JP6009837B2 - Laser microscope and photosensitivity extension method - Google Patents

Description

  The present invention relates to a method for extending the light receiving sensitivity of a laser microscope and a photodetector.

  Conventionally, a scanning laser microscope that performs real-time sensitivity correction of a multi-CH photodetector composed of a plurality of photodetectors is known (see, for example, Patent Document 1). The scanning laser microscope described in Patent Document 1 includes a spectroscopic element that divides fluorescence generated in a specimen into spectral components, and a plurality of light receiving elements that collectively detect the spectral components of the fluorescence dispersed by the spectroscopic elements. When there is a variation in the light receiving sensitivity of each light receiving element, the sensitivity variation of each light receiving element is corrected by a circuit-based correction method that changes the applied voltage of the multi CH light detector. I am going to do that.

JP 2006-58237 A

  However, the light receiving element that has deteriorated due to use does not return to its natural sensitivity after that, and if the deterioration progresses, the correction by changing the applied voltage exceeds the range that can be handled, and the light receiving sensitivity of the light receiving element is no longer available. There is a problem that cannot be corrected. Further, the sensitivity correction of the light receiving element by changing the applied voltage also amplifies the noise component, so that there is a problem that excessive correction becomes an obstacle to a clear image.

  An object of the present invention is to provide a laser microscope and a method for extending the light receiving sensitivity that can extend the light receiving sensitivity of the light receiving element and can be used for a long period of time.

In order to achieve the above object, the present invention provides the following means.
The present invention irradiates a sample with laser light emitted from a light source, collects return light returning from the sample, a photodetector having a light receiving element that receives the return light and performs photoelectric conversion, wherein the incident optical system for entering said return beam focused on the light receiving element of the photodetector by the objective lens, prior SL other of said return beam identical the light receiving element the incident position of the incident on the light-receiving element Provided is a laser microscope including an incident position changing unit that changes the position.

  According to the present invention, the return light returning from the sample by irradiating the sample with laser light emitted from the light source is collected by the objective lens and received by the light receiving element of the photodetector through the incident optical system. Is done. Then, the return light is photoelectrically converted by the light receiving element, whereby the sample image information can be acquired and the sample can be observed.

  Here, if the light receiving element continues to receive light, the composition may be destroyed at the incident position of the light and the light receiving sensitivity may be deteriorated. However, the range necessary for receiving light is the light in the light receiving element. It is a part of the entire incident range. Therefore, by changing the incident position of the return light incident on the light receiving element by the incident optical system by the incident position changing unit, it is possible to effectively use an unused range in which the light receiving element is not deteriorated. As a result, the light receiving sensitivity of the light receiving element can be extended and used for a long time. For example, FIG. 5 is a diagram showing a change in luminance value when the relative position of the light receiving surface is moved in a direction perpendicular to the spectral dispersion direction in the light receiving element, but the light receiving sensitivity in the light receiving element is uniform. It can be seen that the error is at a level that does not cause a problem even if the incident position of the return light is changed.

  In the above invention, a spectroscopic element that divides the return light collected by the objective lens into a spectral component is provided, and the incident position changing unit determines the incident position of the return light in the light receiving element as a spectral component. It is good also as changing to the direction orthogonal to a direction.

With this configuration, it is possible to detect the spectral component of the return light dispersed by the spectroscopic element and detect the amount of return light for each wavelength. In this case, the incident position changing unit effectively utilizes the unused range existing in the direction orthogonal to the spectral direction of the spectral component within the range in which the return light from the light receiving element can be incident. Sensitivity can be extended.
The present invention relates to an objective lens that irradiates a sample with laser light emitted from a light source and collects return light returning from the sample, and a spectroscopic element that splits the return light collected by the objective lens into spectral components A photodetector having a light receiving element that receives the return light and performs photoelectric conversion, an incident optical system that causes the return light collected by the objective lens to enter the light receiving element of the photodetector, and An incident position changing unit that changes an incident position of the return light incident on the light receiving element, and the incident position changing unit is configured to make the incident position of the return light in the light receiving element perpendicular to the spectral direction of the spectral component. A laser microscope to be changed to is provided.

Moreover, in the said invention, the said photodetector is good also as providing the said several light receiving element arranged along the spectral direction of the said spectral component by the said spectroscopic element.
With this configuration, it is possible to make the return light split into spectral components by the spectroscopic elements incident on a plurality of light receiving elements without waste and to selectively detect a desired spectral component at one time by these light receiving elements. it can. Thereby, spectral detection can be performed quickly and efficiently.

  Moreover, in the said invention, the said incident position change part is good also as functioning as an optical path switching part which switches the optical path of the return light split into the said spectral component by the said spectroscopic element into two optical paths from which optical path length differs. .

