JP6128862B2 - Microscope device and microscope system - Google Patents

Description

  The present invention relates to a microscope apparatus and a microscope system.

  Conventionally, a confocal scanning microscope (LSM) has been widely used as a life science research tool. In LSM, in many cases, a photomultiplier tube (PMT) is used as a detector (see, for example, Patent Document 1). The microscope apparatus described in Patent Document 1 deteriorates the sensitivity of the PMT when excessive light such as excitation light is incident on the PMT. Therefore, when a predetermined amount or more of excessive light is incident on the PMT, the voltage applied to the PMT ( HV) is set to zero to turn off the sensitivity of the PMT, thereby reducing the incidence of excessive light and preventing the sensitivity of the PMT from deteriorating.

JP 2004-069752 A

  However, since the microscope apparatus described in Patent Document 1 cannot detect fluorescence from the sample while the HV applied to the PMT is zero, observation data during that time is lost, which is desired. There is an inconvenience that it cannot be observed.

  An object of the present invention is to provide a microscope apparatus and a microscope system capable of observing a specimen while preventing deterioration of a detector without losing observation data.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a light source that emits laser light, a scanning unit that has a scanning mirror that can be swung around a predetermined rocking axis that scans the laser light emitted from the light source on a sample, and the laser that is scanned by the scanning unit. A light detection unit for detecting return light returning from the sample by scanning the light, and whether the amount of the return light incident on the light detection unit is greater than a predetermined threshold or not by the laser by the scanning unit When the incident light quantity determination unit that is determined during light scanning and the incident light quantity determination unit determine that the amount of the return light incident on the light detection unit is greater than a predetermined threshold value, The power and / or the voltage applied to the light detection unit is reduced by a predetermined amount, and the control for causing the scanning unit to scan the laser beam again from the initial position of the oscillation range of the scanning mirror is performed. Is the threshold Providing a microscope apparatus and a control unit which is repeated until the scanning of the laser beam is completed without being determined to be greater than.

  According to the present invention, when the laser beam is emitted from the light source, the laser beam is scanned on the sample by the scanning unit, and the return light returning from the sample is detected by the light detection unit. In this case, when the incident light amount determination unit determines that the amount of return light incident on the light detection unit is greater than a predetermined threshold, the control unit applies the laser light power and / or the light detection unit. By reducing the applied voltage by a predetermined amount, it is possible to prevent excessive light having a large light amount from being incident on the light detection unit any more.

In addition, the scanning unit scans the laser beam again from the initial position of the swing range of the scanning mirror while reducing the power of the laser beam and / or the voltage applied to the light detection unit by a predetermined amount. The desired range of the specimen can be re-observed by a laser beam with an appropriate power that does not cause deterioration of the photodetection unit and a photodetection unit with an appropriate sensitivity.
Therefore, even when the amount of the return light incident on the light detection unit changes significantly, the specimen can be observed while preventing deterioration of the light detection unit without losing observation data.

  In the above invention, the control unit changes the irradiation position of the laser beam irradiating the specimen in the optical axis direction, and the incident light amount determination unit performs the irradiation position different in the optical axis direction of the laser beam. According to the determination result, the power of the laser beam and / or the applied voltage applied to the light detection unit and the scanning of the laser beam by the scanning unit may be controlled.

  With this configuration, it is possible to prevent deterioration of the light detection unit due to the influence of excessive light, and to observe different observation positions in the depth direction of the specimen. As a result, the depth of the object being viewed is in mm units, as in the case of a Scale specimen with a transparent living body, and the brightness of the object being viewed according to the depth is different because the tissue differs for each depth. Even if it changes, the desired position in the depth direction can be observed without losing the observation data and preventing the photodetection unit from deteriorating.

  In the above invention, each time the control unit changes the irradiation position of the laser beam applied to the specimen in the optical axis direction, the power of the laser beam and / or the applied voltage applied to the photodetection unit is initialized. Returning to the value, the incident light quantity determination unit sets the initial value of the power of the laser beam and / or the applied voltage applied to the light detection unit by the control unit for each irradiation position different in the optical axis direction of the laser beam. It is good also as determining the light quantity of the said return light which injects into the said light detection part in the state returned to (2).

  With this configuration, even when the power of the laser light and / or the applied voltage applied to the light detection unit is reduced at a plurality of irradiation positions in the optical axis direction of the laser light, irradiation different in the optical axis direction of the laser light. It is possible to prevent the luminance information of the return light detected by the light detection unit from changing greatly for each position, and to observe the sample with the luminance information approximated to some extent.

