried out to the sub area | region of FIG. It is a figure which shows a mode that the 1st sub area | region is raster-scanned by the laser scanning microscope which concerns on 2nd Embodiment of this invention. It is a figure which shows a mode that the 2nd sub area | region is raster-scanned. It is a figure which shows a mode that the sub area | region where the brightness | luminance of fluorescence exceeds a predetermined threshold value was selected. It is a figure which shows a mode that the mapping scan of the selected 1st sub area | region of FIG. 12 is carried out. It is a figure which shows a mode that the mapping 2nd sub area | region of FIG. 12 is mapping-scanned. It is a schematic block diagram which shows the laser scanning microscope which concerns on 3rd Embodiment of this invention. (A) is a figure which shows the irradiation range of the laser light source when not restrict | limited by LCOS, (b) is a figure which shows the state which made the irradiation range of the laser light source correspond to a sub-region by LCOS. It is a schematic block diagram which shows a laser scanning microscope as the modification of 2nd Embodiment of this invention. It is a schematic block diagram which shows a laser scanning microscope in the modification of 3rd Embodiment of this invention.

[First Embodiment]
A laser scanning microscope according to a first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the laser scanning microscope 100 according to this embodiment includes a stage 1 on which a specimen S is placed, a light source unit 3 that generates laser light, and laser light emitted from the light source unit 3. A reflecting mirror 5 that reflects the light, a dichroic mirror 7 that transmits the laser light reflected by the reflecting mirror 5, a scanning optical unit (scanning unit) 9 that scans the sample S through the laser light transmitted through the dichroic mirror 7, and scanning While the sample S is irradiated with the laser light scanned by the optical unit 9, the objective lens 11 that condenses the fluorescence generated in the sample S and a photodetector (detection unit) that detects the fluorescence collected by the objective lens 11. 13).

  The stage 1 is provided so as to be movable in XY directions orthogonal to the optical axis of the objective lens 11. As a result, the range in which the laser beam can be scanned on the specimen S by the scanning optical unit 9 can be moved in the XY directions only by moving the stage 1.

  The light source unit 3 includes laser light sources 31A and 31B that emit laser beams having different wavelengths, a reflection mirror 33 that reflects the laser light emitted from one laser light source 31A, and an optical path of the laser light reflected by the reflection mirror 33. And the optical path of the laser light emitted from the other laser light source 31B, ON / OFF of the generation of each laser light whose optical path is synthesized by the dichroic mirror 35, the intensity and wavelength component of the laser light, etc. And an acoustooptic device 37 for controlling the above.

The dichroic mirror 35 reflects the laser light incident from the laser light source 31 </ b> A via the reflection mirror 7 toward the acoustooptic element 37, and transmits the laser light from the laser light source 31 </ b> B to be incident on the acoustooptic element 37. It is like that.
As the acoustooptic device 37, for example, an AOTF (Acoustic Optical Tunable Filter) or an AOM (Acousto-Optic Modulator) is used.

  The dichroic mirror 7 transmits the laser light from the reflection mirror 5 and makes it incident on the scanning optical unit 9, while reflecting the fluorescence from the sample S collected by the objective lens 11 and returning through the scanning optical unit 9. The laser beam is branched from the optical path.

  The scanning optical unit 9 includes a Y-direction scanner 9A and an X-direction scanner 9B that deflect light in two directions orthogonal to each other. The scanning optical unit 9 can scan the sample S two-dimensionally on the sample S with the laser light emitted from the objective lens 11 by these scanners 9A and 9B.

  For example, a photomultiplier tube (PMT) is used as the photodetector 13. The photodetector 13 detects fluorescence generated in the specimen S as a reaction of the specimen S caused by irradiation with laser light. Further, the photodetector 13 outputs a luminance signal corresponding to the detected luminance of the fluorescence.

  Further, the laser scanning microscope 100 includes an input device 15 to which an instruction from a user is input, an A / D converter 17 that performs A / D conversion on a luminance signal output from the photodetector 13, and an A / D. A PC (Personal Computer) 19 that processes the converted luminance signal, instructions input to the input device 15, and controls the irradiation of the sample S with laser light, and a scanner drive that drives the scanning optical unit 9. A unit 21, a stage driving unit 23 that moves the stage 1, and a monitor 25 that displays an instruction input to the input device 15, an image of the sample S, and the like.

  The input device 15 includes, for example, a keyboard, a mouse (pointing device), a GUI (Graphical User Interface), and the like (not shown). The input device 15 allows the user to input instructions such as scanning conditions and laser light output conditions. In addition, the input device 15 allows the user to specify a desired observation range.

  The PC 19 includes a laser output control unit 41 that controls the output of laser light generated from the light source unit 3, a frame memory (storage unit) 43 that stores A / D converted luminance signals, and the like, and scanning setting of the laser light. A control program unit 45 that performs image acquisition and the like, drive waveform memories 47A and 47B that store drive waveform data that drives the scanning optical unit 9, a control unit 49 that controls the scanner drive unit 21, the stage drive unit 23, and the like It has.

