JP5901201B2 - Scanning laser microscope - Google Patents

Description

  The present invention relates to a scanning laser microscope.

A scanning laser microscope irradiates a specimen while scanning laser light two-dimensionally, detects transmitted light from the specimen, reflected light, or fluorescence generated in the specimen, and acquires brightness information. The image is formed for each pixel corresponding to the above to form an image and observe the specimen. In such a scanning laser microscope, a photo-multiplier tube (hereinafter simply referred to as “PMT”) is applied as a photodetector for acquiring luminance information (for example, Patent Document 1). reference).
The PMT may be deteriorated or damaged when excessive light is incident on its light receiving portion. Therefore, the PMT is provided with a protection circuit for preventing this, and the output from the PMT exceeds a certain value due to strong incident light. In this case, the protection circuit stops the operation of the PMT.

JP-A-2005-352100

However, in the above-described conventional PMT, the operation of the PMT is stopped when an output of a certain value or more is obtained for a predetermined time. This “predetermined time” is usually an instantaneous (for example, about 50 nsec) time. In practice, the operation of the PMT is instantaneously stopped when an output of a certain value or more is obtained from the PMT. Therefore, a desired image cannot be acquired, and there is a possibility that observation with a scanning laser microscope is hindered.
That is, when an output of a certain value or more is instantaneously obtained from the PMT, the operation of the PMT is stopped by the protection circuit even though the instantaneous output is less than the luminance information for one pixel as luminance information. The Therefore, the PMT operation is stopped not only by excessive light due to disturbance light, but also by, for example, fluorescence from a small amount of fluorescent reagent adhering to the surface of the specimen, so that a desired image cannot be obtained with a scanning laser microscope. May interfere with observation.

  The present invention has been made in view of the above-described circumstances, and is capable of acquiring a desired image and observing a sample satisfactorily while preventing damage to a photodetector due to excessive incident light. An object of the present invention is to provide a scanning laser microscope.

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 a sample with laser light from a light source, a light detection unit that detects light from the sample and outputs a light intensity signal corresponding to the luminance of the detected light, and the light A protection means for protecting the detection means; and an image generation means for generating an image of the sample by converting the light intensity signal into luminance for each pixel corresponding to a scanning position, and the protection means includes the light A threshold value determination unit that determines whether or not the intensity signal exceeds a threshold value, and a pixel counter that determines whether or not the light intensity signal determined to have exceeded the threshold value by the threshold value determination unit is output over a plurality of consecutive pixels. When it is determined that the light intensity signal determined to have exceeded the threshold value is output over a plurality of continuous pixels by the area determination unit and the area determination unit, the output by the light detection unit is stopped. , Above the threshold If it is not determined that the light intensity signal determined to have been output over a plurality of continuous pixels, generation of the sample image by the image generation unit is continued without stopping output by the light detection unit. There is provided a scanning laser microscope provided with a control unit that performs control.

  According to the present invention, a laser beam from a light source is two-dimensionally scanned on the sample by the scanning unit, and a light intensity signal corresponding to the luminance of the light detected by detecting the light from the sample is output by the light detection unit, A sample image is generated by converting the light intensity signal into luminance for each pixel by the image generation means. In this case, the threshold determination unit of the protection unit determines whether the light incident on the light detection unit is excessive by determining whether the light intensity signal exceeds the threshold. Furthermore, excessive light is detected over a predetermined region on the specimen by determining whether the light intensity signal determined to have exceeded the threshold is output over a plurality of continuous pixels. Can be determined. And when it determines with the control part having output the light intensity signal exceeding a threshold value over several continuous pixels, it controls to stop the output by a photon detection means.