  With this configuration, the optical path switching unit can change the spectral resolution to be incident on the photodetector by changing the focal length of the return light that has been spectrally divided into spectral components by the spectroscopic element. In this case, since the incident position changing unit functions as an optical path switching unit, it is not necessary to provide a special mechanism for switching the optical path of the return light, and the apparatus can be simplified.

Moreover, in the said invention, the said incident position change part is good also as changing the incident position of the said return light in the said light receiving element at the time of starting of the said light source.
By configuring in this way, it is possible to prevent light from being returned to a certain position in the light receiving element and receiving light, and to extend the light receiving sensitivity of the light receiving element more efficiently.

  Further, in the above invention, a sensitivity measuring unit that measures the light receiving sensitivity for each of the light receiving elements, the light receiving sensitivity for each of the light receiving elements measured by the sensitivity measuring unit, and the incident position of the return light in the plurality of light receiving elements And the difference between the light receiving sensitivity for each of the light receiving elements stored in the storage unit and the light receiving sensitivity for each of the light receiving elements newly measured by the sensitivity measuring unit is measured. A measuring unit, and when the difference measured by the measuring unit is larger than a predetermined threshold, the incident position changing unit changes the incident position of the return light in the plurality of light receiving elements, and the storage unit Is the incident position of the return light in the plurality of light receiving elements changed by the incident position changing unit and the reception of each light receiving element measured by the sensitivity measuring unit at the incident position. It may be stored in association with sensitivity.

  With this configuration, when the difference in the light receiving sensitivity of each light receiving element measured by the measuring unit increases, that is, when the light receiving sensitivity of the light receiving element becomes uneven, the deterioration in the light receiving element. It is possible to prolong the light receiving sensitivity of the light receiving element by effectively utilizing the unused range. In addition, the detector is used efficiently over a long period of time by updating the light receiving sensitivity for each light receiving element that is measured by the sensitivity measurement unit and stored in the storage unit each time the incident position of the return light in the light receiving element is changed. can do.

Moreover, in the said invention, the said sensitivity measurement part is good also as measuring the light reception sensitivity of the said light receiving element using the reflected light from the reflective mirror arrange | positioned in the irradiation position of the said laser beam by the said objective lens.
By comprising in this way, the light reception sensitivity for every light receiving element can be measured easily.

Moreover, in the said invention, it is good also as providing the switching mechanism which can insert / remove the folding | turning member which folds back this laser beam on the optical path of the said laser beam.
With this configuration, the sensitivity measurement unit receives light only by placing the folding member on the optical path of the laser light by the switching mechanism without taking the trouble of arranging the reflection mirror at the irradiation position of the laser light by the objective lens. The light receiving sensitivity for each element can be measured.

In the invention described above, the switching mechanism may selectively dispose an optical element that causes the laser light to enter the objective lens and the folding member in an optical path of the laser light.
With this configuration, the switching mechanism switches between the measurement of the light receiving sensitivity of the photodetector and the observation of the sample only by switching whether the folding member or the optical element is arranged on the optical path of the laser beam. be able to.

  In the above invention, the scanning unit that reflects the laser beam emitted from the light source and scans the sample, and the position that is out of the optical path of the laser beam between the scanning unit and the objective lens And a folding member that is capable of folding the laser beam, and the scanning unit may reflect the laser beam toward the folding member.

  With this configuration, the scanning range of the laser light on the sample can be observed by causing the scanning unit to scan the sample with the laser light. In this case, the detection sensitivity of the light receiving element can be easily measured by simply reflecting the laser beam toward the folded member by the scanning unit.

  According to the present invention, a laser beam is emitted from a light source and irradiated on a specimen so that return light returned from the specimen is incident on a light receiving element of a photodetector, and the specimen is observed by receiving the return light by the light receiving element. A method for extending the light receiving sensitivity of the light receiving element used in a laser microscope, wherein when the light receiving sensitivity of the light receiving element deteriorates, the incident position of the return light in the light receiving element is changed to a different position. I will provide a.

  According to the present invention, by changing the incident position of the return light in the light receiving element of the photodetector to a different position, it is possible to effectively use an unused range that has not deteriorated in the light receiving element. Therefore, the light receiving sensitivity of the light receiving element can be extended and used for a long time.

  According to the present invention, there is an effect that the light receiving sensitivity of the light receiving element can be extended and used for a long time.

