JP5718012B2 - Scanning laser microscope - Google Patents

Description

  The present invention relates to a scanning laser microscope.

  When it is desired to obtain a wide-range, high-resolution microscope image, a method is generally used in which a plurality of high-magnification partial images are acquired and then bonded together. However, since there is an error in the movement of the microscope stage, there is a possibility that duplication or loss of the observation area may occur if the adjacent partial images are simply connected. Therefore, a method is used in which a plurality of partial images are acquired while providing overlapping regions, and images are synthesized by performing pattern matching using the overlap between the partial images.

  There are two methods for setting the partial image observation area. In the first method, as shown in Patent Document 1, when the user designates the range of the attention area and the magnification of the objective lens, the overlapping area of the predetermined size is overlapped so as to cover the attention area without shortage. In this method, a plurality of partial image observation areas are set. The size of one partial image observation area is the size of the observation area at the specified magnification of the objective lens.

  The second method is a method in which when the user designates the number of partial images and the magnification of the objective lens, the designated number of partial image observation areas are set while providing overlapping areas, and each partial image is acquired. The size of one partial image observation area is the size of the observation area at the specified magnification of the objective lens.

Japanese Patent No. 3824116

  A problem that arises with the first method is that the number of partial images increases by the amount of overlap. For example, if the overlapping area of adjacent partial image observation areas is 20% of the size of one partial image observation area, the number of images is approximately 1.6 times that required when not overlapping. As a result, the time taken to capture all partial images increases. In many cases, the stage movement is more dominant than the image acquisition itself for the time required to acquire the images of a plurality of regions. That is, even if the size of the attention area is the same, if the number of images is large, the number of times the stage is moved increases, and the time for capturing all partial images increases. Also, if the number of images is large, the number of times that pattern matching is performed increases, so the time required for the image composition processing also increases.

  Further, in the second method, there is a problem in that the entire shooting range is reduced by the overlapping area, and the area requested by the user may be taken.

  The present invention has been made in view of the above-described circumstances, and an object of the present invention is to provide a scanning laser microscope capable of acquiring a wide range and a high resolution image at high speed without taking it.

In order to achieve the above object, the present invention employs the following means.
The present invention includes a scanning unit that two-dimensionally scans laser light on a specimen, an objective lens that focuses the laser light scanned by the scanning part on the specimen, and a light detection unit that detects light from the specimen. Based on the intensity of the light detected by the light detection unit and the scanning position information of the scanning unit, the enlarged part includes a partial image of the adjacent sample that does not overlap each other and is larger than the partial image. An enlarged partial image generation unit that generates an image with the same pixel resolution as the partial image, an enlarged partial image storage unit that accumulates the plurality of enlarged partial images generated by the enlarged partial image generation unit, and the enlarged partial image An image synthesis unit that synthesizes the plurality of enlarged partial images accumulated in the accumulation unit so as to partially overlap each other and generates an entire image of the specimen, and the enlarged partial image generation unit includes the scanning unit Scan range by By expanding, employing a laser scanning microscope that generates the enlarged partial image.

  According to the present invention, the laser light that is two-dimensionally scanned on the sample by the scanning unit is condensed on the sample by the objective lens, and the light from the sample is detected by the light detection unit. Then, the enlarged partial image generation unit generates an enlarged partial image with the same pixel resolution as the partial image of the sample based on the light intensity detected by the light detection unit and the scanning position information of the scanning unit. Here, the enlarged partial image is an image that includes partial images of adjacent specimens that do not overlap each other and is larger than the partial image. The plurality of enlarged partial images generated in this way are accumulated in the enlarged partial image accumulating unit, and synthesized by the image synthesizing unit so as to partially overlap each other, thereby generating an entire sample image.

  Here, when the partial images are combined so as to partially overlap each other as in the prior art, the imaging range is reduced by the overlapping area in the entire image. On the other hand, according to the present invention, an enlarged partial image larger than the partial image is synthesized so as to partially overlap each other to generate the entire sample image, thereby preventing a reduction in the shooting range of the entire image Therefore, the desired shooting range is not taken. In this case, since the enlarged partial image is generated with the same pixel resolution as that of the partial image, the entire image can be obtained without reducing the resolution.