  That is, when excessive light such as disturbance light is incident on the light detection means, a light intensity signal exceeding a threshold is obtained over a plurality of continuous pixels when generating an image. Control to stop the output to protect. On the other hand, even if a light intensity signal exceeding the threshold value is output instantaneously due to a minute drug or dust on the sample, the instantaneous light intensity signal does not reach a plurality of continuous pixels. Is not stopped. Therefore, it is possible to obtain a desired image and to observe the sample satisfactorily while preventing damage to the light detection means due to excessive incident light.

In the present invention described above, it is preferable that the control unit stops output from the light detection unit by stopping supply of a driving voltage for driving the light detection unit.
By doing in this way, the output of a photon detection means can be stopped simply and reliably and damage to a photon detection means can be prevented.

In the present invention described above, a shutter is provided in front of the light detection means and allows the light from the sample to be incident or blocked, and the control unit closes the shutter to block the light from the sample. It is preferable to stop the output by the light detection means.
By doing so, since the incidence of light on the light detection means is blocked, the output of the light detection means can be simply and reliably stopped to prevent damage to the light detection means.
In the present invention described above, the control unit includes a timer that measures a time during which the output of the light detection unit is stopped, and a determination unit that determines whether a predetermined time has elapsed. It is preferable that the operation of the light detection means is restored when the determination unit determines that the predetermined time has elapsed.

  In the present invention described above, a stimulus light source that irradiates a stimulus laser beam for applying light stimulus to the specimen, and the image while the stimulus laser light is radiated to the specimen by the stimulus light source. A luminance monitoring unit that calculates an average luminance of the image of the sample generated by the generation unit, and stops the irradiation of the stimulation laser beam by the light source when the average luminance is lower than a predetermined average luminance threshold; And when the control unit stops the output of the light detection unit, the control unit outputs a stop signal indicating that the output of the light detection unit is stopped to the luminance monitoring unit, and the luminance monitoring unit However, it is preferable to continue the irradiation of the stimulation laser beam based on the stop signal.

According to the present invention, in order to observe the behavior of the specimen, a light stimulus is given by irradiating the specimen with a stimulation laser beam from the light source for stimulation, and the specimen gradually fades when this light stimulation is continued. Since the luminance of the obtained image decreases, the luminance is monitored by appropriately calculating the average luminance of the sample image by the luminance monitoring means and comparing it with the average luminance threshold. In this case, if the output of the light detection means is stopped, the brightness of the image generated by the image generation means cannot be obtained. Therefore, there is a possibility that the brightness monitoring means erroneously stops the irradiation of the stimulation laser light as a sample fading. is there. For this reason, when the control unit of the protection unit outputs a stop signal indicating that the output of the light detection unit is stopped to the luminance monitoring unit, as a result, the average luminance falls below the average luminance threshold In addition, the irradiation of the stimulation laser beam is continued by the luminance monitoring means.
Therefore, it is possible to obtain a desired image and to observe the sample satisfactorily while preventing damage to the light detection means due to excessive incident light.

  According to the present invention, there is an effect that a desired image can be acquired and a sample can be favorably observed while preventing damage to the photodetector due to excessive incident light.

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 with the scanning laser microscope which concerns on embodiment of this invention. It is a flowchart which shows the process performed by the scanning laser microscope which concerns on the modification of embodiment of this invention.

[Embodiment]
A scanning laser microscope 100 according to an embodiment of the present invention will be described with reference to the drawings.
As shown in FIG. 1, a scanning laser microscope 100 according to this embodiment includes a light stimulation optical system 1, an observation optical system 2, a light detection unit 3, a controller 4, a stage 5, an objective lens 6, and light detection. A dichroic mirror 8 provided in front of the unit 3 is provided. In the figure, reference numeral 7 denotes a mirror.

The photostimulation optical system 1 includes a photostimulation light source 10 that emits photostimulation laser light, and a scanner 11 that two-dimensionally moves the position of the spot of the photostimulation laser light emitted from the photostimulation light source 10. And.
The observation optical system 2 returns from the observation light source 20 that emits the observation laser light, the scanner 21 that two-dimensionally scans the observation laser light emitted from the observation light source 20, and the specimen A through the scanner 21. And a dichroic mirror 22 for branching the fluorescence from the optical path of the observation laser beam.
The lenses in the optical stimulation optical system 1, the observation optical system 2, and the light detection unit 3 are not shown.