  The present invention includes any one of the above-described microscope apparatuses, an image generation unit that generates an image of the specimen based on the intensity of the return light detected by the light detection unit, and the laser generated by the image generation unit Provided is a microscope apparatus system including an image processing unit that performs image processing so as to reduce variation in luminance variation between images with respect to images having different irradiation positions in the optical axis direction of light.

  According to the present invention, the power of the laser beam and the HV applied to the light detection unit are changed even when the amount of the return light incident on the light detection unit changes significantly at each observation position that differs in the depth direction of the specimen. Therefore, variation in luminance between images can be suppressed.

  According to the present invention, it is possible to observe a specimen while preventing deterioration of the detector without losing observation data.

1 is a schematic configuration diagram showing a microscope apparatus according to a first embodiment of the present invention. It is a flowchart explaining observation of the sample by the microscope apparatus of FIG. It is a flowchart explaining observation of the sample by the microscope apparatus which concerns on the modification of 1st Embodiment of this invention. It is a schematic block diagram which shows the microscope system which concerns on 2nd Embodiment of this invention. It is a graph which shows the state which prepared the balance of the brightness | luminance of the image of each observation position which differs in the depth direction of a sample.

[First Embodiment]
A microscope apparatus according to a first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, a microscope apparatus 10 according to the present embodiment includes a stage 1 on which a specimen is placed, a light source 3 that emits laser light, a scanner (scanning unit) 5 that scans the laser light on the specimen, An objective lens 7 that irradiates the sample with laser light scanned by the scanner 5 while condensing the fluorescence returning from the sample, and a detector (light detector) 9 that detects the fluorescence collected by the objective lens 7; An incident light quantity determination unit 11 that determines whether or not the return light including fluorescence incident on the detector 9 is larger than a predetermined threshold, and the light source 3, the scanner 5, and the detection based on the determination result by the incident light quantity determination unit 11. And a controller (control unit) 13 for controlling the device 9.

The light source 3 generates a laser beam having a power set by the controller 13.
The scanner 5 includes a pair of galvanometer mirrors (not shown, scanning mirrors) that can swing around a predetermined swing axis (not shown) that intersects each other. Each galvanometer mirror is disposed at a position optically conjugate with the pupil position of the objective lens 7. The scanner 5 can scan the laser beam two-dimensionally (X-axis direction and Y-axis direction) on the specimen by reflecting the laser light while the pair of galvanometer mirrors changes the swing angle. It has become.

  For example, a photomultiplier tube (PMT) is used as the detector 9. The detector 9 is configured to determine the sensitivity for detecting fluorescence according to the drive voltage (applied voltage, HV) applied by the controller 13. The return light incident on the detector 9 includes, for example, reflected light of the laser light reflected on the sample, in addition to fluorescence generated in the sample when the sample is irradiated with laser light.

  The incident light quantity determination unit 11 measures the light quantity of the return light incident on the detector 9 and compares it with a predetermined threshold value set in advance. The incident light amount determination unit 11 does not send determination information to the controller 13 when the light amount of the return light incident on the detector 9 is less than or equal to the threshold value, for example, and the light amount of the return light incident on the detector 9 is the threshold value. If it is determined that the return light is excessive light with a large amount of light, determination information indicating that the return light is excessive light is sent to the controller 13.

  When the determination information that the return light is excessive light is sent from the incident light amount determination unit 11, the controller 13 adjusts the power of the light source 3 to reduce the laser light power (laser power) by a predetermined amount. The drive voltage applied to the detector 9 is reduced by a predetermined amount to lower the sensitivity of the detector 9. The laser power reduction range and the drive voltage reduction range can be determined in advance and set in the controller 13. Further, the controller 13 returns the swing angle of the galvanometer mirror of the scanner 5 to the initial position when the determination information that the return light is excessive light is sent.

The operation of the microscope apparatus 10 configured as described above will be described with reference to the flowchart of FIG.
In order to observe a sample with the microscope apparatus 10 according to the present embodiment, first, the sample is placed on the stage 1, and the initial value of the drive voltage applied to the detector 9 and the initial power of the laser light generated from the light source 3 are detected. A value is set in the controller (step SA1).

  Next, laser light is generated from the light source 3 with the set power, the laser light is reflected while changing the swing angle of the pair of galvanometer mirrors of the scanner 5, and the sample is irradiated by the objective lens 7. Thereby, the laser beam is scanned two-dimensionally on the specimen according to the swing angle of each galvanometer mirror (step SA2).