  The laser output control unit 41 has a memory 41 a that stores output conditions of laser light sent from the input device 15. The laser output control unit 41 reads out the output conditions stored in the memory 41 a and can control ON / OFF of the generation of laser light by the AOTF 37.

The frame memory 43 stores, in association with the luminance signal sent from the A / D converter 17, scanning position information relating to the position where the scanning optical unit 9 scans the laser light in association with each other. The frame memory 43 stores coordinate information related to the position coordinates of the stage 1 in association with the luminance signal and the scanning position information.
The drive waveform memory 47A stores drive waveform data for the Y-direction scanner 9A, and stores drive waveform data for the drive waveform memory 47BX-direction scanner 9B.

  As shown in FIG. 2, the control program unit 45 includes a scanning condition input unit 51 to which a scanning condition is input from the input device 15, and an observation range designating unit (observation range dividing unit) 53 for designating an observation range on the sample S. And a scanning order determining unit 55 that determines the scanning order of the irradiation region of the laser light that is scanned by the scanning optical unit 9, and an image constructing unit 57 that constructs an image of the specimen S.

  The scanning condition input unit 51 includes, for example, an XY scan size for determining an imaging region (for example, 512 × 512 points) and a sampling speed for determining a sampling interval of detection signals in the A / D converter 17 as scanning conditions. The data acquisition time per irradiation area within the visual field range is input.

  For example, as shown in FIG. 3, the observation range designation unit 53 displays an observable region 53A and an ROI creation tool 53B (ROI: Region Of Interest, region of interest) on the monitor 25. The user can operate the mouse or the like of the input device 15 to specify the observation range using the observable area 53A on the monitor 25 and the ROI creation tool 53B. In addition, the observation range designating unit 53 sets the observation range designated by the user for the sample S.

  As the ROI creation tool 53B, “Rectangle ROI” that creates a rectangular ROI with the down position and the up position of the left mouse button as diagonal lines, and the click position of the button with the mouse as vertices, the vertices are straight lines in the order of click. There is a “Polygon ROI” for creating a connected polygonal ROI. In “Polygon ROI”, double-clicking the left button of the mouse ends the hitting point.

  When the observation range is set, as shown in FIG. 4, the observation range designating unit 53 uses a plurality of sub-regions (scannable ranges) in which the scanning optical unit 9 can scan the laser beam with the stage 1 fixed. The observation range is divided.

  The scanning order determination unit 55 determines a random scanning order for each sub-region so that the irradiation regions of the laser light continuously irradiated onto the specimen S are not spatially adjacent to each other. For example, a random number is used to determine the scanning order.

  The random scanning order of the irradiation area is determined as shown in FIG. 5, for example. That is, one sub-region is divided into eight divided regions A to H having the same size. And the irradiation area | region of the upper left of each division area is irradiated in order of ACEGBDFH. After one round, the irradiation region adjacent to the X-line direction is irradiated in the same order as ACEGBD. When one line is completed, each irradiation area of the second line of each divided area is irradiated in the same manner. That is, it is determined so that the divided regions A to H are irradiated with laser light in a fixed order according to the same rule.

  FIG. 5 shows the scan order in a 64 × 64 pixel sub-region. The number of each square indicates the scanning order. In the example of FIG. 5, since the irradiation area next to the first irradiation area is the ninth irradiation area, the laser irradiation time interval between adjacent irradiation areas is a data acquisition time for eight pixels.

  In addition, the scanning order determination unit 55 checks whether there is an irradiation area spatially adjacent to each other according to the determined scanning order, and performs processing for replacing with another irradiation area if there is any. Further, the scanning order determining unit 55 sends the coordinates of each irradiation region according to the determined scanning order to the frame memory 43 as scanning position information, and converts the time change of the coordinates into driving waveform data to convert the driving waveform memory 47A. , 47B.

  The image construction unit 57 arranges the luminance signal stored in the frame memory 43 in accordance with the coordinates of each irradiation area as scanning position information, and constructs a two-dimensional image of the sub-area. Further, the image construction unit 57 arranges the constructed sub-region images according to the coordinate information of the stage 1 stored in the frame memory 43, and constructs a two-dimensional image of the entire observation range. ing.

  The control unit 49 sets movement data for sequentially moving the stage 1 so that all the sub-regions divided by the observation range specifying unit 53 are arranged in the visual field range in the spatially adjacent order, and according to the movement data. The stage 1 is moved by the stage driving unit 23.

  The movement data is, for example, that the sub-region is moved in the observation range in the order adjacent to the X direction, and when one line is completed, the line is shifted in the Y direction, and each line is moved in the order adjacent to the X direction. May be. In addition, the control unit 49 sends the time change of the coordinates of the stage 1 according to the set movement data to the frame memory 43 as coordinate information.

  Further, the control unit 49 reads the scanning order of each irradiation region stored in the drive waveform memories 47A and 47B for each sub-region, and drives the scanning optical unit 9 by the scanner driving unit 21 according to the scanning order. It has become.