  The light detection unit 3 detects a light branched by the dichroic mirror 22 and controls a PMT (Photomultiplier Tube) 32 and a PMT 32 that outputs a current signal as a light intensity signal corresponding to the luminance of the detected light. The PMT controller (protection means) 33 is provided for protection according to the above conditions. In addition, a confocal pinhole 30 disposed at a position optically conjugate with the focal position of the objective lens 6 is disposed before the PMT 32 of the light detection unit 3. The PMT controller 33 includes a threshold determination unit 34, a region determination unit 35, and a PMT control unit 36.

  The threshold determination unit 34 determines whether or not the current signal of the PMT 32 exceeds the threshold. That is, the threshold value determination unit 34 stores a threshold value for the current signal from the PMT 32 in advance. When the current signal is obtained from the PMT 32, the obtained current signal is compared with the threshold value to determine whether the current signal exceeds the threshold value. Determine whether. The threshold value is preferably set to a value exceeding the rated current of the PMT to be applied.

  The region determination unit 35 determines whether or not the current signal determined to have exceeded the threshold by the threshold determination unit 34 is output over a plurality of continuous pixels. That is, when the current signal for each pixel forming the image exceeds a threshold value and the pixels are continuous with each other, the pixel corresponding to the current signal exceeding the threshold value is counted. When the counted number of pixels exceeds a predetermined pixel threshold, it is determined that the current signal determined to exceed the threshold is output over a plurality of consecutive pixels.

  The PMT control unit 36 stops the output of the PMT 32 when the region determination unit 34 determines that the current signal as the light intensity signal determined to exceed the threshold is output over a plurality of continuous pixels. To control. The PMT control unit 36 includes a timer (not shown) that counts the time during which the output of the PMT 32 is stopped. The PMT control unit 36 allows the PMT control unit 36 to count the PMT after a predetermined time has elapsed. Control to resume output.

  In order to stop the output of the PMT, for example, the supply of the drive voltage (HV) from a high voltage power source (not shown) that drives the PMT 32 can be stopped. In addition, a shutter is provided in front of the PMT 32 to allow the fluorescence returning from the specimen to enter or block, and this shutter is closed to block the fluorescence from the specimen A so that the fluorescence does not enter the PMT 32. Can be stopped.

  The controller 4 controls the optical stimulation optical system 1, the observation optical system 2, and the light detection unit 3, converts the current signal obtained from the light detection unit 3 into luminance, and corresponds to the scanning position of the scanner 21. A sample image is generated for each pixel.

  The stage 5 places the specimen A. The objective lens 6 is disposed so as to face the stage 5, and irradiates the specimen A with the light stimulation laser light and the observation laser light combined by the dichroic mirror 8 when the specimen A is placed on the stage 5. At the same time, the fluorescence returning from the specimen A is collected.

  In the scanning laser microscope 100 configured as described above, the specimen A is observed as follows. The observation light source 20 is operated to emit observation laser light, and the scanner 21 is operated to scan the observation laser light on the specimen A. That is, the observation laser light emitted from the observation light source 20 is reflected by the dichroic mirror 22, scanned two-dimensionally by the scanner 21, reflected by the dichroic mirror 8 and the mirror 7, and then sample A by the objective lens 6. Focused on top.

  On the other hand, the laser light for light stimulation emitted from the light source 10 for light stimulation is adjusted in two-dimensional position by the scanner 11, then transmitted through the dichroic mirror 8, reflected by the mirror 7, and sampled by the objective lens 6. Focused on A. As a result, the specimen A can be stimulated by irradiating the focal position of the objective lens 6 with the laser light for light stimulation.