  When fluorescence is generated by irradiating the sample with laser light, the fluorescence is collected by the objective lens 7, returns to the optical path of the laser light, and enters the detector 9 through the scanner 5. Thereby, the fluorescence is detected by the detector 9, and the luminance information is obtained. Therefore, for example, the specimen can be observed on the image by generating the specimen image based on the fluorescence luminance information obtained by the detector 9.

  In this case, the reflected light of the laser beam from the specimen and the like enter the detector 9 together with the fluorescence. When return light such as fluorescent light or reflected light of laser light enters the detector 9, the incident light quantity determination unit 11 measures the incident light quantity and compares it with a preset threshold value (step SA3).

  When the incident light amount determination unit 11 determines that the light amount of the return light is larger than the threshold value, determination information that the return light is excessive light is sent to the controller 13 (step SA3 “YES”). In the controller 13, when the determination information that the return light is excessive light is input from the incident light amount determination unit 11, the drive voltage applied to the detector 9 is reduced and the laser power of the light source 3 is reduced ( Step SA4). Then, the controller 13 returns the swing angle of each galvanometer mirror of the scanner 5 to the initial position of the swing range, and returns to step SA2.

  As a result, the sensitivity of the detector 9 is reduced, and the laser light is scanned again from the initial position of the swing range of each galvanometer mirror by the scanner 5 with the power of the laser light emitted from the light source 3 reduced. (Step SA2). Then, steps SA2 to SA4 are repeated until the laser beam is scanned over the entire scanning range of the sample without the incident light amount determination unit 11 determining that the return light is excessive light.

  When the incident light quantity determination unit 11 does not determine that the return light is excessive light and the entire scanning range of the sample is scanned with the laser light (step SA3 “NO”), the observation ends.

  As described above, according to the present embodiment, when the incident light amount determination unit 11 determines that the light amount of the return light incident on the detector 9 is larger than the predetermined threshold, the controller 13 controls the laser power and By reducing the driving voltage of the detector 9 by a predetermined amount, it is possible to prevent excessive light with a large amount of light from entering the detector 9 any more. In addition, when the laser power and the driving voltage of the detector 9 are reduced by a predetermined amount, the laser beam is scanned again from the initial position of the oscillating range of the galvanometer mirror by the scanner 5, so that the detector 9 is deteriorated due to excessive light. The desired range of the specimen can be re-observed with a laser beam having an appropriate power that does not occur and a detector 9 having an appropriate sensitivity.

  Therefore, even when the amount of return light incident on the detector 9 changes greatly, the specimen can be observed while preventing the detector 9 from being deteriorated without losing observation data.

This embodiment can be modified as follows.
In the present embodiment, the case where observation is performed at a certain depth without changing the observation position in the depth direction (Z-axis direction) (XY scan) has been described. While changing, it is good also as performing XY scan of this embodiment (XYZ scan) about each observation position (Z position) which differs in a depth direction.

  In this case, the stage 1 and / or the objective lens 7 are provided so as to be movable in the optical axis direction, and the controller 13 moves the stage 1 and / or the objective lens 7 in the optical axis direction to thereby move the laser light applied to the specimen. The irradiation position may be changed in the optical axis direction (Z-axis direction). Further, every time the controller 13 finishes scanning on a two-dimensional plane, the stage 1 and / or the objective lens 7 may be moved in the optical axis direction.

  The controller 13 can set a start position and an end position for switching the observation position of the specimen in the depth direction, and a number of steps for switching the observation position of the specimen in the depth direction.

  The controller 13 controls the power of the laser light and the drive voltage of the detector 9 according to the determination result by the incident light amount determination unit 11 for each irradiation position different in the optical axis direction of the laser light, that is, for each Z position. The laser beam scanning by the scanner 5 may be controlled.

In this modification, every time the controller 13 changes the irradiation position of the laser beam irradiated to the specimen in the optical axis direction, the laser beam power generated from the light source 1 and the drive voltage applied to the detector 9 are initialized. It is desirable to return to.
In addition, the incident light quantity determination unit 11 applies the detection light to the detector 9 in a state where the laser light power and the driving voltage of the detector 9 are returned to the initial values by the controller 13 for each irradiation position different in the optical axis direction of the laser light. It is desirable to determine the amount of incident return light.

Hereinafter, the operation of the microscope apparatus 10 according to this modification will be described with reference to the flowchart of FIG.
In order to observe the sample with the microscope apparatus 10 according to this modification, the sample is placed on the stage 1 and the initial value of the drive voltage applied to the detector 9 and the initial value of the power of the light source 3 are set in the controller 13. (Step SA1), the start position and end position for switching the specimen observation position in the depth direction, and the number of steps for switching the specimen observation position in the depth direction are set (step SB2, Z scan setting). ).