The scanner drive unit 21 scans the laser beam by swinging the scanners 9A and 9B under the control of the control unit 49.
The stage drive unit 23 moves the stage 1 under the control of the control unit 49.

The operation of the laser scanning microscope 100 configured as described above will be described.
In order to observe the sample S with the laser scanning microscope 100 according to the present embodiment, first, the user places the sample S on the stage 1 and operates the input device 15 to designate the observation range by the observation range designation unit 53. To do.

  For example, when the user designates the observation range by “Polygon ROI” as shown in FIG. 3, the observation range is set for the sample S by the observation range designation unit 53, and the setting is made as shown in FIG. The observed range is divided into a plurality of sub-regions.

  Next, when the user inputs scanning conditions for each sub-region to the input device 15, the scanning conditions are sent from the input device 15 to the scanning condition input unit 51 of the control program unit 45. Then, as shown in FIG. 5, the scanning order determination unit 55 determines a random scanning order in which laser light is not continuously irradiated to adjacent irradiation regions in the sub-region. Further, the number of samplings per irradiation area is calculated based on the sampling speed and the data acquisition time per irradiation area. In the present embodiment, the number of samplings per irradiation area is set to one.

  Next, the scanning order determination unit 55 sends the coordinates of each irradiation area according to the determined scanning order to the frame memory 43 as scanning position information, and the time change of the coordinates is converted into driving waveform data to drive waveform memory 47A. , 47B and stored.

  Further, the control unit 49 sets movement data for sequentially moving the stage 1 in the spatially adjacent order for all the sub-regions divided by the observation range specifying unit 53, and the stage 1 according to the movement data. The time change of the coordinate is sent to the frame memory 43 as coordinate information.

  Next, when an instruction to start scanning is input from the user to the input device 15, the stage drive unit 23 moves the stage 1 as shown in FIG. Is arranged in the field of view. The controller 49 reads the drive waveform data in the drive waveform memories 47A and 47B and controls the scanner drive unit 21, and the Y-direction scanner 9A and the X-direction scanner 9B are swung according to the drive waveform data. At the same time, the laser output control unit 41 is controlled by the control unit 49, and laser light is emitted from the light source unit 3.

  The laser light emitted from the light source unit 3 is reflected by the reflection mirror 5, passes through the dichroic mirror 7, is deflected by the Y direction scanner 9 A and the X direction scanner 9 B of the scanning optical unit 9, and is sampled S by the objective lens 11. The upper irradiation area is irradiated.

  Then, the scanners 9A and 9B change the swing angle according to the drive waveform data indicating the time change of the coordinates of the irradiation region according to the scanning order randomly determined by the scanning order determination unit 55, so that the irradiation regions that are not adjacent to each other. Are sequentially irradiated with laser light. As a result, the laser beam is scanned two-dimensionally on the specimen S as shown in FIG. Hereinafter, scanning in which laser beams are irradiated onto irradiation regions that are not spatially adjacent to each other on the specimen S is referred to as mapping scanning.

  When the fluorescent light is emitted in each irradiation region of the specimen S by being irradiated with the laser light, the fluorescent light is condensed by the objective lens 11 and branched from the optical path of the laser light by the dichroic mirror 35 via the scanning optical unit 9. And detected by the photodetector 13.

  When fluorescence is detected by the photodetector 13, a luminance signal corresponding to the luminance of the fluorescence is output from the photodetector 13, A / D converted by the A / D converter 17, and sent to the frame memory 43. The luminance signal of each irradiation area sent to the frame memory 43 is stored in association with scanning position information indicating the coordinates of each irradiation area according to the scanning order sent from the scanning order determining unit 55.

  Next, the image construction unit 57 arranges the luminance signals stored in the frame memory 43 in accordance with the coordinates of the associated irradiation region, and constructs an image of the sub region. The constructed image is displayed on the monitor 25.

  In this case, by performing mapping scanning according to the random scanning order determined by the scanning order determining unit 55 for each sub-region, the spatially adjacent irradiation regions can be irradiated with laser light continuously in time. Absent. Therefore, even when the reaction of the irradiation region irradiated with the laser beam reaches the adjacent surrounding irradiation region, the laser beam was irradiated immediately before the detection of the fluorescence from the irradiation region irradiated with the laser beam. It can prevent that the detection of the fluorescence of an irradiation area affects. Thereby, it is possible to obtain a high-resolution image in which the luminance of each irradiation area is correctly detected.

  When the mapping scan of the first sub-region is completed, the stage drive unit 23 moves the stage 1 under the control of the control unit 49 as shown in FIG. A sub-region of the eye is placed in the field of view. As shown in FIG. 9, the second sub-region is subjected to mapping scan by the control unit 49 in the same manner as the first sub-region, and the image construction unit 57 constructs an image. Similarly, all the sub-regions are sequentially arranged in the visual field range such as the third sub-region and the fourth sub-region, and the mapping scan is performed.