In the above invention, the enlarged partial image generating unit, by extending the scanning range by the scanning unit, in a Turkey generate the enlarged partial image, to generate the magnified partial image at the same pixel resolution as the partial image The entire image can be acquired without reducing the resolution.

In the above invention, the enlarged partial image generation unit may generate the enlarged partial image by enlarging the partial image by half the size of the overlapping region.
By enlarging the partial images by half the size of the overlapping area, when multiple enlarged partial images are arranged later, the arrangement is the same as when the partial images before enlargement are arranged so as to touch each other. It becomes easy to understand how the enlarged partial images are arranged.

In the above invention, a stage for moving the sample relative to the objective lens in a direction orthogonal to the optical axis of the objective lens is provided, and the enlarged partial image generation unit is based on a movement error of the stage. The partial image may be enlarged and an overlapping area of the enlarged partial image may be set.
By using the movement error of the stage as a parameter for calculating the overlapping area, it is possible to capture images while reliably overlapping adjacent partial images, and to prevent the observation area from being lost in the entire image.

In the above invention, the enlarged partial image generation unit may calculate a movement error of the stage from a synthesis position obtained as a result of image synthesis.
In this way, the stage movement error can be calculated from the combined position obtained as a result of the image composition, and the size of the overlapping area can be calculated based on the stage movement error. An enlarged partial image can be acquired in the overlapping region.

In the above invention, a stage for moving the sample relative to the objective lens in a direction orthogonal to the optical axis of the objective lens is provided, and the image composition unit is configured to detect an image based on a movement error of the stage. It is good also as setting the range which searches a synthetic | combination position.
By setting the search range during image composition based on the stage movement error, it is possible to ensure that the correct composition position is included in the range where the composition position is searched, and to prevent the observation area from being lost in the entire image. be able to.

In the above invention, the image composition unit may perform image composition after removing an outer peripheral portion of the enlarged partial image in which light from the specimen has not reached the light detection unit.
By doing this, it is possible to generate an entire image by synthesizing only areas effective for image synthesis, except for areas where the light from the specimen did not reach the light detection unit. Defects can be prevented.

In the above invention, the image composition unit may adjust the size of the whole image.
In this way, the size of the entire image obtained by the image composition can be adjusted to a size desired by the user as one image, and the user can obtain the entire image of the desired size. it can.

In the above invention, a control unit is provided for controlling the scanning unit and the light detection unit so as to change a range in which the laser beam is irradiated and an imaging range on the specimen while maintaining the scanning range of the scanning unit. It is good.
In this way, even when it is difficult to change the scanning range halfway, such as when using a scanning unit that cannot easily change the scanning range, or when performing time-lapse observation at high speed, the laser beam The range for irradiating and the range for imaging can be changed.

  According to the present invention, there is an effect that it is possible to acquire a wide range and high resolution images at high speed without taking them.

1 is a block diagram illustrating a schematic configuration of a scanning laser microscope according to an embodiment of the present invention. It is a flowchart which shows the process performed by the scanning laser microscope which concerns on the 1st Embodiment of this invention. It is a figure explaining the relationship between the partial image observation area | region at the time of registration, the partial image observation area | region after an expansion, and an overlapping area | region. It is a figure which shows a state when the partial image observation area | region is arrange | positioned. It is a figure explaining the relationship between a partial image observation area | region and a visual field circle. It is a figure explaining the method of image composition. It is a flowchart which shows the process performed by the scanning laser microscope which concerns on the 2nd Embodiment of this invention.

[First Embodiment]
A scanning laser microscope 100 according to a first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, a scanning laser microscope 100 according to this embodiment includes a laser light source 1, a galvanometer mirror 2 (scanning unit), an objective lens 3, a specimen 4, an XY stage 5, and a dichroic mirror 6. A pinhole 7, a PMT 8 (light detection unit), and a PC 15.

The galvanometer mirror 2 scans the laser beam from the laser light source 1 two-dimensionally on the specimen 4 placed on the XY stage 5.
The objective lens 3 is disposed so as to face the specimen 4, condenses the laser light on the specimen 4 and illuminates the specimen 4.
The XY stage 5 relatively moves the specimen 4 and the objective lens 3 in a direction (XY direction) orthogonal to the optical axis of the objective lens 3.