  Next, laser light is generated from the light source 3 with the set power, and XYZ scanning is started (step SB3). Since the XYZ scan has not been completed yet (step SB4 “NO”), the controller 13 moves the observation position of the specimen to the start position (first Z position) in the depth direction (step SB5), and the scanner 5 is moved. The XYZ scan is performed by swinging (step SB6).

  Laser light emitted from the light source 3 is irradiated onto the first Z position of the specimen through the scanner 5 and the objective lens 7, and the laser light is emitted on the first Z position of the specimen according to the swing angle of each galvanometer mirror. Scanned two-dimensionally. Fluorescence generated at the first Z position of the sample is incident on the detector 9 via the objective lens 7 and the scanner 5. Thereby, the luminance information of the fluorescence in the first Z position is obtained by the detector 9.

  In this case, the light quantity of the return light incident on the detector 9 is measured by the incident light quantity determination unit 11 and compared with a threshold value. If it is determined that the light quantity of the return light is larger than the threshold value (step SB7 “YES”). The determination information that the return light is excessive light is sent to the controller 13. Thereby, the drive voltage applied to the detector 9 by the controller 13 is reduced, and the laser power of the light source 3 is reduced (step SB8).

  Then, the controller 13 returns the swing angle of each galvanometer mirror of the scanner 5 to the initial position of the swing range (step SB9). Thereby, at the first Z position of the sample, the laser beam is scanned again from the initial position of the swing range of each galvanometer mirror by the scanner 5 (step SB6), and steps SB6 to SB9 are repeated.

  In step SB7, when the incident light amount determination unit 11 does not determine that the return light is excessive light and the entire scanning range at the first Z position of the sample is scanned with laser light (step SB7 “NO”), step SB7 is performed. Return to SB4.

Also in this case, since the XYZ scan is not completed (step SB4 “NO”), the position of the stage 1 and / or the objective lens 7 in the optical axis direction is adjusted by the controller 13, and the irradiation position of the laser beam on the specimen is deep. It is moved to the next step in the direction (referred to as the second Z position) (step SB5).
Steps SB6 to SB9 are repeated for the second Z position of the sample.
In this way, the observation position of the sample is switched in the depth direction by the number of steps set in advance in the controller 13, and Steps SB4 to SB9 are repeated at each Z position. When the XYZ scan is completed at the end position in the depth direction of the specimen observation position (step SB4 “YES”), the observation is finished.

  As described above, according to the microscope apparatus 10 according to this modification, the detector 9 can be prevented from being deteriorated due to the influence of excessive light, and a plurality of observation positions different in the depth direction of the specimen can be observed. . As a result, the depth of the object being viewed is in mm units, as in the case of a Scale specimen with a transparent living body, and the brightness of the object being viewed according to the depth is different because the tissue differs for each depth. Even if it changes, the desired position in the depth direction can be observed by preventing the detector 9 from deteriorating without losing the observation data.

[Second Embodiment]
Next, a microscope apparatus and a microscope system according to the second embodiment of the present invention will be described.
As shown in FIG. 4, the microscope system 100 according to the present embodiment includes a microscope apparatus 10 according to a modification example of the first embodiment and a sample intensity based on the fluorescence intensity detected by the detector 9 of the microscope apparatus 10. An image generation unit 20 that generates an image and an image processing unit 30 that performs image processing on an image at each Z position of the specimen are provided.
In the following, portions having the same configuration as those of the microscope apparatus 10 according to the first embodiment are denoted by the same reference numerals and description thereof is omitted.

The stage 1 can send position information regarding the position of the stage 1 in the optical axis direction to the image generation unit 20.
The scanner 5 can send the swing angle information regarding the swing angle of each galvanometer mirror to the image generation unit 20.
The detector 9 sends luminance information related to the detected fluorescence luminance to the image generation unit 20.

  The image generation unit 20 includes the position information of the stage 1 sent from the stage 1, the swing angle information of each galvanometer mirror sent from the scanner 5, and the luminance information of the fluorescence sent from the detector 9. Based on the above, a three-dimensional image of the specimen S is generated.

  The image processing unit 30 performs image processing on a plurality of images with different irradiation positions in the optical axis direction of the laser light generated by the image generation unit 20 so as to reduce variation in luminance variation between images. It is like that.

The operation of the microscope system 100 according to the present embodiment configured as described above will be described.
The process up to detecting the fluorescence from the specimen for each Z position by the detector 9 while switching the observation position of the specimen in the depth direction is the same as in the first embodiment, and thus the description thereof is omitted.
When the fluorescence is detected by the detector 9, the luminance information is sent to the image generation unit 20 together with the swing angle information of each galvanometer mirror of the scanner 5 and the position information of the stage 1 in the optical axis direction. Then, the image generation unit 20 generates a three-dimensional image of the specimen based on these pieces of information and sends it to the image processing unit 30.