1 is a schematic configuration diagram illustrating a laser microscope according to a first embodiment of the present invention. It is a top view which shows the photodetector of FIG. It is a schematic block diagram which shows the laser microscope which concerns on 2nd Embodiment of this invention. It is a top view which shows the photodetector of FIG. When the relative position of the light receiving surface is moved (horizontal axis) in the direction perpendicular to the spectral dispersion direction (spectral direction of spectral components) in the light receiving element of the photodetector in FIG. It is the figure which showed the change of a luminance value (vertical axis). It is a schematic block diagram which shows the laser microscope which concerns on 3rd Embodiment of this invention. It is a schematic block diagram which shows the laser microscope which concerns on the 1st modification of 3rd Embodiment of this invention. It is a schematic block diagram which shows the laser microscope which concerns on the 2nd modification of 3rd Embodiment of this invention. It is a schematic block diagram which shows the laser microscope which concerns on the 3rd modification of 3rd Embodiment of this invention. It is a perspective view which shows the turret of FIG. It is a schematic block diagram which shows the laser microscope which concerns on the 4th modification of 3rd Embodiment of this invention.

[First Embodiment]
A laser microscope and a light receiving sensitivity extending method 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 100 according to the present embodiment reflects a stage 1 on which a sample S is placed, a laser unit 10 that emits laser light, and laser light emitted from the laser unit 10. An illumination optical system 20 having a scanning unit 23 that scans on the specimen S; an objective lens 5 that irradiates the specimen S with laser light from the illumination optical system 20 and collects fluorescence (return light) generated in the specimen S; , A detection optical system 30 having a detector (light detector) 45 for detecting fluorescence condensed by the objective lens 5, and a control unit (incident position changing unit) 7 for controlling the detection optical system 30. . In FIG. 1, reference numeral 3 indicates a reflection mirror that reflects the laser light from the illumination optical system 20 toward the objective lens 5.

  The laser unit 10 includes a laser light source 11 that generates laser light, and an AOTF (Acousto-Optic Tunable Filter) 13 that controls wavelength selection and intensity adjustment of the laser light generated by the laser light source 11. . The laser unit 10 can emit laser light having a predetermined intensity in a predetermined wavelength range by generating laser light from the laser light source 11 and controlling the wavelength selection and intensity adjustment by the AOTF 13. Yes.

  The illumination optical system 20 reflects a laser beam incident from the laser unit 10 toward the scanning unit 23, while scanning an excitation dichroic mirror (excitation DM) 21 that transmits fluorescence from the sample S and a galvano mirror. , A pupil projection lens (PL) 25 that condenses the laser light reflected by the scanning unit 23, and an imaging lens (TL) 27 that collimates the laser light collected by the pupil projection lens 25. ing.

  The scanning unit 23 includes, for example, a pair of galvanometer mirrors (not shown) arranged close to each other. Each of the pair of galvanometer mirrors is provided so as to be swingable about a swing axis that intersects the optical axis of the laser beam. The scanning unit 23 can deflect the laser light in accordance with the swing angle of the pair of galvanometer mirrors.

  In addition to the detector 45, the detection optical system 30 includes a condenser lens 31 that condenses the fluorescence transmitted through the excitation dichroic mirror 21, and a pinhole 33 that allows a part of the fluorescence condensed by the condenser lens 31 to pass therethrough. The reflection mirrors 35 and 37 that reflect the fluorescence that has passed through the pinhole 33, the condenser lens 39 that collects the fluorescence reflected by the reflection mirror 37, and the fluorescence that is collected by the condenser lens 39 are detected by the detector 45. An incident position switching mirror (incident optical system) 41 that reflects toward the light source, and a reflection mirror 43 that reflects the fluorescence from the incident position switching mirror 41 and makes it incident on the detector 45.

  As the detector 45, for example, a side-on type PMT (Photomultiplier Tube) as shown in FIG. 2 or a head-on type PMT (not shown) is used. The detector 45 includes a light receiving element 46 that receives fluorescence and performs photoelectric conversion.

  The pinhole 33 has a pinhole diameter of 50 to 800 um, for example. The pinhole 33 is arranged at a position conjugate with the focal position of the objective lens 5 so that only the fluorescence generated at the focal position of the objective lens 5 in the sample S is allowed to pass through.

  The incident position switching mirror 41 can change the angle around an axis that intersects the optical axis of fluorescence. The incident position switching mirror 41 can change the incident position of the fluorescence incident on the light receiving element 46 of the detector 45 in the direction along the optical axis by changing the angle around the axis.

  The control unit 7 changes the angle around the axis of the incident position switching mirror 41 in accordance with an instruction from a user input to an input unit (not shown). Thereby, the control unit 7 can change the incident position of the fluorescence incident on the light receiving element 46 of the detector 45 according to the angle of the incident position switching mirror 41. The incident position of the fluorescence in the light receiving element 46 may be changed to a predetermined position, or may be changed to a position determined at random when the laser microscope 100 is activated.

A method for extending the light receiving sensitivity life according to the present embodiment will be described.
In the light receiving sensitivity extending method according to the present embodiment, when the light receiving sensitivity of the light receiving element 46 of the detector 45 is deteriorated, the user inputs an incident position switching instruction to the input unit, and the control unit 7 controls the incident position switching mirror 41. By changing the angle around the axis, the incident position of the fluorescence in the light receiving element 46 of the detector 45 is changed to a different position.