  When the mapping scan is performed in all the sub-regions and the image is constructed, the image construction unit 57 arranges and pastes the images of all the sub-regions according to the coordinate information of the stage 1 stored in the frame memory 43. It is done. Thereby, a two-dimensional image of the entire observation range including the outside of the sub-region, that is, the outside of the visual field range is constructed. The constructed image of the observation range is displayed on the monitor 25. The user can observe the specimen S by looking at the image of the entire observation range.

  As described above, according to the laser scanning microscope 100 according to the present embodiment, an image is constructed by mapping scanning for each sub-region, and an image of the entire observation range is constructed by combining the images of each sub-region. By doing so, it is possible to acquire an image of the reaction of the specimen S in a wide range including the outside of the visual field range without reducing the resolution while reducing the influence between spatially adjacent irradiation areas in the specimen S.

[Second Embodiment]
Next, a laser scanning microscope according to the second embodiment of the present invention will be described.
In the laser scanning microscope 100 according to the present embodiment, the control unit 49 reacts by simple detection that can irradiate substantially the entire sub-region in a shorter time than the mapping scan prior to the main detection by the mapping scan. It is different from the first embodiment in that a certain sub-region is selected and only the selected sub-region is detected by a mapping scan.
In the following, portions having the same configuration as those of the laser scanning microscope 100 according to the first embodiment are denoted by the same reference numerals and description thereof is omitted.

The control unit 49 is configured to perform the main detection by mapping scanning according to the scanning order (main scanning order) determined by the scanning order determination unit 55.
In addition, the control unit 49 controls the scanner driving unit 21 to perform simple detection by raster scanning in which the scanning optical unit 9 sequentially scans the irradiation regions spatially adjacent to each other. .

  The mapping scan reduces the influence between the spatially adjacent irradiation regions in the sample S, but the entire sub-region is as much as the distance between the irradiation regions irradiated with laser light continuously in time is separated. It takes time to scan. On the other hand, raster scan irradiates laser light to substantially the entire sub-region in a shorter time than mapping scan by the amount of adjacent irradiation regions that are continuously irradiated with laser light. Can do.

  In the raster scan, for example, the laser beam may be scanned in the X direction for each line in the sub-region, and the line may be shifted in the Y direction when one line is completed. Further, the simple detection by the raster scan may use a laser beam having the same wavelength as the main detection by the mapping scan.

  The control unit 49 performs simple detection by sequentially moving the stage 1 with respect to all the sub-regions. In the simple detection, the control unit 49 determines whether or not the luminance of the fluorescence from any one irradiation region of the sample S detected by the photodetector 113 has exceeded a predetermined threshold value. .

  In addition, the control unit 49 selects a sub-region where the luminance of the fluorescence exceeds a predetermined threshold as a sub-region having a reaction. Then, the control unit 49 moves the stage 1 again for only the selected sub-region and performs the main detection by the mapping scan. For example, the predetermined threshold value may be set in advance by the user inputting to the input device 15.

The operation of the laser scanning microscope 100 configured as described above will be described.
When the specimen S is observed with the laser scanning microscope 100 according to the present embodiment, first, simple detection is performed. Since the user places the specimen S on the stage 1 and the stage 1 is moved by the control unit 49 and the first sub-region is arranged in the visual field range, it is the same as in the first embodiment. Is omitted.

  When the first sub-region is arranged in the visual field range, the scanner drive unit 21 is controlled by the control unit 49, and the entire sub-region is raster scanned by the scanning optical unit 9 as shown in FIG. When the fluorescence generated in the sample S by the raster scan is detected by the photodetector 13, a luminance signal corresponding to the luminance of the fluorescence is sent to the frame memory 43 via the A / D converter 17, and each irradiation region is detected. It is stored in association with the coordinates.

  When the raster scan of the first sub-region is finished, the stage drive unit 23 moves the stage 1 under the control of the control unit 49, and the second sub-region adjacent to the first sub-region is the visual field range. Placed in. Then, as shown in FIG. 11, the second sub-region is raster scanned in the same manner as the first sub-region, and the fluorescence from the specimen S is detected. Similarly, all the sub-regions are sequentially arranged in the visual field range, and fluorescence is detected by raster scanning.

  Next, the main detection is performed. First, the control unit 49 determines whether or not the luminance of fluorescence from any one irradiation region exceeds a predetermined threshold for each sub-region, and as shown in FIG. A sub-region is selected. FIG. 12 exemplifies two selected sub-regions and is indicated by “reaction A” and “reaction B”, respectively.

  Next, the stage drive unit 23 moves the stage 1 by the control unit 49, and as shown in FIG. 13, the first sub-region with reaction (A with reaction) is arranged in the visual field range. Then, mapping scanning is performed on the sub-region by the control unit 49 according to the scanning order randomly determined by the scanning order determination unit 55, and an image is constructed by the image construction unit 57.

