JP2016071160A - Laser scanning type microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a laser scanning type microscope designed to prevent an optical detector from becoming hot and reducing the size of the device body.SOLUTION: The laser scanning type microscope comprises: a scanner 33 for scanning a laser beam; an objective lens 23 for irradiating a sample S with the laser beam scanned by the scanner 33 and collecting fluorescent light from the sample S; an excitation dichroic mirror 41 for branching the fluorescent light collected by the objective lens 23 from the optical path of the laser beam between the objective lens 23 and the scanner 33; a liquid light guide 7 for guiding the fluorescent light branched off by the excitation dichroic mirror 41; and PMTs 59A, 59B for detecting the fluorescent light guided by the liquid light guide 7. A culture space 10 accommodating the sample S and whose internal temperature and humidity are maintainable and an optical space 20 for accommodating the objective lens 23 are disposed adjacent to each other, and an optical detection space 50 accommodating the PMTs 59A, 59B is disposed separately from the culture space 10 and the optical space 20.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a laser scanning microscope.

  Conventionally, a laser scanning microscope provided with a culture space for storing a specimen under a specific temperature and humidity, and an optical space in which an optical system such as an objective lens and a photodetector for observing the specimen in the culture space is arranged Is known (for example, see Patent Document 1).

JP 2011-102970 A

  The laser scanning microscope described in Patent Document 1 has one photodetector installed in the optical space. However, a large number of photodetectors are used to detect different wavelengths simultaneously, or photodetectors having different characteristics are used. When trying to adopt a plurality, peripheral constituent units including the photodetectors are enlarged. Further, if the fluorescence generated in the specimen is to be taken into the photodetector without loss, the optical detection optical system such as a lens, a barrier filter, or a spectroscopic dichroic mirror that guides the fluorescence to the photodetector tends to increase in size.

In addition, the culture space is only a few φ35 dishes and one microplate, and it is not necessary to increase the projection area on the desk surface so much. It is feared that the entire device including the device will be enlarged. Furthermore, in the optical space, a focus mechanism that moves the objective lens along the optical axis and a stage drive mechanism that moves the specimen in a direction crossing the optical axis of the objective lens are arranged. Occupies the space.
Therefore, in the conventional laser scanning microscope, there is a problem that it is difficult to downsize the apparatus main body and to thermally separate the photodetector from the optical space.

  The present invention has been made in view of the above-described circumstances, and an object of the present invention is to provide a laser scanning microscope that prevents the temperature of the photodetector from being increased and reduces the size of the apparatus main body.

In order to achieve the above object, the present invention provides the following means.
The present invention provides a scanning unit that two-dimensionally scans a laser beam, an objective lens that irradiates the sample with the laser beam scanned by the scanning unit, and condenses the light from the sample, and the objective lens. Observation light branching portion for branching the condensed light from the specimen from the optical path of the laser light between the objective lens and the scanning portion, and observation for guiding the light branched by the observation light branching portion A light guide fiber, and an observation light detection unit that detects light guided by the observation light guide fiber; a culture space that accommodates the specimen and maintains an internal temperature and humidity; and the objective lens Provided is a laser scanning microscope in which an optical space to be accommodated is disposed adjacent to each other, and a light detection space for accommodating the observation light detection unit is disposed apart from the culture space and the optical space.

  According to the present invention, the specimen is maintained in a healthy state for a long time by maintaining the inside of the culture space at a specific temperature and humidity. Laser light is scanned on the specimen by the scanning unit and irradiated by the objective lens, and the light from the specimen is collected by the objective lens and branched from the optical path of the laser light by the observation light branching unit. By being guided by the fiber and detected by the observation light detection unit without passing through the scanning unit, the sample in a healthy state can be observed by two-photon excitation.

In this case, the light detection space is disposed away from the optical space and the culture space, thereby preventing the observation light detection unit from being thermally separated from the optical space and the culture space and being heated to a high temperature. Noise can be suppressed. Further, the optical space can be reduced in size compared to the case where the observation light detection unit is accommodated in the optical space.
Therefore, it is possible to prevent the observation light detection unit from becoming high temperature and to reduce the size of the apparatus main body.

In the above-described invention, there is a through hole into which the objective lens can be inserted, a partition wall that separates the culture space and the optical space, and an optical axis direction of the objective lens inserted into the through hole of the partition wall And a seal part capable of closing the gap between the objective lens and the partition wall part, and a part of the objective lens is inserted into the culture space through the through-hole. Also good.
By comprising in this way, the objective lens with a short distance from a front-end | tip to a sample can be used.

In the said invention, it is good also as providing the light-shielding wall which encloses this photon detection space and interrupts | blocks the light from the exterior to the inside of this photon detection space.
With such a configuration, since the environmental light is blocked by the light shielding wall, the observation light detection unit can eliminate the influence of the environmental light and perform stable observation.

In the said invention, it is good also as having the thermostat in the said optical space which thermostats the inside of this optical space substantially equal to the temperature in the said culture space.
By comprising in this way, it can prevent that the temperature gradient based on the temperature of culture | cultivation space generate | occur | produces in the optical system and optical system in optical space by the action | operation of a thermostat. Thereby, it is possible to prevent distortion from occurring in the optical system and the mechanical system, and to effectively prevent the brightness and blurring of the obtained image.

