JP5185771B2 - microscope - Google Patents

Description

  The present invention relates to a microscope that performs observation using fluorescence and transmitted light.

Conventionally, a method using both fluorescence observation and transmission observation such as differential interference observation and phase difference observation is known (for example, see Patent Document 1). For example, in a laser scanning microscope (LSM), a fluorescence observation detector and a transmission observation detector are provided, and images of both fluorescence and transmitted light can be acquired simultaneously by a single scan of laser light. it can.
JP 9-138353 A

  However, the brightness of the fluorescent image in the LSM varies depending on the intensity of the laser light, the confocal pinhole, and the setting of the detector multiplication factor, and the brightness varies depending on the sample. In general, it is necessary to adjust the acquired image. However, when both the fluorescent image and the transmitted image are acquired simultaneously with the same laser light source, the brightness of the fluorescent image is adjusted by the intensity of the laser light. This inevitably changes the brightness of the transmitted image. Therefore, in accordance with the brightness adjustment of the fluorescent image, the multiplication factor of the transmission detector must be changed each time to adjust the brightness of the transmission image, resulting in inconvenience that the adjustment of the image becomes complicated. .

  The present invention has been made in view of the above-described circumstances, and an object of the present invention is to provide a microscope that can prevent fluctuations in the brightness of a transmitted image even when the brightness of a fluorescent image is adjusted. .

In order to achieve the above object, the present invention provides the following means.
The present invention provides an excitation light source that emits excitation light, an input unit that inputs an intensity setting of the excitation light, an illumination optical system that irradiates the sample with the excitation light having an intensity according to the intensity setting, and the sample A fluorescence detection optical system for detecting emitted fluorescence, a transmitted light detection optical system for detecting excitation light transmitted through the specimen, and an intensity setting input to the input means when the intensity setting is changed. A calculation unit that determines the detection sensitivity of the transmitted light detection optical system so that the brightness of the transmitted image obtained by the transmitted light detection optical system does not vary based on the value , and the transmitted light based on the determination of the calculation unit A microscope provided with a control part which adjusts detection sensitivity of a detection optical system .
Is adopted.

According to the present invention, the excitation light emitted from the excitation light source is irradiated onto the specimen by the illumination optical system, and the fluorescence emitted from the specimen is detected by the fluorescence detection optical system, while the excitation light transmitted through the specimen is transmitted light. It is detected by a detection optical system. Here, when the brightness of the fluorescence image detected by the fluorescence detection optical system is adjusted, the intensity of the excitation light emitted from the excitation light source is changed. In this case, based on the intensity of the excitation light emitted from the excitation light source, the control unit does not change the brightness of the transmitted image obtained by the transmitted light detection optical system regardless of the change in the intensity of the excitation light. The sensitivity of the transmitted light detection optical system is adjusted.

  As a result, even when the brightness of the fluorescent image is adjusted, the sensitivity of the transmitted light detection optical system is adjusted according to the intensity of the excitation light emitted from the excitation light source, so that it is detected by the transmitted light detection optical system. Variation in the brightness of the transmitted image can be prevented.

The present invention also provides an excitation light source that emits excitation light, an illumination optical system that irradiates the specimen with excitation light from the excitation light source, a fluorescence detection optical system that detects fluorescence emitted from the specimen, and the specimen A transmitted light detection optical system that detects excitation light transmitted through the excitation light source, and a control unit that adjusts the detection sensitivity of the transmitted light detection optical system based on the intensity of the excitation light emitted from the excitation light source. The optical detection optical system includes a photomultiplier tube, the voltage applied to the photomultiplier tube is V, the desired brightness of the transmission image detected by the photomultiplier tube is I, and the excitation light source emits the light. When the intensity of the excitation light is P, the multiplication coefficient of the photomultiplier tube is α, and the transmitted light detection sensitivity coefficient is β, the control unit applies the photomultiplier tube to the photomultiplier tube based on the equation (1). to adopt a microscope that controls the voltage V to be applied.
V = (I / βP) ^{1 / α} (1)

  By controlling the voltage applied to the photomultiplier tube based on the formula (1), the sensitivity of the photomultiplier tube can be easily adjusted, and the brightness of the transmission image detected by the photomultiplier tube can be adjusted. Variation can be effectively prevented.