  When the mapping scan of the first sub-region is finished, the stage drive unit 23 moves the stage 1 by the control unit 49, and as shown in FIG. 14, the second luminance whose fluorescence intensity exceeds a predetermined threshold value The sub-region (with reaction B) is arranged in the visual field range. Then, a mapping scan is performed on the second sub-region in the same manner as the first sub-region, and an image is constructed. In this way, the main detection by the mapping scan is performed on all the selected sub-regions, and an image is constructed.

  When the main detection is performed in all the sub-regions and the image is constructed, the image constructing unit 57 arranges the images of all the sub-regions according to the coordinate information of the stage 1 and generates a two-dimensional image of the entire observation range. Built. The user can observe the specimen S by looking at the image of the entire observation range.

  As described above, according to the laser scanning microscope 100 according to the present embodiment, sub-regions in which the specimen S reacts are selected in a short time by simple detection by raster scan, and only the selected sub-region is subjected to mapping scan. By performing this detection, it is possible to efficiently observe a sub-region having a large response in the specimen S in a shorter time while reducing the influence between spatially adjacent irradiation regions.

[Third Embodiment]
Next, a laser scanning microscope according to the third embodiment of the present invention will be described.
As shown in FIG. 15, the laser scanning microscope 200 according to the present embodiment includes a wide field light source unit 103 (hereinafter referred to as a wide light source unit 103) that irradiates the entire sub-region with laser light at a time. The control unit 49 is different from the second embodiment in that the controller 49 performs simple detection by irradiating laser light from the wide light source unit 103 instead of raster scanning.
In the following, portions having the same configuration as those of the laser scanning microscope 100 according to the second embodiment are denoted by the same reference numerals and description thereof is omitted.

  The wide light source unit 103 includes a laser light source (wide-area illumination light source) 131 that generates laser light that can be irradiated at once on substantially the entire sub-region, and an LCOS (Liquid Crystal) that performs pattern irradiation with the laser light emitted from the laser light source 131. On Silicon, irradiation area limiting unit) 132.

  The LCOS 132 is configured to restrict the laser light emitted from the laser light source 131 from being irradiated to other spatially adjacent subregions. When the irradiation range of the laser light emitted from the laser light source 131 is not limited, for example, as shown in FIG. 16A, the entire visual field range becomes the irradiation range of the laser light. ), The laser light irradiation range can be limited to a sub-region. Thus, it is possible to prevent a reaction from occurring by irradiating the laser beam outside the sub-region even within the visual field range and erroneously detecting the reaction.

  On the optical path between the scanning optical unit 9 and the objective lens 11, a dichroic mirror (optical path combining unit) 133 that combines the optical paths of the laser beams emitted from the wide light source unit 103 is detachably provided. When the dichroic mirror 133 is inserted in the optical path, the wide light source unit 103 irradiates the sample S with laser light, and when the dichroic mirror 133 is removed from the optical path, the light source unit 3 can irradiate the sample S with laser light. .

The PC 19 includes a wide field illumination control unit 149 (hereinafter referred to as a wide illumination control unit 149) that controls the wide light source unit 103.
Prior to the main detection by the mapping scan, the control unit 49 performs simple detection by the wide light source that irradiates the entire sub-region with the laser light at once by the wide light source unit 103.

The operation of the laser scanning microscope 200 configured as described above will be described.
When the sample S is observed with the laser scanning microscope 200 according to the present embodiment, first, simple detection is performed. The user places the sample S on the stage 1, removes the dichroic mirror 133 from the optical path of the laser light, and sets the objective lens 11 at a low magnification.

  Next, laser light is generated from the light source unit 3, the scanner driving unit 21 is controlled by the control unit 49, and the laser light is raster-scanned on the specimen S by the scanning optical unit 9. Then, fluorescence is detected by the photodetector 13 and the luminance signal is imaged by the image construction unit 57 to obtain a raster scan image.

  Next, the observable area 53A and the ROI creation tool 53B are displayed on the monitor 25 by the observation range designating unit 53, and the raster scan image is superimposed on the observable area 53A. When the user designates the observation range by “Polygon ROI” with reference to the raster scan image acquired at a low magnification, the observation range is set for the sample S by the observation range designation unit 53, and the observation range includes a plurality of observation ranges. Divided into sub-regions. Hereinafter, the process up to the point where the user inputs a scan start instruction to the input device 15 is the same as that in the first embodiment, and a description thereof will be omitted.

  Next, the user switches the objective lens 11 to high magnification, inserts the dichroic mirror 133 in the optical path of the laser light, and inputs an instruction to start scanning to the input device 15. Then, under the control of the control unit 49, the stage driving unit 23 moves the stage 1, and the first sub-region is arranged in the visual field range.

  Next, a laser beam is emitted from the wide light source unit 103 by the wide illumination control unit 149. The laser light emitted from the wide light source unit 103 is reflected by the dichroic mirror 133 and is irradiated onto the entire sub-region of the sample S by the objective lens 11 at a time.

  With wide illumination, compared to the case of mapping scan on the specimen, it is affected by the spatially adjacent irradiation areas, but the laser beam is not scanned for the laser beam over the entire sub-area rather than the mapping scan or raster scan. Light can be irradiated in a short time.