The dichroic mirror 6 reflects the laser light from the laser light source 1 and transmits the observation light from the specimen 4. Thereby, the dichroic mirror 6 separates the laser light and the observation light from the specimen 4.
The pinhole 7 blocks light from the observation light transmitted through the dichroic mirror 6 from other than the focal point of the objective lens 3.
A PMT (Photomultiplier Tube) 8 detects observation light that has passed through the pinhole 7, converts it into an electrical signal, and outputs it to the PC 15.

  The PC 15 includes a CPU 9 (enlarged partial image generation unit, image synthesis unit, control unit), a memory 10, a hard disk 11 (enlarged partial image storage unit), a monitor 12, a mouse 13, and a keyboard 14. .

  The CPU 9 generates a partial image based on information from the PMT 8, gives a control command to the galvanometer mirror 2, the PMT 8, and the XY stage 5, and performs image composition from the partial images to generate an entire image.

Specifically, the CPU 9 generates an enlarged partial image with the same pixel resolution as the partial image of the sample 4 based on the intensity of the observation light from the sample 4 detected by the PMT 8 and the scanning position information of the galvanometer mirror 2. Here, as shown in FIG. 3, the enlarged partial image is an image that includes a partial image of the adjacent specimen 4 without overlapping each other and is larger than the partial image.
Further, the CPU 9 combines the plurality of enlarged partial images generated in this way so as to partially overlap each other as shown in FIG.

The memory 10 temporarily stores partial image acquisition conditions and partial image information.
The hard disk 11 stores the enlarged partial image generated by the CPU 9.
The monitor 12 displays an image acquisition condition setting screen and the entire image generated by the CPU 9.

The mouse 13 and the keyboard 14 allow the user to specify a partial image acquisition condition, a partial image observation area, and an attention area to be acquired as one image.
The user can perform various settings by operating the mouse 13 and the keyboard 14 while referring to the monitor 12.

The operation of the scanning laser microscope 100 having the above configuration will be described with reference to the flowchart of FIG.
First, a partial image observation area is set (step S1). The user operates the mouse 13 and the keyboard 14 while referring to the monitor 12 to set partial image acquisition conditions. Specifically, the magnification of the objective lens 3, zoom magnification, pixel resolution, image size, laser intensity, and PMT8 sensitivity are designated. In addition, the user moves the XY stage 5 to the center of a range desired to be acquired as one image, and registers the observation area at that time as a partial image observation area using the mouse 13 and the keyboard 14. The partial image acquisition conditions at the time of registration are stored in the memory 10. In the present embodiment, the pixel resolution is set to 1 μm / pixel and the image size is set to 512 pixels × 512 pixels as partial image acquisition conditions.

  Next, the attention area is set (step S2). The user designates the number of partial images to be expanded vertically and horizontally around the registered partial image observation area so that the entire range to be acquired as one image can be photographed using a mouse or a keyboard. In the present embodiment, as shown in FIG. 4, settings are made so that a total of nine partial images are acquired in a length of 3 and a width of 3.

  Next, the size of the overlapping area is calculated by the CPU 9 (step S3). The size of the overlapping area means the length of the short side of the rectangular area that overlaps between adjacent images. First, the number of pixels on one side of the overlapping area necessary for pattern matching is determined appropriately. If the numerical value is large, the accuracy of pattern matching becomes high, but considering that processing takes time, an appropriate numerical value is set. In this embodiment, it is set to 30 pixels.

  The maximum error in stage control is 10 μm. The size [μm] of the overlapping region is obtained by the maximum error [μm] of stage control + the number of pixels necessary for pattern matching × pixel resolution. In this embodiment, 10 + 30 × 1 = 40 [μm]. By calculating the overlapping area in this way, it is possible to reliably overlap the area corresponding to the number of pixels necessary for pattern matching in the acquired partial image.