The operation of the thus configured laser microscope 100 and light receiving sensitivity life extension method will be described.
In order to observe the specimen S with the laser microscope 100 according to the present embodiment, first, the specimen S is placed on the stage 1 and laser light having a predetermined intensity is emitted from the laser unit 10 in a predetermined wavelength range.

  The laser light emitted from the laser unit 10 is reflected by the excitation dichroic mirror 21, then reflected by the scanning unit 23, and condensed by the pupil projection lens 25. Then, the laser light is collimated by the imaging lens 27 and irradiated on the sample S by the objective lens 5 through the reflection mirror 3.

  When fluorescence is generated in the specimen S by being irradiated with the laser light, the fluorescence is collected by the objective lens 5, and the fluorescence of the laser light is transmitted through the reflection mirror 3, the imaging lens 27, the pupil projection lens 25, and the scanning unit 23. It returns along the optical path, passes through the excitation dichroic mirror 21, and is separated from the laser light.

  The fluorescence transmitted through the excitation dichroic mirror 21 is collected by the condenser lens 31, and only the fluorescence generated at the focal position of the objective lens 5 in the specimen S passes through the pinhole 33. The fluorescence that has passed through the pinhole 33 is reflected by the incident position switching mirror 41 via the reflection mirrors 35 and 37 and the condenser lens 39, and is incident on the detector 45 via the reflection mirror 43. In the detector 45, the fluorescence is received by the light receiving element 46 and photoelectrically converted. Thereby, the image generation part (not shown) can acquire the image information of the sample S and observe the sample.

Here, if the light receiving element 46 continues to receive the fluorescence, the composition may be destroyed at the fluorescence incident position and the light receiving sensitivity may be deteriorated.
When the light receiving sensitivity of the light receiving element 46 deteriorates, the user inputs an instruction to switch the incident position of the laser light on the light receiving element 46 through the input unit.

  When an instruction to switch the incident position is input from the user, the controller 7 changes the angle around the axis of the incident position switching mirror 41. Thereby, the incident position of the fluorescence reflected by the incident position switching mirror 41 and incident on the light receiving element 46 is set to another unused position determined in advance or another unused position randomly determined at the time of activation. Be changed.

  In the light receiving element 46, the range necessary for receiving the fluorescence is a part of the entire range in which the fluorescence can be incident. It is possible to effectively use unused unused ranges. As a result, the fluorescence from the sample S can be received by the light receiving element 46 with normal sensitivity, and a clear image can be acquired.

  As described above, according to the laser microscope 100 and the light receiving sensitivity extension method according to the present embodiment, when the sensitivity of the light receiving element 46 of the detector 45 deteriorates, the incident light is incident on the light receiving element 46 by the incident position switching mirror 41. The light receiving sensitivity of the light receiving element 46 can be extended by changing the incident position of the fluorescent light to be unused.

[Second Embodiment]
Next, a laser microscope and a light receiving sensitivity life extension method according to a second embodiment of the present invention will be described.
In the laser microscope 200 according to the present embodiment, as shown in FIG. 3, the detection optical system 30 includes a spectroscopic element 136 such as a diffraction grating that separates fluorescence into spectral components (Grating), and instead of the detector 45, The second embodiment is different from the first embodiment in that a detector 145 having a plurality of light receiving elements 146 arranged along the spectral direction of the spectral component by the spectroscopic element 136 is provided.
In the following, portions having the same configuration as those of the laser microscope 100 and the light receiving sensitivity life extension method according to the first embodiment are denoted by the same reference numerals, and description thereof is omitted.

The spectroscopic element 136 is disposed between the reflection mirrors 35 and 37, and spectrally separates the fluorescent spectral component reflected by the reflection mirror 35 in one direction.
As the detector 145, for example, a multi-channel PMT (Photomultiplier Tube) is used.

  As shown in FIG. 4, the detector 145 has a plurality of light receiving elements 146 (for example, 32 (CH (channel) 1 to 32), 32 elements)) along the spectral direction of the spectral component of fluorescence by the spectroscopic element 136. Are arranged. Each light receiving element 146 detects the spectral component of the fluorescence reflected by the incident position switching mirror 41 and collected on the respective light receiving surfaces.

  By changing the angle around the axis, the incident position switching mirror 41 changes the incident position of the fluorescence in each light receiving element 146 in the direction orthogonal to the spectral direction of the spectral component, that is, the optical axis direction of the fluorescence in each light receiving element 146. It can be changed.