  When the fluorescence generated in the specimen S due to the wide illumination is detected by the photodetector 13, a luminance signal corresponding to the luminance of the fluorescence is sent to the frame memory 43 via the A / D converter 17, and each of the irradiation regions It is stored in association with the coordinates.

When the first sub-region is illuminated wide, the control of the control unit 49 causes the stage drive unit 23 to move the stage 1 so that the adjacent sub-regions are sequentially arranged in the visual field range. In the same manner as in the first sub-region, the fluorescent light is detected by wide illumination.
Next, the main detection is performed. Since this detection is the same as in the second embodiment, a description thereof will be omitted.

  As described above, according to the laser scanning microscope 200 according to the present embodiment, it is possible to identify a sub-region having a reaction earlier than the raster scan by the amount that the laser light is not scanned by the wide illumination. Thereby, simple detection can be performed more rapidly.

In the present embodiment, the laser light source 131 is exemplified as the wide range illumination light source. However, instead of this, for example, a mercury lamp may be employed.
In the present embodiment, the LCOS 132 has been described as an example of the irradiation region limiting unit. However, instead of this, a DMD (Digital Micromirror Device: registered trademark) or an optical aperture may be employed.

  Further, in the present embodiment, the adjacent sub-regions are sequentially subjected to wide illumination when performing simple detection, but instead, for example, the scanning order determination unit 55 includes sub-spaces that are spatially adjacent to each other. The irradiation order of the sub-regions to be irradiated with the laser light is determined by the wide light source unit 103 so that the laser light is not continuously irradiated onto the regions, and the control unit 49 determines the irradiation determined by the scanning order determination unit 55. Simple detection may be performed by moving the stage 1 according to the order.

  By doing in this way, the laser beam emitted from the wide light source unit 103 is not continuously irradiated temporally to the spatially adjacent sub-regions. Therefore, even when the reaction of the sub-region irradiated with the laser light by the wide light source unit 103 reaches the surrounding sub-regions adjacent spatially, the laser light is immediately before the reaction of the sub-region irradiated with the current laser light. It is possible to prevent the reaction of the sub-region irradiated with. Thereby, while reducing the influence of spatially adjacent sub-regions in the sample S, sub-regions having a large response in the sample S can be selected with higher accuracy.

The second embodiment and the third embodiment can be modified as follows.
In the second embodiment and the third embodiment, whether or not the luminance of fluorescence from any one irradiation region in the sub-region exceeds a predetermined threshold when selecting the sub-region that has reacted by simple detection. However, as a first modification, it may be determined whether or not the average of the luminance of fluorescence in a plurality of irradiation regions in the sub-region exceeds a predetermined threshold value.

  In the second embodiment and the third embodiment, it is determined whether or not there is a reaction for each sub-region after simple detection of all the sub-regions. It may be determined whether or not there is a reaction in the sub-region after the simple detection every time. Then, when there is no reaction, the process moves to the next sub-region, and when there is a reaction, the mapping scan may be performed in the sub-region as it is before moving to the next sub-region.

The first to third embodiments can be modified as follows.
In each of the above embodiments, the photodetector 13 is exemplified as the detection unit, and the fluorescence generated in the sample S by being irradiated with the laser light is detected as a reaction of the sample S. However, as a first modification example, For example, as shown in FIGS. 17 and 18, a current value detector 113 that detects a current value of the sample S irradiated with the laser light as a reaction of the sample S may be adopted as the detection unit. FIG. 17 shows a configuration in which the current value detector 113 is applied to the laser scanning microscope 100 of the first and second embodiments, and FIG. 18 shows the current value detector 113 to the laser scanning microscope 200 of the third embodiment. The structure which applied is shown.

  The current value detector 113 is inserted into the sample S on the stage 1 to detect the current value of the sample S, converts the detected current value into a current value signal, and sends it to the A / D converter 17. Yes. In this case, the current value may be detected by the current value detector 113 while irradiating the sample S with laser light.

  The current value signal detected by the current value detector 113 is A / D converted by the A / D converter 17 in the same manner as the luminance signal detected by the light detector 13, and laser light is emitted by the scanning optical unit 9. The image is stored in the frame memory 43 in association with the scanning position information regarding the scanned position, and the image is constructed by being arranged according to the coordinates of each irradiation region by the image construction unit 57.

  For example, in the laser scanning microscope 200 according to the third embodiment shown in FIG. 18, the current value of the sample S is detected by the current value detector 113 while performing wide illumination on the sub-region of the sample S during simple detection. You can do that. Further, the control unit 49 may determine whether or not the current value signal exceeds a predetermined threshold and select a sub-region where the current value signal exceeds the predetermined threshold.

  In this modification, the current value detector 113 is inserted into one place of the sample S and the current value is measured for one part. However, the current value detector 113 is inserted into a plurality of places, The current value may be measured for the part.