In the image processing unit 30, the average luminance of the image of each frame for each Z position in the sample is calculated. Next, the image processing unit 30 performs a moving average process on the calculated average brightness data of each image to calculate the target brightness of each frame. Then, the image processing unit 30 performs the following processing on the frame data of each frame image for each Z position of the specimen.
Luminance value of each pixel of each frame x (target luminance / average luminance of frame)

  Thereby, as shown in FIG. 5, with respect to the series data obtained by the XYZ scan, the amount of change in the luminance of the data between the frames can be smoothed. In FIG. 5, the vertical axis indicates the luminance value, and the horizontal axis indicates each observation position (Z position) that differs in the depth direction of the sample.

  As described above, according to the microscope system 100 according to the present embodiment, even when the amount of the return light incident on the detector 9 greatly changes at each observation position that differs in the depth direction of the specimen, It is possible to suppress variations in luminance between images due to changing the power and the driving voltage of the detector 9.

  In this modification, the stage 1 is moved in the optical axis direction and a three-dimensional image is generated based on the position information of the stage 1. Instead, the stage 1 is fixed. Then, the objective lens 7 is moved in the optical axis direction, and a three-dimensional image of the specimen is generated based on the positional information regarding the position of the objective lens 7 in the optical axis direction, the swing angle information of each galvanometer mirror, and the fluorescence luminance information. It is good as well. Further, both the stage 1 and the objective lens 7 are moved in the optical axis direction, and a three-dimensional image of the sample S is obtained based on the position information, the swing angle information of the galvanometer mirrors, and the fluorescence luminance information. It is good also as producing | generating.

  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 each of the above embodiments, and may be applied to embodiments in which these embodiments are appropriately combined, and is not particularly limited.

  Further, for example, in each of the above embodiments, when it is determined that the amount of the return light incident on the detector 9 is larger than a predetermined threshold, both the laser light power and the drive voltage of the detector 9 are determined. Instead of this, instead of this, the controller 13 reduces at least one of the laser light power and the driving voltage of the detector 9 by a predetermined amount so that excessive light is incident on the detector 9 and detected. The device 9 may be prevented from deteriorating.

3 Light source 5 Scanner (scanning unit)
9 Detector (light detector)
DESCRIPTION OF SYMBOLS 10 Microscope apparatus 11 Incident light quantity determination part 13 Controller (control part)
20 Image generation unit 30 Image processing unit 100 Microscope system

Claims (4)

A light source that emits laser light;
A scanning unit having a scanning mirror capable of swinging around a predetermined swinging axis for scanning the sample with laser light emitted from the light source;
A light detection unit for detecting return light returning from the sample by the laser beam being scanned by the scanning unit;
An incident light amount determination unit that determines whether or not the light amount of the return light incident on the light detection unit is larger than a predetermined threshold value during scanning of the laser light by the scanning unit;
When the incident light amount determination unit determines that the amount of the return light incident on the light detection unit is larger than a predetermined threshold, the laser light power and / or the applied voltage applied to the light detection unit Is controlled by the scanning unit to scan the laser beam again from the initial position of the swing range of the scanning mirror without determining that the amount of the return light is larger than the threshold value. And a control unit that repeats until the scanning of light is completed .   The control unit changes the irradiation position of the laser light that irradiates the specimen in the optical axis direction, and for each irradiation position different in the optical axis direction of the laser light, according to the determination result by the incident light amount determination unit, 2. The microscope apparatus according to claim 1, wherein the power of the laser light and / or an applied voltage applied to the light detection unit and scanning of the laser light by the scanning unit are controlled. Each time the control unit changes the irradiation position of the laser beam that irradiates the specimen in the optical axis direction, the power of the laser beam and / or the applied voltage applied to the light detection unit is returned to the initial value,
The incident light quantity determination unit returns the power of the laser beam and / or the applied voltage applied to the light detection unit to the initial value by the control unit for each irradiation position different in the optical axis direction of the laser beam. The microscope apparatus according to claim 2, wherein the amount of the return light incident on the light detection unit in a state is determined. The microscope apparatus according to claim 2 or claim 3,
An image generation unit that generates an image of the specimen based on the intensity of the return light detected by the light detection unit;
A microscope comprising: an image processing unit that performs image processing so as to reduce variation in luminance variation between images with respect to images generated by the image generation unit with different irradiation positions in the optical axis direction of the laser light Equipment system.

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