The operation of the thus configured laser microscope 200 and light receiving sensitivity life extension method will be described.
When observing the specimen S with the laser microscope 200 according to the present embodiment, laser light is emitted from the laser unit 10 and irradiated onto the specimen S, as in the first embodiment. The fluorescence generated in the sample S is collected by the objective lens 5, passes through the pinhole 33 through the illumination optical system 20, is reflected by the reflection mirror 35, and is split into spectral components by the spectroscopic element 136.

  The fluorescence separated into spectral components by the spectroscopic element 136 is reflected by the reflection mirror 37 and collected by the condensing lens 39, and is received by each light receiving element 146 of the detector 145 via the incident position switching mirror 41 and the reflection mirror 43. Each is incident on the light receiving surface. Thereby, a spectrum component is detected for each light receiving element 146.

  Therefore, the fluorescence separated into the spectral components by the spectroscopic element 136 can be incident on the plurality of light receiving elements 146 without waste, and the desired spectral components can be selectively detected at once by the light receiving elements 146. Thereby, spectral detection can be performed quickly and efficiently.

  In this case, when the light receiving sensitivity of the light receiving element 146 of the detector 145 deteriorates, first, the laser microscope 200 is activated and initialized. Then, the user inputs an incident position switching instruction via the input unit.

  When an instruction to switch the incident position is input from the user, the controller 7 changes the angle around the axis of the incident position switching mirror 41. As a result, the fluorescence incident position in each light receiving element 146 is changed to a direction orthogonal to the spectral direction of the spectral component. In this case, as shown in FIG. 5, it can be seen that the uniformity of light receiving sensitivity (Uniformity) in the same channel (light receiving element 146) is not a problem level.

  Thereby, according to the laser microscope 200 and the light receiving sensitivity extending method according to the present embodiment, an unused range that exists in a direction perpendicular to the spectral direction of the spectral component in the range in which fluorescence in each light receiving element 146 can enter. It is possible to extend the life of the light receiving sensitivity of the light receiving element 146 by effectively utilizing the above.

[Third Embodiment]
Next, a laser microscope and a light receiving sensitivity life extension method according to a third embodiment of the present invention will be described.
As shown in FIG. 6, the laser microscope 300 according to the present embodiment includes a movable mirror 237 that can move in the dispersion direction of the spectral component of the fluorescence dispersed by the spectroscopic element 136, instead of the reflection mirror 37. The control unit 7 is different from the first and second embodiments in that a control device 250 that corrects the sensitivity of the plurality of light receiving elements 146 of the detector 145 is provided instead of the control unit 7.
In the following, portions having the same configurations as those of the laser microscopes 100 and 200 and the light receiving sensitivity life extension method according to the first and second embodiments are denoted by the same reference numerals and description thereof is omitted.

  The movable mirror 237 reflects the fluorescence separated into spectral components by the spectroscopic element 136 toward the incident position switching mirror 41. The movable mirror 237 moves in the dispersion direction of the spectral component of the fluorescence so that the fluorescence can be scanned in one direction on the photocathode of each light receiving element 146 from CH1 to CH32 of the detector 145. It has become.

  The control device 250 includes a control unit (incident position changing unit) 207 that controls the scanning unit 23, the spectroscopic element 136, the movable mirror 237, the incident position switching mirror 41, the detector 145, and the like, a storage unit 261, and a PC. (Personal Computer, sensitivity measuring unit, measuring unit) 263 and an analog arithmetic circuit 265 are provided.

The detector 145 sends an electrical signal obtained by photoelectric conversion of the fluorescence received by each light receiving element 146 to the analog arithmetic circuit 265 together with the incident position information regarding the incident position of the fluorescence in each light receiving element 146. .
The analog arithmetic circuit performs an arithmetic process on the electric signal sent from the detector 145 and sends it to the PC 263 as image information together with the incident position information.

  The PC 263 is controlled by the control unit 207, and generates an image based on image information sent from the analog arithmetic circuit 265. In addition, the PC 261 acquires image luminance information for each light receiving element 146. The PC 261 calculates a sensitivity correction gain corresponding to each light receiving sensitivity based on the acquired luminance information for each light receiving element 146.

  The sensitivity correction gain of each light receiving element 146 calculated by the PC 261 is stored in the storage unit 261 in association with the fluorescence incident position in each light receiving element 146. Furthermore, the PC 261 measures the difference between the sensitivity correction gain of each light receiving element 146 stored in the storage unit 261 and the newly measured sensitivity correction gain of each light receiving element 146, and whether the difference is greater than a predetermined threshold value. It is determined whether or not.

The operation of the thus configured laser microscope 300 and light receiving sensitivity life extension method will be described.
The method for observing the specimen S with the laser microscope 300 according to the present embodiment is the same as the laser microscope 200 according to the second embodiment except that the fluorescence is reflected by the movable mirror 237 instead of the reflection mirror 37. Is omitted.