  In each of the above embodiments, the stage 1 is moved. However, as a second modification, the microscope main body having the objective lens 11 and the scanning optical unit 9 is connected to the stage 1 with light from the objective lens 11. A microscope stage (not shown) that is movably supported in a direction orthogonal to the axis may be employed, and the microscope main body may be moved relative to the stage 1 by the microscope stage instead of moving the stage 1.

  The microscope stage includes, for example, a fixed plate fixed on a surface plate (not shown) and two movable plates movable in directions orthogonal to each other in the XY direction with respect to the fixed plate in a stacked state in the plate thickness direction, The microscope main body may be mounted on the movable plate. In this case, if the stage 1 and the microscope stage are mounted on the surface plate, the microscope main body is mounted on the microscope stage, and the stage 1 is fixed, the microscope main body is moved in the XY directions by the microscope stage. Good.

  By doing in this way, the position of the objective lens 11 can be moved on the horizontal plane with respect to the stage 1 by moving the microscope stage. Therefore, by moving the microscope stage instead of moving the stage 1 in each of the above embodiments, the same operation as in each of the above embodiments can be realized. In this case, the image construction unit 57 may construct a two-dimensional image of the observation range by arranging the images of the sub-regions using the position coordinates of the microscope body as coordinate information instead of the coordinates of the stage 1.

  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. For example, the present invention is not limited to those applied to the above-described embodiments and modifications, but may be applied to embodiments in which these embodiments and modifications are appropriately combined, and is not particularly limited. .

1 stage (specimen stage)
9 Scanning optical unit (scanning unit)
11 Objective Lens 13 Photodetector (Detector)
43 Frame memory (storage unit)
49 Control unit 53 Observation range designating unit (observation range dividing unit)
55 Scanning Order Determination Unit (Order Determination Unit)
57 Image construction unit 100, 200 Laser scanning microscope 131 Laser light source (wide range illumination light source)
132 LCOS (irradiation area restriction part)
S specimen

Claims (14)