Furthermore, the present invention provides an excitation light source that emits excitation light, an illumination optical system that irradiates the specimen with excitation light from the excitation light source, a fluorescence detection optical system that detects fluorescence emitted from the specimen, and the specimen A transmitted light detection optical system that detects excitation light transmitted through the excitation light source, and a control unit that adjusts the detection sensitivity of the transmitted light detection optical system based on the intensity of the excitation light emitted from the excitation light source. The light detection optical system includes a photoelectric conversion element, the exposure time of the photoelectric conversion element is τ, the desired brightness of the transmission image detected by the photoelectric conversion element is I, and the excitation light emitted from the excitation light source the intensity P, and the transmitted light detection sensitivity coefficient when and beta, wherein the control unit is to adopt a microscope that controls the exposure time τ of the photoelectric conversion elements on the basis of the equation (2).
τ = (I / βP) (2)

  By controlling the exposure time of the photoelectric conversion element based on the formula (2), it is possible to easily adjust the sensitivity of the photoelectric conversion element and effectively change the brightness of the transmitted image detected by the photoelectric conversion element. Can be prevented.

  In the above invention, the control unit is configured to detect the transmitted light detection sensitivity based on transmittances of the illumination optical system and the transmitted light detection optical system and sensitivity characteristics of the photomultiplier tube or the photoelectric conversion element. The coefficient may be determined.

In the above invention, the control unit may include a storage unit that stores a transmittance table associated with each inserted optical element.
In this way, even when there are a plurality of optical elements to be inserted, the control unit reads the transmittance associated with each inserted optical element from the storage unit, and the sensitivity coefficient of the transmitted light detection optical system Can be requested. Thereby, the sensitivity adjustment of the transmitted light detection optical system according to each optical element can be easily performed.
In the above invention, the image processing apparatus further includes an input unit that receives an input of a desired brightness of a transmission image obtained by the transmitted light detection optical system, and the calculation unit includes the input value of the excitation light intensity setting and the input transmission The detection sensitivity of the transmitted light detection optical system may be determined based on the input value of the desired brightness of the image.

  According to the present invention, it is possible to prevent fluctuations in the brightness of a transmitted image even when the brightness of a fluorescent image is adjusted.

[First Embodiment]
A laser microscope apparatus 1 according to a first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the laser microscope apparatus 1 according to the present embodiment includes an excitation light source 2 that emits excitation light, an illumination optical system 3 that irradiates the specimen A with excitation light from the excitation light source 2, and a specimen A. A fluorescence detection optical system 4 that detects fluorescence emitted from the light source, a transmitted light detection optical system 5 that detects excitation light that has passed through the specimen A, and a control device 6 that controls each part.

  The excitation light source 2 includes a first laser light source 11 that emits laser light having a wavelength λ1 as excitation light, a second laser light source 12 that emits laser light having a wavelength λ2, and the first laser light source 11 and the second laser light source 11. Mirrors 27 and 28 for deflecting the laser light emitted from the laser light source 12 are provided.

  The illumination optical system 3 includes a stage 19 on which the specimen A is placed, a dichroic mirror 13 that reflects the laser light from the excitation light source 2, and a scanner that two-dimensionally scans the laser light reflected by the dichroic mirror 13 on the specimen A. 14, lenses 15 and 16 that relay laser light scanned by the scanner 14, a mirror 17 that deflects laser light that has passed through the lenses 15 and 16, and a laser beam deflected by the mirror 17 is irradiated on the sample A On the other hand, an objective system 18 such as an objective lens that condenses the fluorescence generated in the specimen A is provided.