Next, a case where the sensitivity of the light receiving element 146 of the detector 145 is corrected will be described.
In the laser microscope 300 and the light-receiving sensitivity life extension method according to the present embodiment, the following measures are performed by the manufacturer at the time of factory shipment.
First, a mirror sample (reflection mirror) S ′ is placed on the stage 1, and the laser beam emitted from the laser unit 10 is reflected as the excitation dichroic mirror 21, while the reflected light of the laser beam returning from the mirror sample S ′ ( An element having a characteristic of transmitting return light (for example, a dichroic mirror that has no wavelength dependence characteristic, reflects received light at a certain ratio, and transmits the remaining light) is disposed. Further, the pinhole diameter of the pinhole 33 is reduced, and the control unit 207 sets the incident position switching mirror 41 to the initial position (optical axis center).

Next, the sensitivity correction gain of each light receiving element 146 of the detector 145 is acquired.
First, a laser beam is emitted from the laser unit 10 to irradiate the mirror sample S ′, and the control unit 207 controls the scanning unit 23 to scan the laser sample on the mirror sample S ′. Then, the reflected light reflected by the mirror sample S ′ and transmitted through the excitation dichroic mirror 21 is detected and photoelectrically converted by the detector 145, the electric signal is arithmetically processed by the analog arithmetic circuit 265, and the mirror sample S is processed by the PC 263. An XY image of 'is generated.

  Next, the control unit 7 moves the movable mirror 237 in the direction of spectral dispersion of fluorescence by the spectroscopic element 136, and scans the reflected light on the photocathode of each light receiving element 146 from CH1 to CH32. Then, the PC 261 acquires the luminance at the peak time when the image is brightest for each light receiving element 146. In this case, the voltage applied from the control unit 207 to the detector 145 is adjusted, or the laser output of the laser unit 10 is adjusted so that the output of the detector 145 is not saturated.

  Next, the PC 263 normalizes the acquired light receiving element 146 having the highest luminance as 100, and calculates the sensitivity correction gain of each light receiving element 146. For example, among the light receiving elements 146 of CH1 to CH32, the maximum luminance (MaxL) is the luminance of the light receiving element 146 of CH16, and MaxL = 3800. In this case, for example, if the maximum luminance (Max1) of the CH1 light receiving element 146 is 2600, the sensitivity correction gain of the CH1 light receiving element 146 is (100 / MaxL) × Max1 = 68 (rounded off after the decimal point).

The sensitivity correction gain of each light receiving element 146 calculated by the PC 263 is stored in the storage unit 261 in association with the incident position of the reflected light in each light receiving element 146. The same applies to the other light receiving elements 146 of CH2 to CH32.
The work so far is performed by the manufacturer before delivery.

Next, at the time of maintenance of the laser microscope 300 after delivery, the user next performs treatment.
First, the PC 263 acquires the sensitivity correction gain after delivery by the same method as the acquisition of the sensitivity correction gain before delivery. When the sensitivity correction gain after delivery is acquired, the PC 263 compares the acquired sensitivity correction gain after delivery with the sensitivity correction gain before delivery stored in the storage unit 261. When the difference between the two is larger than a predetermined threshold (for example, twice or more), it is considered that the sensitivity is deteriorated, and the method for extending the light receiving sensitivity of the light receiving element 146 is executed.

  Specifically, the mirror specimen S ′ is placed on the stage 1 and the laser beam emitted from the laser unit 10 is reflected as the excitation dichroic mirror 21 while the reflected light of the laser beam returning from the mirror specimen S ′ is transmitted. (For example, a dichroic mirror that has a wavelength-dependent characteristic, reflects received light at a certain ratio, and transmits the remaining light), and narrows the pinhole diameter of the pinhole 33 .

  Next, a laser beam is emitted from the laser unit 10 to irradiate the mirror sample S ′, and the control unit 207 controls the scanning unit 23 to scan the laser beam on the mirror sample S ′. Then, the reflected light reflected by the mirror sample S ′ is detected and photoelectrically converted by the detector 145, and the electric signal is arithmetically processed by the analog arithmetic circuit 265, and an XY image of the mirror sample S ′ is generated by the PC 263. .

  Then, while changing the angle of the incident position switching mirror 41 by the control unit 207, the luminance of the light receiving element 146 where the sensitivity degradation is most advanced is sampled in a direction orthogonal to the dispersion direction of the spectrum component, and the received light is received by the PC 263. The position with the highest luminance (the optimum position) in the element 146 is searched.