  On the focal plane of the objective lens 6, the fluorescent substance in the specimen A is excited by the observation laser light, and fluorescence is generated. The generated fluorescence is collected by the objective lens 6, reflected by the mirror 7 and the dichroic mirror 8, returned through the scanner 21, transmitted through the dichroic mirror 22, reflected by the mirror 31, and then confocal pinhole. Only the signal that has passed through 30 is detected as a light intensity signal (current signal) by the PMT 32. The detected current signal is output to the controller 4 via the PMT controller 33 and stored in association with the scanning position of the observation laser beam by the scanner 21. In the controller 4, the current signal is integrated for each pixel corresponding to the scanning position of the scanner 21 to construct a two-dimensional image of the sample.

  Here, the PMT 32 is driven or controlled by the PMT controller 33, and when detecting the light intensity signal, the PMT controller 33 determines whether or not the PMT 32 should be protected based on the detected current signal.

Hereinafter, the operation of the PMT 32 and the PMT controller 33 will be described with reference to the flowchart of FIG.
When starting or resuming the detection of the current signal by the PMT 32, the count value of the number of pixels in the area determination unit 35 is reset (step S11). Subsequently, the incident fluorescence is detected in the PMT 32 and is output to the PMT controller 33 as a current signal as a light intensity signal. The threshold value determination unit 34 of the PMT controller 33 measures the current value of the input current signal (step S12), compares the measured current value with a predetermined threshold value, and determines whether or not the current value exceeds the threshold value. Is determined (step S13).

  As a result of the determination, if the current value does not exceed the threshold value, the process returns to step S11 and the area determination unit 35 resets the count value of the number of pixels again. On the other hand, when the current value exceeds the threshold value as a result of the determination, the threshold value determination unit 34 notifies the region determination unit 35 that the current value of the current signal has exceeded the threshold value. In response to the notification from the threshold determination unit 34, the region determination unit 35 increases the count value of the number of pixels (step S14).

  Subsequently, the area determination unit 35 compares the number of pixels whose count value has been increased with a predetermined pixel threshold value, and determines whether or not the number of pixels has exceeded the pixel threshold value (step S15). As a result of the determination, if the number of pixels does not exceed the pixel threshold value, the process returns to step S12, and measurement of the current value of the current signal from the PMT 32 is continued. On the other hand, if the number of pixels exceeds the pixel threshold as a result of the determination, the process proceeds to the next step S16, and the PMT controller 36 stops the supply of the drive voltage (HV) of the PMT 32 (HV = 0).

  When the output of the PMT 32 is stopped due to the stop of the supply of the drive voltage, the timer of the PMT control unit 36 is operated to measure the time during which the output of the PMT 32 is stopped (step S17). In the next step S18, it is determined whether or not a predetermined time has elapsed. This determination is continued until the time measured by the timer elapses a predetermined time, and when it is determined that the predetermined time has elapsed, the PMT control unit 36 resumes the supply of the drive voltage of the PMT 32. The PMT 32 restores its operation when the supply of the drive voltage is resumed, and again detects the light intensity signal (step S19).

  As described above, according to the scanning laser microscope 100 according to the present embodiment, the threshold value determination unit 34 determines whether or not the current value of the current signal exceeds the threshold value, so that the light incident on the PMT 32 is excessive. It is determined whether or not. Further, the region determination unit 35 detects excessive light over a predetermined region on the specimen A by determining whether the current signal determined to exceed the threshold is output over a plurality of continuous pixels. Can be determined. And when it determines with the electric current signal exceeding a threshold value being output over several continuous pixels, the PMT control part 36 stops the output by PMT32.

  That is, when excessive light such as disturbance light is incident on the PMT 32, a current signal exceeding a threshold value is obtained over a plurality of continuous pixels when generating an image. Control to stop. On the other hand, even if a current signal exceeding the threshold value is output instantaneously due to a minute drug or dust on the specimen A, the output of the PMT 32 is not stopped because the instantaneous current signal does not reach a plurality of continuous pixels. . Therefore, it is possible to obtain a desired image and observe the sample satisfactorily while preventing damage to the PMT 32 due to excessive incident light.