  The dichroic mirror 13 transmits the fluorescence condensed by the objective system 18 and passing through the lenses 15 and 16 and the scanner 14. Thereby, the dichroic mirror 13 branches the optical path of the fluorescence from the specimen A from the optical path of the laser light.

  The scanner 14 has a pair of galvanometer mirrors (not shown) coated with aluminum, for example, and is driven by a raster scan method by changing the angle of the galvanometer mirrors. Thereby, the laser beam from the excitation light source 2 can be scanned two-dimensionally on the specimen A.

  The fluorescence detection optical system 4 includes a lens 24 that collects fluorescence generated on the specimen A and transmitted through the dichroic mirror 13, a pinhole 25 that allows only fluorescence generated on the focal plane of the specimen A to pass, and a pinhole 25. A dichroic mirror 26 that disperses the fluorescence that has passed through, and a first PMT 21 and a second PMT 22 that detect the fluorescence dispersed by the dichroic mirror 26 are provided.

  The first PMT 21 and the second PMT 22 are, for example, photomultiplier tubes (PMT: Photo Multiplier Tube), which photoelectrically convert fluorescence dispersed by the dichroic mirror 26 and control the obtained light intensity signal. 6 is output.

The transmitted light detection optical system 5 includes a capacitor 20 that condenses the laser light emitted from the excitation light source 2 and transmitted through the specimen A, and a third PMT 23 that detects the laser light (transmitted light) collected by the capacitor 20. It has.
The third PMT 23 is, for example, a photomultiplier tube (PMT), which photoelectrically converts the transmitted light collected by the capacitor 20 and outputs the obtained light intensity signal to the control device 6. It has become.

The control device 6 includes, for example, an input / output device 38 that performs operations such as changing the intensity of laser light emitted by the excitation light source 2 and a control unit 30 that controls each unit.
The control unit 30 includes a laser control unit 31 that controls the first laser light source 11 and the second laser light source 12, a transmission image PMT control unit 32 that controls the third PMT 23, the first PMT 21 and the second PMT 21. The PMT control unit 33 for fluorescent image that controls the PMT 22, a calculation unit 34 that performs various calculations described later, and a storage unit 35 that stores parameters used by the calculation unit 34.

The calculation unit 34 adjusts the sensitivity of the third PMT 23 based on the intensity of the laser light emitted from the excitation light source 2.
Specifically, the calculation unit 34 calculates the voltage V applied to the third PMT 23 based on the following equation (1).
V = (I / βP) ^{1 / α} (1)
Here, V is a voltage applied to the third PMT 23, I is a desired brightness of a transmission image generated using the laser light detected by the third PMT 23, and P is a laser emitted from the excitation light source 2. The light intensity, α represents the multiplication factor of the third PMT 23, and β represents the sensitivity coefficient of transmitted light detection.

  Here, as in the present embodiment, a plurality of laser light sources that emit laser light are provided (first laser light source 11 and second laser light source 12), and laser light is emitted from these laser light sources simultaneously. , (1) is transformed into the following equation (1-1), and the voltage V applied to the third PMT 23 is calculated based on the equation (1-1).

In the expression (1-1), V is a voltage applied to the third PMT 23, I is a desired brightness of a transmission image generated using the laser light detected by the third PMT 23, and P (λ1) is The intensity of laser light emitted from the first laser light source 11, P (λ2) is the intensity of laser light emitted from the second laser light source 12, α is a multiplication factor of the third PMT 23, and β (λ1) Represents a transmitted light detection sensitivity coefficient corresponding to the wavelength λ1 of the first laser light source 11, and β (λ2) represents a transmitted light detection sensitivity coefficient corresponding to the wavelength λ2 of the second laser light source 12.
The transmitted light detection sensitivity coefficient includes the transmittance of the optical system (including the illumination optical system 3, the transmitted light detection optical system 5, and the optical system in the excitation light source 2) from the laser light sources 11 and 12 to the PMT 23, and the sensitivity characteristics of the PMT 23. To be determined.