Next, the partial image observation area is enlarged and arranged (steps S4 and S5).
Here, the expansion and arrangement of the partial image observation area will be described with reference to FIGS. 3 and 4. FIG. 3 shows the relationship between the partial image observation area 21 (partial image), the enlarged partial image observation area 22 (enlarged partial image), and the overlapping area 23 when registered in step S1. The shaded area is the overlapping area 23, and the length of the arrow indicates the size of the overlapping area. As shown in FIG. 3, the partial image observation area is enlarged by half the size of the overlapping area, and the overlapping area is set in the peripheral portion.

  FIG. 4 shows a state when nine partial image observation areas are arranged. Based on the number of vertical and horizontal partial images, the CPU 9 arranges the enlarged partial image observation areas 32 so that the overlapping areas 31 overlap, and calculates the stage coordinates of all the partial image observation areas. When the partial image observation area is enlarged so as to be half the size of the overlapping area and overlapped in the overlapping area in this way, the arrangement is the same as when the partial image observation areas 33 at the time of registration are arranged so as to contact each other. Thus, it is easy for the user to predict how the partial image observation area is arranged.

  Next, the CPU 9 moves the XY stage 5 to the stage coordinates of the first partial image observation area (step S6). As the order of acquiring the partial images, for example, a method of acquiring in order from the upper left to the lower right, or a method of acquiring from the center to the outside can be considered.

  Next, the CPU 9 sends a control command to the galvanometer mirror 2, the PMT 8, etc., and images the partial image observation area (step S7). As the image acquisition condition, the condition at the time of registering the partial image observation area stored in the memory 10 is used. The partial image obtained in this way is stored in the hard disk 11.

Next, it is determined whether or not all partial images have been acquired (step J1), and the processes in steps S6 to S7 are repeated until all partial images are acquired.
When all the partial images have been acquired, the CPU 9 reads the partial images from the hard disk 11 into the memory 10 (step S8).

  Next, from the size of one partial image observation area and the size of the field circle of the optical system, it is confirmed whether the partial image observation area is within the field circle (step J2). As shown in FIG. 5, if the partial image observation region 41 is larger than the field circle 42, the outer peripheral portion 43 of the partial image observation region where the observation light is expected not to reach the PMT 8 is cut off, and the next template matching is performed. It is made not to use (step S9).

Next, the CPU 9 performs pattern matching using the overlapping area, and synthesizes all partial images to generate an entire image (step S10).
Here, as shown in FIG. 6, an image composition method will be described by taking an example of pattern matching between a partial image A51 and a partial image B52 obtained by photographing a region immediately adjacent to the partial image A51. First, only the overlapping area of the partial image A51 or the partial image B52 is cut out. The overlapping area in this case is the same as the size of the overlapping area at the time of image acquisition. This time, an overlapping area of the partial image A51 is cut out and called a template image 53 (shaded portion in FIG. 6).

  Then, the template image 53 is moved on the partial image B52, and the similarity is calculated using the sum of squares of the differences, the cross-correlation coefficient, or the like. A known technique is used for the template matching algorithm. The search range 54 in which the template image 53 is moved is a range that is widened from the overlapping region 55 of the partial image B52 by left and right and up and down (maximum stage control error [μm] / pixel resolution). Is moved so that the template image 53 does not protrude, and the similarity is calculated at each location. The position with the highest similarity is defined as the composite position. If the search range at the time of pattern matching is set based on the movement error of the XY stage 5 in this way, the correct synthesis position is surely included in the search range 54, and the possibility of failure in image synthesis is reduced.

  Next, the CPU 9 makes the size of the entire image equal to the size of the attention area designated by the user (step S11). In the present embodiment, since the partial image acquisition condition is 512 pixels × 512 pixels and the number of partial images of 3 pixels in the vertical direction and 3 in the horizontal direction is specified, the surrounding area is cut off if it is excessive so that the entire image has an image size of 1536 pixels × 1536 pixels. By this processing, the user can obtain an entire image of the size requested at the time of registering the attention area.