Even when the detection time (sampling time) of the signal current per pixel changes depending on the scanning speed of the scanner, the image resolution, the observation range, etc., when excessive light is detected for a plurality of predetermined pixels. Since only the output of the PMT 32 is stopped, the PMT can be protected easily and reliably.
The return of the PMT 32, that is, the resumption of output, may be realized by the user operating an operation interface such as a GUI for operating the scanning laser microscope 100, for example.

[Modification]
Hereinafter, as a modification of the scanning laser microscope 100 according to the above-described embodiment, a case where the controller 4 controls the optical system 1 for light stimulation by monitoring the luminance of the specimen A will be described.

  When observing the specimen A with the scanning laser microscope 100, in order to observe the behavior of the specimen, the stimulating light source 10 irradiates the specimen A with the stimulating laser light to give a light stimulus. If the operation is continued, the luminance of the image obtained by fading the specimen A gradually decreases. Therefore, the controller 4 calculates the average brightness of the generated image of the specimen A while irradiating the specimen A with the stimulation laser light, and when the average brightness falls below a predetermined average brightness threshold, The luminance of the image of the specimen A is monitored by controlling so as to stop the irradiation of the stimulation laser beam by (luminance monitoring function).

  Here, if the output of the PMT 32 is stopped for some reason, a light intensity signal related to the brightness of the specimen A cannot be obtained. Therefore, the brightness information is stored in the pixel corresponding to the scanning position by the scanner 21 after the output of the PMT 32 is stopped. It does not exist, and the average brightness of the generated image of the specimen A is significantly reduced.

  In the controller 4, it is difficult to specify whether the decrease in the average luminance of the image of the specimen A is caused by the stoppage of the output of the PMT 32 or the fading of the specimen A. There is a risk of stopping the irradiation of the laser beam. For this reason, when the output of the PMT 32 is stopped, the PMT control unit 36 outputs a stop signal indicating that the output of the PMT 32 is stopped to the controller 4. Similarly, when the PMT 32 restarts the output, the PMT control unit 36 outputs a restart signal indicating that the output is restarted to the controller 4.

  Therefore, in the controller 4, even when the average luminance of the image of the sample A is below the average luminance threshold, when the stop signal is input from the PMT control unit 36, the luminance is based on the stop signal. The monitoring function is stopped and the optical system 1 for light stimulation is controlled to continue irradiation with the laser light for stimulation. When a restart signal is input from the PMT control unit 36, the luminance monitoring function is operated, and the luminance of the generated image of the specimen A is monitored again.

  Hereinafter, the operation of the controller 4 during the specimen A observation in the scanning laser microscope according to the present modification will be described with reference to the flowchart of FIG.

  When irradiation of the light stimulation laser beam from the light stimulation light source 10 is started (step S21), the luminance monitoring function starts operating in the controller 4 (step S22). That is, the controller 4 calculates the average luminance of the generated image of the specimen A while the specimen A is irradiated with the stimulation laser light, and determines whether or not the average luminance exceeds a predetermined average luminance threshold. The determination is made at a predetermined cycle.

  While the controller 4 operates the brightness monitoring function to monitor the brightness of the image of the specimen A, the controller 4 determines whether or not a stop signal is input from the PMT control unit 36 (in parallel with this) ( Step S23) If a stop signal is input, the luminance monitoring function is stopped (step S24).

  When the luminance monitoring function is stopped, the controller 4 stops the calculation of the average luminance of the image of the specimen A, and whether or not a restart signal indicating that the output of the PMT 32 is restarted is input from the PMT control unit 36. Is determined (step S25). As a result of the determination, when a restart signal is input from the PMT control unit 36, the operation of the luminance monitoring function is restarted (step S26).