The operation of the laser microscope apparatus 1 according to this embodiment configured as described above will be described below.
First, the specimen A stained with the fluorescent dye is placed on the stage 19 and the input / output device 38 is operated to output the outputs of the first laser light source 11 and the second laser light source 12 suitable for exciting the fluorescence. Are set as P (λ1) and P (λ2), respectively.
Similarly, by operating the input / output device 38, the sensitivity (voltage) of the first PMT 21 and the second PMT 22 is set to an appropriate value, and the desired brightness I of the transmission image to be acquired is input. .

  Then, the first laser light source 11 and the second laser light source 12 are operated to emit laser light. Laser light from the first laser light source 11 and the second laser light source 12 is reflected by the mirrors 27 and 28 and the dichroic mirror 13 and guided to the scanner 14. The scanner 14 scans the sample A two-dimensionally on the specimen A. The laser beam thus scanned passes through the lenses 15 and 16, is deflected by the mirror 17, and is irradiated onto the specimen A by the objective system 18.

  In the focal plane of the objective system 18 on the specimen A, the fluorescent substance in the specimen A is excited and fluorescence is generated. The generated fluorescence is collected by the objective system 18 and deflected by the mirror 17. The deflected fluorescence passes through the lenses 15 and 16 and the scanner 14, passes through the dichroic mirror 13, and is guided to the fluorescence detection optical system 4. The guided fluorescence is collected by the lens 24, and only the fluorescence generated in the focal plane of the specimen A passes through the pinhole 25. The fluorescence that has passed through the pinhole 25 is split by the dichroic mirror 26 into fluorescence (fluorescence image 1) by the laser light from the first laser light source 11 and fluorescence (fluorescence image 2) by the second laser light source 12. , Detected by the first PMT 21 and the second PMT 22, respectively.

  Thus, by storing the intensity information of the fluorescence detected by the first PMT 21 and the second PMT 22 and the irradiation position of the laser beam by the scanner 14 in association with each other, the two-dimensional fluorescence image 1 and the fluorescence image 2 are stored. Can be built.

On the other hand, the laser beam irradiated onto the specimen A by the objective system 18 and transmitted through the specimen A is condensed by the capacitor 20 and detected by the third PMT 23. Then, by storing the intensity information of the transmitted light detected by the third PMT 23 and the irradiation position of the laser light from the scanner 14 in association with each other, it is possible to construct a two-dimensional transmitted image.
Thus, the fluorescence image 1, the fluorescence image 2, and the transmission image are acquired simultaneously by one scan.

  At this time, while viewing the acquired image, the outputs of the first laser light source 11 and the second laser light source 12 and the sensitivities of the first PMT 21 and the second PMT 22 are repeatedly changed to obtain the fluorescence image 1. Adjustment is performed so that the fluorescent image 2 becomes an appropriate image.

  Here, the brightness of the fluorescent image 1 is determined by the intensity P (λ1) of the first laser light source 11 and the sensitivity of the first PMT 21. Similarly, the brightness of the fluorescent image 2 is determined by the intensity P (λ 2) of the second laser light source 12 and the sensitivity of the second PMT 22. On the other hand, the transmitted image has the intensity P (λ1) of the first laser light source 11, the intensity P (λ1) of the second laser light source 12, and the transmittance of the illumination optical system 3 and the transmitted light detection optical system 5. It is determined by T (λ1), T (λ2) and the sensitivity of the third PMT 23.

Therefore, for example, if the intensity of the first laser light source 11 is changed in order to adjust the brightness of the fluorescent image 1, the amount of light received by the third PMT 23 also changes.
Therefore, the calculation unit 34 includes the parameters α, β (λ1), β (λ2) stored in the storage unit 35 and the intensity values P (λ1), P of the intensity of the laser beam input by the input / output device 38. (Λ2) and the desired brightness I of the transmitted image are read out, and the required sensitivity of the third PMT 23 is calculated by the above-described equation (1-1). The transmission image PMT controller 32 receives the calculated applied voltage (sensitivity) V from the calculator 34 and changes the sensitivity of the third PMT 23.
Even when the intensity of the second laser light source 12 is changed to adjust the fluorescent image 2, the same processing is performed, and the sensitivity of the third PMT 23 is changed.