As described above, according to the scanning laser microscope 100 according to the present embodiment, a wide range and high resolution image can be acquired, and the partial image observation area is enlarged, so that the area to be imaged is reduced by the overlapping area. And the user's requested range is not taken. Specifically, in this embodiment, since one side of the partial image is 512 μm and the number of partial images of 3 vertical and 3 horizontal is specified, it is guaranteed that one side of the entire image is 1536 μm. On the other hand, when image acquisition is performed by the conventional technique, the image is reduced by the overlap region (40 μm), so one side of the entire image is 1536−40 × 2 = 1456 [μm]. As a result, when the end of the observation object is cut, an image must be acquired again.
When an image is acquired at a high magnification or when the stage movement error is large and the ratio of the overlapping area to the partial image is increased, a more remarkable effect can be obtained.

  Also, by enlarging the partial image observation area by a size that is ½ of the overlapping area, when a plurality of partial image observation areas are arranged later, the partial image observation areas before enlargement are arranged so as to contact each other It becomes the same arrangement as the time, and it becomes easy for the user to understand how the partial image observation area is arranged.

Moreover, since the partial image observation area is enlarged while maintaining the pixel resolution, the entire image can be acquired without reducing the resolution.
Further, by using the stage movement error as a parameter for calculating the overlapping area, it is possible to ensure the overlapping area necessary for pattern matching, thereby reducing the possibility of image synthesis failure.

In addition, since the search range at the time of pattern matching is set based on the movement error of the stage, the correct synthesis position is surely included in the search range, and the possibility of failure in image synthesis is reduced.
In addition, since the region where the observation light does not reach the detection means that may occur when the partial image observation region is enlarged is excluded, the possibility of failure in image composition is reduced.
Further, since the size of the entire image is adjusted to be equal to the size of the attention area designated by the user, the user can obtain the entire image of the requested size.

[First Modification]
Below, the 1st modification of the scanning laser microscope 100 which concerns on this embodiment is demonstrated.
As a first modification of the scanning laser microscope 100 according to the present embodiment, when the user sets the attention area, the range of the attention area may be specified instead of specifying the number of partial images. In that case, the minimum necessary number of partial images is calculated from the size of the region of interest and the partial image acquisition conditions (the magnification of the objective lens 3 and the zoom magnification). For example, if the pixel resolution of the partial image acquisition condition is 1 μm / pixel, the image size is 512 pixels × 512 pixels, the size of the overlapping region is 40 μm, the size of the region of interest is 3000 μm in length, and 4000 μm in width, the length is 3000 / (512 × 1 + 40) is approximately 5.4, and the horizontal is 4000 / (512 × 1 + 40), which is approximately 7.2. Therefore, if a total of 48 partial images are arranged in a vertical direction of 8 and a horizontal size of 8, a region of interest can be captured without a shortage.

  In this way, in the method of designating the range of the attention area, if the enlargement of the partial image observation area is applied, the number of partial images is reduced and the number of movements of the XY stage 5 is reduced. The time to do can be reduced. Further, when the specimen 4 is fluorescently stained, the total number of overlapping regions is reduced, so that the region scanned with the laser beam twice, that is, the region where the fading of fluorescence is likely to proceed can be reduced.

[Second Modification]
Below, the 2nd modification of the scanning laser microscope 100 which concerns on this embodiment is demonstrated.
In the above-described embodiment, the generation of the partial image, the control of the galvano mirror 2, the PMT 8, and the XY stage 5 and the image synthesis process are performed by the CPU 9 of the same PC 15. However, in this modification, different PCs (PC-1, PC-2) may be used. For example, PC-1 in charge of generating partial images and controlling the galvanomirror 2, PMT8, and XY stage 5 and PC-2 in charge of image composition processing are connected by a network, and PC-2 is connected to PC-1. The entire image may be generated with reference to the partial image stored in the hard disk.

[Second Embodiment]
A scanning laser microscope 101 according to a second embodiment of the present invention will be described below with reference to the drawings.
In the case where the specimen 4 is fluorescently stained, fading of the overlapping region is likely to proceed, so it is preferable for the user to make the overlapping region as small as possible. In the first embodiment, the overlap area is calculated using the maximum error of the stage control. However, since the stage movement error varies depending on various conditions, the overlap area is actually reduced with a smaller error. Even if it is calculated, it is possible that the pasting will succeed. In particular, it is expected that the errors in acquiring images of the same region under the same conditions are all close values.