  By doing so, the controller 4 is prevented from erroneously stopping the irradiation of the stimulation laser beam as a discoloration of the specimen, and a desired image is obtained while preventing damage to the light detection means due to excessive incident light. It is possible to obtain and observe the specimen satisfactorily.

  In the above-described modification, the stop signal is output when the output of the PMT 32 is stopped and the restart signal is output when the output of the PMT 32 is restarted. The output timing can be determined as appropriate according to the response speed of the device to be applied. For example, the output timing can be configured prior to stopping or restarting the PMT 32.

In this embodiment, the laser scanning confocal microscope has been described as an example. However, an ultrashort pulse laser light source is used as an observation light source, the mirror 7 is replaced with a dichroic mirror, and the laser beam is branched by the dichroic mirror. Further, by arranging a PMT for detecting the fluorescence, it may be applied to a multi-photon excitation type laser scanning microscope.
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.

DESCRIPTION OF SYMBOLS 1 Optical system for light stimulation 2 Optical system for observation 3 Light detection part 4 Controller (Image generation means, brightness | luminance monitoring means)
5 Stage 6 Objective lens 7 Mirror 8 Dichroic mirror 10 Light source for light stimulation (light source for stimulation)
11 Scanner 20 Light source for observation 21 Scanner (scanning means)
22 Dichroic mirror 30 Pinhole 31 Mirror 32 PMT (light detection means)
33 PMT controller (protection means)
34 threshold determination unit 35 region determination unit 36 PMT control unit (control unit)
100 Scanning laser microscope

Claims (5)

Scanning means for two-dimensionally scanning the sample with laser light from a light source;
A light detection means for detecting light from the specimen and outputting a light intensity signal corresponding to the brightness of the detected light;
Protection means for protecting the light detection means;
Image generation means for converting the light intensity signal into luminance for each pixel corresponding to a scanning position and generating an image of the specimen;
The protective means is
A threshold determination unit that determines whether or not the light intensity signal exceeds a threshold;
An area determination unit having a pixel counter for determining whether the light intensity signal determined to exceed the threshold by the threshold determination unit is output over a plurality of continuous pixels;
When it is determined by the region determination unit that the light intensity signal determined to exceed the threshold is output over a plurality of continuous pixels, the output by the light detection unit is stopped, and it is determined that the threshold is exceeded. If it is not determined that the output light intensity signal is output over a plurality of continuous pixels, the image generation unit continues to generate the sample image without stopping the output of the light detection unit. A scanning laser microscope.   The scanning laser microscope according to claim 1, wherein the control unit stops output from the light detection unit by stopping supply of a driving voltage for driving the light detection unit. A shutter that is arranged in front of the light detection means and that allows the light from the sample to enter or be blocked;
The scanning laser microscope according to claim 1, wherein the control unit stops the output of the light detection unit by closing the shutter and blocking light from the specimen. A timer for measuring the time when the output of the light detection means is stopped by the control unit;
A determination unit that determines whether a predetermined time has elapsed from the time measured by the timer;
2. The scanning laser microscope according to claim 1, wherein the operation of the light detection unit is restored when the determination unit determines that the predetermined time has elapsed. A stimulation light source for irradiating a stimulation laser beam for applying a light stimulus to the specimen;
While the sample is irradiated with the stimulation laser beam by the stimulation light source, an average luminance of the sample image generated by the image generation unit is calculated, and the average luminance is a predetermined average luminance threshold value. A luminance monitoring means for stopping the irradiation of the stimulation laser beam by the light source when it falls below,
When the protection means stops the output of the light detection means, outputs a stop signal indicating that the output of the light detection means is stopped to the luminance monitoring means,
The scanning laser microscope according to any one of claims 1 to 3, wherein the luminance monitoring unit continues the irradiation of the stimulation laser beam based on the stop signal.

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