As described above, according to the laser microscope apparatus 1 according to the present embodiment, even when the brightness of the fluorescent image 1 and / or the fluorescent image 2 is adjusted, the intensity of the laser light emitted from the excitation light source 2 is increased. Accordingly, since the sensitivity of the third PMT 23 is adjusted, it is possible to prevent fluctuations in the brightness of the transmitted image generated using the transmitted light detected by the third PMT 23.
Further, the sensitivity of the third PMT 23 can be easily adjusted by controlling the voltage applied to the third PMT 23 based on the above-described equation (1-1).

The storage unit 35 may store a transmittance table associated with each inserted optical element.
By doing in this way, even when there are a plurality of optical elements to be inserted, the calculation unit 34 reads the transmittance associated with each optical element to be inserted from the storage unit 34 and sets the transmitted light detection sensitivity coefficient. Can be sought. Thereby, the sensitivity adjustment of the 3rd PMT23 according to the use condition of each optical element can be performed easily.

[Second Embodiment]
Next, a microscope apparatus according to a second embodiment of the present invention will be described with reference to the drawings.
The microscope apparatus 50 according to this embodiment is different from the first embodiment in that a CCD (photoelectric conversion element) is provided as means for detecting fluorescence and transmitted light. Hereinafter, with respect to the microscope apparatus 50 of the present embodiment, description of points that are common to the first embodiment will be omitted, and different points will be mainly described.

  As shown in FIG. 2, the microscope apparatus 50 according to the present embodiment includes an excitation light source 42 that emits excitation light, an illumination optical system 43 that irradiates the specimen A with excitation light from the excitation light source 42, and a specimen A A fluorescence detection optical system 44 that detects emitted fluorescence, a transmitted light detection optical system 45 that detects excitation light transmitted through the specimen A, and a control device 46 that controls each unit are provided.

The excitation light source 42 includes a light source 51 and an excitation filter 52 that extracts excitation light necessary for excitation of the specimen A from the light from the light source 51.
The illumination optical system 43 includes a stage 19 on which the specimen A is placed, and a capacitor 20 that irradiates the specimen A with excitation light from the excitation light source 42.

  The fluorescence detection optical system 44 and the transmitted light detection optical system 45 have, as common parts, an objective system 18 that collects the fluorescence and excitation light transmitted through the sample A, and a mirror 17 that deflects the fluorescence and excitation light transmitted through the sample A. And the lens 53 and the dichroic mirror 13 that transmits the fluorescence generated from the specimen A and reflects the excitation light that has passed through the specimen A.

The transmitted light detection optical system 45 includes a first CCD 61 that detects excitation light that is transmitted through the specimen A and reflected by the dichroic mirror 13.
The fluorescence detection optical system 44 includes a barrier filter 54 that transmits only the fluorescence generated in the specimen A, and a second CCD 62 that detects the fluorescence transmitted through the barrier filter 54.
The first CCD 61 and the second CCD 62 photoelectrically convert the detected excitation light and fluorescence and output the obtained light intensity signal to the control device 46.

The control device 46 includes an input / output device 78 and a control unit 70 that controls each unit.
The control unit 70 includes a filter control unit 71 that controls the transmittance of the excitation filter 52 and the barrier filter 54, a light source control unit 72 that controls the light source 51, and a transmission image CCD control unit 73 that controls the first CCD 61. , A fluorescent image CCD control unit 74 for controlling the second CCD 62, a calculation unit 75 for performing various calculations described later, and a storage unit 76 for storing parameters used by the calculation unit 75.