Therefore, in the second embodiment, in the case of performing time-lapse observation in which an image of the same region is acquired a plurality of times under the same conditions, the movement error of the stage obtained as a result of acquiring the partial images and performing the image synthesis is determined as the next partial image. It is reflected in the calculation of the overlap area at the time of acquisition. Since a cell may be deformed when observing a living cell, time-lapse observation of such a wide area is often performed.
Hereinafter, with respect to the scanning laser microscope 101 of the present embodiment, description of points that are common to the laser microscope apparatus 100 of the first embodiment will be omitted, and different points will be mainly described.

  The scanning laser microscope 101 according to the present embodiment has the same configuration as the scanning laser microscope 100 according to the first embodiment. As shown in FIG. 1, the laser light source 1 and the galvanometer mirror 2 ( A scanning unit), an objective lens 3, a specimen 4, an XY stage 5, a dichroic mirror 6, a pinhole 7, a PMT 8 (light detection unit), and a PC 15.

The operation of the scanning laser microscope 101 according to this embodiment will be described with reference to FIG.
First, a partial image observation area is set (step S21). The user operates the mouse 13 and the keyboard 14 while referring to the monitor 12 to set partial image acquisition conditions. Specifically, the magnification of the objective lens 3, the zoom magnification, the pixel resolution, the image size, the laser intensity, the sensitivity of the PMT 8, and the number of series of time lapse are designated. In addition, the user moves the XY stage 5 to the center of a range to be acquired as one image, and registers the current observation area as a partial image observation area using the mouse 13 and the keyboard 14. The partial image acquisition conditions at the time of registration are stored in the memory 10.

Next, similarly to the first embodiment, the attention area is set (step S22), and it is determined that the first series or the second series is selected (step J21).
First, the first series of processes will be described.
In the case of the first series of processing, the size of the overlapping area is calculated (step S23), the partial image observation area is enlarged (step S24), the partial image observation area is arranged (step S25), and the stage is moved (step S26). ), Acquisition and storage of partial images (step S27), determination of whether all images have been acquired (step J22), reading of partial images (step S28), determination of whether the partial image observation area is larger than the viewing circle (step S22) Step J23), removal of the outer peripheral portion of the partial image observation area (step S29), and image composition processing (step S30) are performed.
Since these processes are the same as those in the first embodiment, description thereof is omitted here.

  Next, it is determined that it is the first series (step J24), and the CPU 9 determines the difference between the composition position obtained by the image composition processing and the ideal composition position when there is no stage movement error (that is, The movement error of the XY stage 5 is calculated (step S31). For example, if there is a deviation of 2 μm in the X direction and 3 μm in the Y direction, the difference from the ideal synthesis position is approximately 3.6 μm. This calculation is performed between all adjacent partial images, and the maximum value is substituted into the maximum error of the stage control stored in the memory 10 or the hard disk 11.

Next, as in the first embodiment, the size of the entire image is adjusted (step S32).
The entire image is stored in the hard disk 11 (step S33). This completes the processing of the first series.

Next, the processing of the second series will be described.
First, the CPU 9 performs the calculation of the size of the overlapping area using the updated maximum error of the stage control (step S23).
Next, the CPU 9 performs enlargement of the partial image observation area (step S24) and arrangement of the partial image observation area (step S25) using the updated size of the overlapping area.

  Then, movement of the stage (step S26), acquisition and storage of the partial image (step S27), reading of the partial image (step S28), removal of the outer peripheral portion of the partial image observation area (step S29), and image composition processing (step S30) ), Adjustment of the size of the entire image (step S32), and storage of the entire image (step S33) are performed in the same manner.

  In the third and subsequent series, stage movement (step S26), acquisition and storage of partial images (step S27), determination of whether all images have been acquired (step J22), reading of partial images (step S28), partial images Judgment whether the observation area is larger than the field circle (step J23), removal of the outer peripheral portion of the partial image observation area (step S29), image composition processing (step S30), size adjustment of the whole image (step S32), whole image (Step S25) is repeated (step J25), and all series images are acquired.