  When the optimum position in the light receiving element 146 is determined, the sensitivity correction gain of each light receiving element 146 is acquired again by the same method as the acquisition of the sensitivity correction gain of the laser microscope 300 before delivery. The PC 263 compares the newly acquired sensitivity correction gain with the pre-delivery sensitivity correction gain stored in the storage unit 261 to determine whether or not the sensitivity is restored, that is, the difference is equal to or less than a predetermined threshold value. Check whether or not. When the brightness is restored, the sensitivity correction gain of each light receiving element 146 and the new incident position of the reflected light at each light receiving element 146 are associated with each other and stored in the storage unit 261, and the maintenance after delivery ends.

  In the present embodiment, the acquisition of a new incident position of the reflected light in the light receiving element 146 may be performed by using means for moving a specified amount obtained from the design spot size of the reflected light without performing sampling.

This embodiment can be modified as follows.
For example, as a first modified example, as shown in FIG. 7, the incident position switching mirror 41 switches the optical path of the fluorescence separated into spectral components by the spectroscopic element 136 into two optical paths having different optical path lengths. It is good also as functioning as.

  In this case, by rotating the incident position switching mirror 41 by 90 ° around the axis, the optical path for reflecting the fluorescence incident from the condenser lens 39 toward the reflection mirror 43 and the reflection mirror 43 are reflected in the opposite direction. What is necessary is just to switch to an optical path.

  Further, in the optical path, the relay lenses 251 and 259 that relay fluorescence and the fluorescence relayed by the relay lenses 251 and 259 are reflected and returned to the incident position switching mirror 41 from different directions. Three reflecting mirrors 253, 255, and 257 that are reflected again toward 43 may be arranged.

  By doing so, the focal length from the spectroscopic element 136 is increased by the amount that passes through the relay lenses 251 and 259 and the reflection mirrors 253, 255, and 257. Therefore, the spectral resolution of the fluorescence incident on the detector 145 can be increased compared to the short optical path.

  According to the present modification, the incident position switching mirror 41 can change the spectral resolution of the fluorescence incident on the detector 145 by changing the focal length of the fluorescence separated into the spectral components by the spectroscopic element 136. In this case, since the incident position switching mirror 41 functions as an optical path switching unit, it is not necessary to provide a special mechanism for switching between the optical path of the fluorescence and the apparatus can be simplified.

  As a second modification, as shown in FIG. 8, a switching mechanism 273 capable of inserting / removing a total reflection mirror (folding member) 271 that folds the laser light along the same optical path on the optical path of the laser light. It is good also as providing.

  In this case, for example, the total reflection mirror 271 is inserted on the optical path of the laser light between the pupil projection lens 25 and the imaging lens 27 by the switching mechanism 273 and is incident from the pupil projection lens 25 by the total reflection mirror 271. What is necessary is just to enable it to be able to return | fold the laser beam to be turned along the same optical path.

  When the laser beam is turned back by the total reflection mirror 271, the excitation dichroic mirror 21 reflects the laser beam emitted from the laser unit 10 and transmits the reflected beam of the laser beam returning from the mirror sample S ′. An element (for example, a dichroic mirror that has no wavelength-dependent characteristics, reflects received light at a certain ratio, and transmits the remaining light) may be disposed.

  In this way, the switching mechanism 273 merely places the total reflection mirror 271 on the optical path of the laser light without taking the trouble of arranging the mirror sample S ′ at the irradiation position of the laser light by the objective lens 5. The light receiving sensitivity of each light receiving element 146 can be measured by the PC 263.

  As a third modification, as shown in FIGS. 9 and 10, a reflection mirror 3 and a corner cube (folding member) 275 that folds the laser light along the same optical path are selected on the optical path of the laser light. It is good also as providing the turret (switching mechanism) 277 which can be arrange | positioned automatically. FIG. 10 illustrates an 8-series turret 277.

  In this case, the reflecting mirror 3 and the corner cube 275 are attached to the turret 277 at a circumferential interval around a predetermined rotation axis, and the turret 277 is rotated about the rotation axis, thereby allowing the laser light and the fluorescence light path to be on the optical path. The reflecting mirror 3 and the corner cube 275 may be selectively arranged.

  In this way, the turret 277 can be used to measure the light receiving sensitivity of the detector 45 and observe the sample S only by switching whether the reflecting mirror 3 or the corner cube 275 is disposed on the optical path of the laser light. Can be switched easily.

  Further, as a fourth modified example, as shown in FIG. 11, a corner cube 275 disposed at a position off the optical path of the laser light between the scanning unit 23 and the objective lens 5 may be provided. In this case, the laser beam incident from the excitation dichroic mirror 21 may be reflected by the scanning unit 23 toward the corner cube 275. In addition, the corner cube 275 may return the laser light incident from the scanning unit 23 to the scanning unit 23 along the same optical path.

  By doing so, the light receiving element can be obtained by simply reflecting the laser light toward the corner cube 275 by the scanning unit 23 without taking the trouble of arranging the mirror sample S ′ at the irradiation position of the laser light by the objective lens 5. The detection sensitivity of 46 can be easily measured.