The computing unit 75 adjusts the sensitivity of the first CCD 61 based on the intensity of light emitted from the light source 51.
Specifically, the calculation unit 75 calculates the exposure time τ of the first CCD 61 based on the following equation (2).
τ = (I / βP) (2)
Here, τ is the exposure time of the first CCD 61, I is the desired brightness of the transmitted image detected by the first CCD 61, P is the intensity of light emitted from the light source 51, and β is the transmitted light detection sensitivity coefficient. Represents.

The operation of the microscope apparatus 50 according to this embodiment configured as described above will be described below.
First, the specimen A stained with a fluorescent dye is placed on the stage 19 and the input / output device 78 is operated to set the appropriate transmittances of the excitation filter 52 and the barrier filter 54 for exciting and transmitting the fluorescence.
Similarly, by operating the input / output device 78, the output of the light source 51 is set to an appropriate value, and the exposure time of the second CCD 62 is set to an appropriate value. Further, the desired brightness I of the transmission image to be acquired is input.

Then, the light source 51 is activated to emit light. Light from the light source 51 passes through the excitation filter 52 and is irradiated onto the specimen A by the capacitor 20.
The fluorescence generated on the specimen A and the excitation light transmitted through the specimen A are collected by the objective system 18 and dispersed by the dichroic mirror 13 through the mirror 17 and the lens 53.

  The excitation light reflected by the dichroic mirror 13 is detected by the first CCD 61, and a transmission image is generated. On the other hand, the fluorescence transmitted through the dichroic mirror 13 is detected by the second CCD 62, and a fluorescence image is generated.

At this time, while changing the output of the light source 51 and the exposure time of the second CCD 62 while watching the acquired image, adjustment is performed so that the fluorescent image becomes an appropriate image.
Here, in both the fluorescent image and the transmission image, the brightness of the image is determined by the output of the light source 51 and the exposure time of each CCD. Therefore, if the intensity of the light source 51 is changed to adjust the fluorescence image, the amount of light received by the second CCD 62 also changes.

  Therefore, the calculation unit 75, the transmitted light detection sensitivity coefficient β stored in the storage unit 76, the intensity P of the light emitted from the light source 51 input by the input / output device 78, and the desired brightness I of the transmitted image. And the required exposure time of the first CCD 61 is calculated by the above-described equation (2). The transmission image CCD control unit 73 receives the calculated exposure time τ from the calculation unit 75 and changes the exposure time of the first CCD 61.

As described above, according to the microscope apparatus 50 according to the present embodiment, even when the brightness of the fluorescent image is adjusted, the exposure time of the first CCD 61 depends on the intensity of the light emitted from the light source 51. Since the adjustment is made, fluctuations in the brightness of the transmission image generated by the first CCD 61 can be prevented.
Further, the brightness of the transmission image generated by the first CCD 61 can be easily adjusted by controlling the exposure time of the first CCD 61 based on the expression (2).

The storage unit 76 may store a transmittance table associated with each inserted optical element.
By doing in this way, even when there are a plurality of optical elements to be inserted, the calculation unit 75 reads out the transmittance associated with each optical element to be inserted from the storage unit 76 and sets the transmitted light detection sensitivity coefficient. Can be sought. Thereby, the exposure time of the first CCD 61 corresponding to each optical element can be easily adjusted.

As mentioned above, although each embodiment of the present invention has been described in detail with reference to the drawings, the specific configuration is not limited to this embodiment, and includes design changes and the like without departing from the gist of the present invention. .
For example, in each embodiment, since the fluorescent specimen is often almost transparent, it has been described that the transmittance of the specimen A is not considered. However, when the transmittance of the specimen A becomes a problem, a desired brightness is obtained. What is necessary is just to set I high.
As the excitation light intensity P, an intensity instruction value input from the input unit may be used, or a means for actually measuring the intensity of the excitation light may be provided, and a measurement value obtained therefrom may be used. In the transmitted light detection, differential interference microscopy or phase difference microscopy can be used. In this case, the transmitted light detection sensitivity coefficient β is assumed to reflect the transmittance of the optical element used in each microscopic method.