  As described above, according to the scanning laser microscope 101 according to the present embodiment, the movement error of the XY stage 5 obtained by performing the image composition process is used for the next calculation of the size of the overlapping region. Partial images can be acquired with an overlap area of the minimum necessary size. Further, in the case where the specimen 4 is fluorescently stained, it is possible to reduce the area where the fading of the fluorescent light easily proceeds. Moreover, the damage by a laser can be reduced in the observation of a living cell.

[First Modification]
Below, the 1st modification of the scanning laser microscope 101 which concerns on this embodiment is demonstrated.
The movement error of the XY stage 5 is considered to strongly depend on the position of the XY stage 5 and the moving distance. Therefore, in this modification, when an image is acquired from the same area under the same conditions, an error may be calculated for each location and used for the next calculation of the size of the overlapping area. Thereby, a partial image can be acquired with a smaller overlapping area.

[Second Modification]
Below, the 2nd modification of the scanning laser microscope 101 which concerns on this embodiment is demonstrated.
In this modification, when the partial image observation area is enlarged in the second series and the sizes of the first series and the partial image observation area are changed, the laser beam is emitted while maintaining the driving range of the galvanometer mirror 2. The irradiation range and the imaging range may be changed. This eliminates the need to generate an electric signal for changing the drive range, and a resonant galvanometer mirror, which is difficult to change the drive range in the middle, can be used in place of the galvanometer mirror 2.

  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, and may be applied to embodiments in which these embodiments and modifications are appropriately combined, and is particularly limited. is not.

1 Laser light source 2 Galvano mirror (scanning part)
3 Objective Lens 4 Specimen 5 XY Stage 6 Dichroic Mirror 7 Pinhole 8 PMT (Photodetection Unit)
9 CPU (enlarged partial image generation unit, image composition unit, control unit)
10 memory 11 hard disk (enlarged partial image storage unit)
12 Monitor 13 Mouse 14 Keyboard 15 PC
100, 101 Scanning laser microscope

Claims (9)

A scanning unit for two-dimensionally scanning laser light on the specimen;
An objective lens that focuses the laser beam scanned by the scanning unit on the specimen;
A light detection unit for detecting light from the specimen;
Based on the intensity of the light detected by the light detection unit and the scanning position information of the scanning unit, the enlarged partial image includes a partial image of the adjacent sample that does not overlap each other and is larger than the partial image. An enlarged partial image generating unit that generates the same pixel resolution as the partial image,
An enlarged partial image accumulating unit for accumulating a plurality of the enlarged partial images generated by the enlarged partial image generating unit;
An image synthesis unit that synthesizes the plurality of enlarged partial images stored in the enlarged partial image storage unit so as to partially overlap each other, and generates an entire image of the specimen ;
The magnified partial image generating unit, by extending the scanning range by the scanning unit, the scanning laser microscope that generates the enlarged partial image. The scanning laser microscope according to claim 1, wherein the enlarged partial image generation unit generates the enlarged partial image by enlarging the partial image by half the size of the overlapping region. A stage for moving the specimen relative to the objective lens in a direction perpendicular to the optical axis of the objective lens;
The magnified partial image generating unit, based on the movement error of the stage, scanning laser microscope according to claim 1 or claim 2 for setting the overlap region of expansion and the enlarged image segments of the partial image. The scanning laser microscope according to claim 3 , wherein the enlarged partial image generation unit calculates a movement error of the stage from a synthesis position obtained as a result of image synthesis. A stage for moving the specimen relative to the objective lens in a direction perpendicular to the optical axis of the objective lens;
The scanning laser microscope according to any one of claims 1 to 4 , wherein the image composition unit sets a range for searching for an image composition position based on a movement error of the stage. The image synthesizing section, scanning according to claims 1 to perform image synthesis from excluding the peripheral portion of the enlarged partial image light is not received from the specimen to said light detector to claim 5 Laser microscope. The image synthesizing section, scanning laser microscope according to any one of claims 1 to 6 for adjusting the size of the entire image. The control part which controls the said scanning part and the said light detection part is provided so that the range which irradiates a laser beam on the said sample, and the range to image may be changed, maintaining the scanning range of the said scanning part. Item 8. The scanning laser microscope according to any one of Items 7 . The scanning laser microscope according to any one of claims 1 to 8 , wherein a user can set a size of a region of interest to be acquired as the entire image based on the partial image.

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