  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 the above-described embodiments and modifications thereof, the incident position of the fluorescence in the light receiving elements 46 and 146 of the detectors 45 and 145 is changed by controlling the incident position switching mirror 41. For example, the detectors 45 and 145 are provided so as to be movable in the direction along the fluorescence optical axis, and the control units 7 and 207 move the detectors 45 and 145 in the direction along the fluorescence optical axis. The incident position of the fluorescence in the light receiving elements 46 and 146 may be changed.

5 Objective lens 7,207 Control unit (incident position changing unit, measuring unit)
11 Laser light source
23 Scanning section 41 Incident position switching mirror (incident optical system)
45,145 Detector (light detector)
46,146 Light receiving element 136 Spectroscopic element 261 Storage unit 263 PC (sensitivity measuring unit)
271 Total reflection mirror (folding member)
273 Turret (switching mechanism)
275 Corner cube
277 Turret 100, 200, 300 Laser microscope

Claims (12)

An objective lens that irradiates the sample with laser light emitted from a light source and collects return light returning from the sample;
A photodetector having a light receiving element that receives the return light and performs photoelectric conversion;
An incident optical system for causing the return light collected by the objective lens to enter the light receiving element of the photodetector;
A laser microscope comprising: an incident position changing unit that changes an incident position of the return light incident on the light receiving element to another position of the same light receiving element . An objective lens that irradiates the sample with laser light emitted from a light source and collects return light returning from the sample;
A spectroscopic element that splits the return light collected by the objective lens into spectral components;
A photodetector having a light receiving element that receives the return light and performs photoelectric conversion;
An incident optical system for causing the return light collected by the objective lens to enter the light receiving element of the photodetector;
An incident position changing unit for changing the incident position of the return light incident on the light receiving element;
The laser microscope in which the incident position changing unit changes the incident position of the return light in the light receiving element in a direction orthogonal to the spectral direction of the spectral component. A spectroscopic element that splits the return light collected by the objective lens into spectral components;
The laser microscope according to claim 1, wherein the incident position changing unit changes the incident position of the return light in the light receiving element in a direction orthogonal to a spectral direction of the spectral component. The laser microscope according to claim 2, wherein the photodetector includes a plurality of the light receiving elements arranged along a spectral direction of the spectral component by the spectral element. The incident position changing unit, the optical path of the spectrally separated return light to the spectral components by the spectroscopic element, claims 2 to the optical path length functions as a light path switching unit for switching the two different optical paths to one of claims 4 The laser microscope described. The incident position changing unit, laser microscope as claimed in any one of claims 5 to change the incident position of the return light in the light receiving element during activation of the light source. A sensitivity measuring unit for measuring the light receiving sensitivity of each light receiving element;
A storage unit for storing the light receiving sensitivity for each of the light receiving elements measured by the sensitivity measuring unit and the incident position of the return light in the plurality of light receiving elements in association with each other;
A measuring unit that measures a difference between the light receiving sensitivity for each light receiving element stored in the storage unit and the light receiving sensitivity for each light receiving element newly measured by the sensitivity measuring unit;
When the difference measured by the measuring unit is larger than a predetermined threshold, the incident position changing unit changes the incident position of the return light in the plurality of light receiving elements, and the storage unit is the incident position changing unit. claim from claim 2 in association with the light receiving sensitivity of each of the light receiving element measured by the sensitivity measuring portions incident position of the return light in the modified plurality of light receiving elements and at its incident position by 5 The laser microscope according to any one of the above. The laser microscope according to claim 7 , wherein the sensitivity measurement unit measures light reception sensitivity of the light receiving element using reflected light from a reflection mirror arranged at a position irradiated with the laser light by the objective lens. The laser microscope according to claim 7 , further comprising a switching mechanism capable of inserting and removing a folding member that folds the laser light on an optical path of the laser light. The laser microscope according to claim 9 , wherein the switching mechanism can selectively arrange an optical element that makes the laser light incident on the objective lens and the folding member in an optical path of the laser light. A scanning unit that reflects the laser light emitted from the light source and scans the sample;
A folding member disposed at a position deviating from the optical path of the laser beam between the scanning unit and the objective lens, and a folding member capable of folding the laser beam;
The laser microscope according to claim 7 , wherein the scanning unit can reflect the laser light toward the folding member. A laser beam is emitted from a light source and irradiated on the specimen. The return light returning from the specimen is incident on a light receiving element of a photodetector, and the light receiving element receives the return light and uses the laser microscope to observe the specimen. A method for extending the light receiving sensitivity of the light receiving element,
A method for extending the life of light receiving sensitivity, wherein when the light receiving sensitivity of the light receiving element deteriorates, the incident position of the return light in the light receiving element is changed to a different position.

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