1 is a block diagram illustrating an overall configuration of a laser microscope apparatus according to a first embodiment of the present invention. It is a block diagram which shows the whole structure of the microscope apparatus which concerns on the 2nd Embodiment of this invention.

Explanation of symbols

A Specimen 1 Laser microscope device 2, 42 Excitation light source 3, 43 Illumination optical system 4, 44 Fluorescence detection optical system 5, 45 Transmitted light detection optical system 6, 46 Control device 11 First laser light source 12 Second laser light source 13 Dichroic mirror 18 Objective 19 Stage 20 Capacitor 21 First PMT
22 Second PMT
23 Third PMT
30, 70 Control unit 34, 75 Calculation unit 35, 76 Storage unit 38, 78 Input / output device 50 Microscope device 61 First CCD
62 Second CCD

Claims (6)

An excitation light source that emits excitation light;
Input means for inputting the intensity setting of the excitation light;
An illumination optical system that irradiates a sample with the excitation light having an intensity according to the intensity setting ;
A fluorescence detection optical system for detecting fluorescence emitted from the specimen;
A transmitted light detection optical system for detecting excitation light transmitted through the specimen;
When the intensity setting input to the input means is changed, the transmitted light detection optical system is controlled so that the brightness of the transmitted image obtained by the transmitted light detection optical system does not fluctuate based on the value of the intensity setting . A calculation unit for determining the detection sensitivity ;
A microscope provided with a control part which adjusts detection sensitivity of the transmitted light detection optical system based on determination of the operation part . An excitation light source that emits excitation light;
An illumination optical system for irradiating the specimen with excitation light from the excitation light source;
A fluorescence detection optical system for detecting fluorescence emitted from the specimen;
A transmitted light detection optical system for detecting excitation light transmitted through the specimen;
A controller that adjusts the detection sensitivity of the transmitted light detection optical system based on the intensity of the excitation light emitted from the excitation light source,
The transmitted light detection optical system includes a photomultiplier tube,
The voltage applied to the photomultiplier tube is V, the desired brightness of the transmission image detected by the photomultiplier tube is I, the intensity of the excitation light emitted from the excitation light source is P, and the photomultiplier tube When the multiplication factor of α is α and the transmitted light detection sensitivity coefficient is β,
Wherein the control unit is, (1) microscope that controls the voltage V to be applied to the photomultiplier on the basis of the equation.
V = (I / βP) ^{1 / α} (1) An excitation light source that emits excitation light;
An illumination optical system for irradiating the specimen with excitation light from the excitation light source;
A fluorescence detection optical system for detecting fluorescence emitted from the specimen;
A transmitted light detection optical system for detecting excitation light transmitted through the specimen;
A controller that adjusts the detection sensitivity of the transmitted light detection optical system based on the intensity of the excitation light emitted from the excitation light source,
The transmitted light detection optical system includes a photoelectric conversion element,
The exposure time of the photoelectric conversion element is τ, the desired brightness of the transmitted image detected by the photoelectric conversion element is I, the intensity of the excitation light emitted from the excitation light source is P, and the transmitted light detection sensitivity coefficient is β. If
Wherein the control unit, (2) microscope that controls the exposure time τ of the photoelectric conversion elements on the basis of the equation.
τ = (I / βP) (2)   The control unit determines the transmitted light detection sensitivity coefficient based on transmittances of the illumination optical system and the transmitted light detection optical system and sensitivity characteristics of the photomultiplier tube or the photoelectric conversion element. The microscope according to claim 2 or claim 3.   The microscope according to any one of claims 1 to 4, wherein the control unit includes a storage unit that stores a transmittance table associated with each inserted optical element. An input unit that receives an input of a desired brightness of a transmission image obtained by the transmitted light detection optical system;
  The calculation unit determines the detection sensitivity of the transmitted light detection optical system based on an input value of intensity setting of the excitation light and an input value of desired brightness of the input transmitted image. The microscope described.

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