An objective lens for irradiating the specimen with laser light emitted from a laser light source;
A scanning unit that scans the sample with laser light irradiated by the objective lens;
A stage on which the specimen is mounted and capable of moving the specimen in a direction intersecting the optical axis of the objective lens;
A detection unit for detecting a reaction of the specimen caused by irradiation with laser light;
A storage unit that stores brightness information corresponding to the reaction of the sample detected by the detection unit and scanning position information related to a position at which the laser beam is scanned by the scanning unit in association with each other;
An observation range dividing unit that sets an observation range to be observed on the specimen and divides the observation range into a plurality of scannable ranges that can be scanned with laser light by the scanning unit in a state where the stage is fixed;
An order in which the scanning order of the irradiation areas to be scanned by the scanning unit is determined for each scannable range so that the irradiation areas of the laser beams irradiated continuously on the specimen are not spatially adjacent to each other. A decision unit;
A laser scanning microscope comprising: a control unit that sequentially moves the stage with respect to all the scannable ranges and causes the scanning unit to scan with laser light in accordance with the scanning order determined by the order determination unit. An objective lens for irradiating the specimen with laser light emitted from a laser light source;
A scanning unit that scans the sample with laser light irradiated by the objective lens;
A stage on which the specimen is mounted and capable of moving the specimen in a direction intersecting the optical axis of the objective lens;
A detection unit for detecting a reaction of the specimen caused by irradiation with laser light;
A storage unit that stores brightness information corresponding to the reaction of the sample detected by the detection unit and scanning position information related to a position at which the laser beam is scanned by the scanning unit in association with each other;
An observation range dividing unit that sets an observation range to be observed on the specimen and divides the observation range into a plurality of scannable ranges that can be scanned with laser light by the scanning unit in a state where the stage is fixed;
The main scanning order of the irradiation areas to be scanned by the scanning unit is determined for each scannable range so that the irradiation areas of the laser beams irradiated continuously on the specimen are not spatially adjacent to each other. An order determination unit;
Prior to the main detection in which the laser beam is scanned in accordance with the main scanning order determined by the order determination unit, the stage is sequentially moved with respect to all the scannable ranges, and each stage is performed in a shorter time than the scanning in the main detection. Performing simple detection of irradiating light to substantially the entire scannable range, the scannable range in which the reaction of the specimen detected by the detection unit in the simple detection exceeds a predetermined threshold at least in part, A laser scanning microscope comprising: a control unit that moves the stage again and scans the laser beam by the scanning unit according to the main scanning order.   3. The laser scanning microscope according to claim 2, wherein the control unit performs the simple detection by causing the scanning unit to sequentially scan the irradiation regions spatially adjacent to each other with laser light. A wide-area illumination light source capable of irradiating illumination light at a time over substantially the entire scanable range;
The laser scanning microscope according to claim 2, wherein the control unit performs the simple detection by irradiating the scannable range with illumination light by the wide range illumination light source.   5. The laser scanning type according to claim 4, further comprising an irradiation area limiting unit configured to limit illumination light irradiated to the scannable range by the wide range illumination light source to other scannable ranges spatially adjacent to each other. microscope. The order determination unit determines an irradiation order of the scannable range in which illumination light is irradiated by the wide range illumination light source so that illumination light is not continuously irradiated onto the scannable ranges spatially adjacent to each other in time. And
The laser scanning microscope according to claim 4 or 5, wherein the control unit performs the simple detection by moving the stage according to the irradiation order determined by the order determination unit. An image construction unit for constructing an image of the specimen;
The storage unit stores coordinate information related to the position coordinates of the stage in association with the luminance information and the scanning position information,
The image construction unit arranges the luminance information stored in the storage unit based on the scanning position information, constructs a two-dimensional image of the scannable range, and converts the image of the scannable range to the coordinates The laser scanning microscope according to any one of claims 1 to 6, wherein a two-dimensional image of the observation range is constructed by arranging according to information. A specimen stage on which the specimen is placed;
A microscope main body including an objective lens that irradiates the specimen with laser light emitted from a laser light source, and a scanning unit that scans the specimen with the laser light emitted by the objective lens;
A microscope stage that supports the microscope body movably in a direction intersecting the optical axis of the objective lens with respect to the specimen stage;
A detection unit for detecting a reaction of the specimen caused by irradiation with laser light;
A storage unit that stores brightness information corresponding to the reaction of the sample detected by the detection unit and scanning position information related to a position at which the laser beam is scanned by the scanning unit in association with each other;
Setting an observation range to be observed on the specimen, and dividing the observation range into a plurality of scannable ranges in which laser light can be scanned by the scanning unit with the microscope main body fixed to the stage And
An order in which the scanning order of the irradiation areas to be scanned by the scanning unit is determined for each scannable range so that the irradiation areas of the laser beams irradiated continuously on the specimen are not spatially adjacent to each other. A decision unit;
A laser including a control unit that sequentially moves the microscope main body by the microscope stage with respect to all the scannable ranges and scans the laser beam by the scanning unit in accordance with the order of the irradiation areas determined by the order determining unit Scanning microscope. A specimen stage on which the specimen is placed;
A microscope main body including an objective lens that irradiates the specimen with laser light emitted from a laser light source, and a scanning unit that scans the specimen with the laser light emitted by the objective lens;
A microscope stage that movably supports the microscope main body in a direction intersecting the optical axis of the objective lens with respect to the specimen on the specimen stage;
A detection unit for detecting a reaction of the specimen caused by irradiation with laser light;
A storage unit that stores brightness information corresponding to the reaction of the sample detected by the detection unit and scanning position information related to a position at which the laser beam is scanned by the scanning unit in association with each other;
Setting an observation range to be observed on the specimen, and dividing the observation range into a plurality of scannable ranges in which laser light can be scanned by the scanning unit with the microscope main body fixed to the stage And
The main scanning order of the irradiation areas to be scanned by the scanning unit is determined for each scannable range so that the irradiation areas of the laser beams irradiated continuously on the specimen are not spatially adjacent to each other. An order determination unit;
Prior to the main detection in which laser light is scanned in accordance with the main scanning order determined by the order determination unit, the microscope light body is sequentially moved by the microscope stage with respect to all the scannable ranges, and scanning in the main detection is performed. The simple detection in which light is applied to substantially the entire scannable range in a shorter time than the above, and the response of the specimen detected by the detection unit in the simple detection exceeds at least a predetermined threshold value. A laser scanning microscope comprising: a control unit configured to move the microscope main body again and scan the laser beam by the scanning unit according to the main scanning order with respect to a scannable range.   10. The laser scanning microscope according to claim 9, wherein the control unit performs the simple detection by causing the scanning unit to sequentially scan the irradiation areas spatially adjacent to each other with laser light. A wide-area illumination light source capable of irradiating illumination light at a time over substantially the entire scanable range;
The laser scanning microscope according to claim 9, wherein the controller performs the simple detection by irradiating the scannable range with illumination light by the wide range illumination light source.   12. The laser scanning type according to claim 11, further comprising an irradiation region limiting unit that limits illumination light applied to the scannable range by the wide-area illumination light source to other scannable ranges spatially adjacent to each other. microscope. The order determination unit determines an irradiation order of the scannable range in which illumination light is irradiated by the wide range illumination light source so that illumination light is not continuously irradiated onto the scannable ranges spatially adjacent to each other in time. And
The laser scanning microscope according to claim 11 or 12, wherein the control unit performs the simple detection by moving the microscope main body by the microscope stage in accordance with the irradiation order determined by the order determination unit. An image construction unit for constructing an image of the specimen;
The storage unit stores coordinate information related to the position coordinates of the microscope body in association with the luminance information and the scanning position information,
The image construction unit arranges the luminance information stored in the storage unit based on the scanning position information, constructs a two-dimensional image of the scannable range, and converts the image of the scannable range to the coordinates The laser scanning microscope according to claim 8, wherein a two-dimensional image of the observation range is constructed by arranging according to information.

Images