In the said invention, it is good also as providing a light source and the laser beam light guide fiber which guides the said laser beam emitted from this light source to the said scanning part.
With this configuration, it is not necessary to arrange a light source in the optical space. Therefore, the optical space can be downsized as compared with the case where the light source is arranged in the optical space. Further, the light source can be exchanged by changing the laser light guide fiber.

In the above invention, the observation light branching section may be disposed adjacent to the objective lens.
With this configuration, light from the specimen can be branched by the observation light branching unit immediately after being emitted from the objective lens. The light from the specimen emitted from the objective lens varies in the position of the light beam according to the scanning of the laser light by the scanning unit, but the light immediately after emitted from the objective lens has a relatively small change in the position of the light beam. Therefore, if the light from the sample is branched by the observation light branching portion immediately after being emitted from the objective lens, and if it is incident on the observation light guiding fiber immediately after branching, it is necessary to occupy a very large space near the objective lens. There is no. Thereby, the temperature of the optical space can be easily controlled by narrowing the optical space while maintaining the detection efficiency of light from the objective lens.

  If the light from the specimen emitted from the objective lens is detected after returning to the scanning unit, it does not occupy the space near the objective lens, but the light emitted from the specimen is scattered, and the scattered light is However, it is necessary to detect light from a wider field of view of the sample (range farther from the optical axis of the sample surface). In this case, in order to secure a light beam larger than the laser beam, the entire detection optical path such as an imaging lens, a pupil projection lens, and a scanning unit mirror is enlarged, which is not preferable because the microscope main body is enlarged. .

In the said invention, it is good also as having a cooling part which cools the temperature in this light detection space in the said light detection space.
With this configuration, the observation light detection unit can be cooled by the cooling unit to reduce dark current noise. Thereby, the S / N ratio can be improved and the sample can be observed.

In the above invention, the observation light guiding fiber may be a liquid light guide.
With this configuration, the liquid light guide has a relatively large core diameter, so that light from the specimen can be efficiently taken into the observation light guide fiber. Even when the pupil diameter of the objective lens is large, it is not necessary to increase the reduction magnification of the lens that allows light to enter the observation light guide fiber from the pupil, and the distance from the objective lens to the observation light guide fiber should be relatively short. Can do. Therefore, the optical space can be reduced in size. Furthermore, since the liquid light guide can be bent relatively easily even when the core diameter is large, the observation light guide fiber can be routed in a narrow space.

  In the above invention, a laser beam transmitted through the sample, a transmission side optical fiber that guides fluorescence emitted from the sample in the same direction as the laser beam, and the laser beam guided by the transmission side optical fiber; A transmission side branching part for branching the fluorescence; a transmission side laser light detection part for detecting a laser beam branched at the transmission side branching part; and a transmission side fluorescence for detecting the fluorescence branched at the transmission side branching part. It is good also as providing a detection part.

  With this configuration, after the laser beam that passes through the specimen and the fluorescence emitted from the specimen to the transmission side are guided by the transmission side optical fiber, each is branched by the transmission side branching section and transmitted through the transmission side laser light. It is detected by the detection unit or the transmission side fluorescence detection unit. Accordingly, it is possible to observe the outline of the specimen with the laser light transmitted through the specimen, and at the same time to observe the microstructure (for example, the organelle in the cell) in the specimen with fluorescence having a wavelength different from that of the laser light. If the observation light detection unit detects fluorescence having the same wavelength as that of the transmission side fluorescence detection unit, brighter fluorescence observation is possible.

  In the above invention, the laser light may have at least one of a characteristic of multiphoton absorption by the specimen and a characteristic of generating a harmonic from the specimen.

  According to the present invention, it is possible to prevent an increase in the temperature of the photodetector and to reduce the size of the apparatus main body. Furthermore, the footprint of the microscope main body, which is easily affected by image deterioration due to external vibration, can be reduced, and the vibration isolation table on which the microscope main body is loaded can be downsized. When the light source is connected to the microscope body with a fiber, even if the light source itself vibrates, the angle and power fluctuations of the laser beam emitted from the fiber are small, the scanning position is shifted, and the laser power varies for each scanning position. There is little worry to go. The observation light detection unit only detects the intensity of light emitted from the sample at a certain position (point) scanned by the scanning unit. It is sufficient that the light flux is not lost, and if the beam bundle has a sufficient margin, the intensity of light hardly changes due to vibration.

1 is a schematic configuration diagram illustrating a laser scanning microscope according to a first embodiment of the present invention. It is a schematic block diagram which shows the permeation | transmission side detection unit of the laser scanning microscope which concerns on 2nd Embodiment of this invention. It is a figure which shows an example of a structure around the objective lens of the laser scanning microscope which concerns on the modification of 1st, 2nd embodiment of this invention. It is a figure which shows another example of a structure around the objective lens of the laser scanning microscope which concerns on the modification of 1st, 2nd embodiment of this invention.

[First Embodiment]
A laser scanning microscope according to a first embodiment of the present invention will be described below with reference to the drawings.
A laser scanning microscope 100 according to the present embodiment includes, for example, a laser light source (light source) 1 that generates laser light and an optical fiber that guides the laser light emitted from the laser light source 1, as shown in FIG. A laser light guide fiber) 3, a microscope main body 5 that irradiates the specimen S with laser light guided by the optical fiber 3, and a liquid light guide (observation light guide fiber that guides fluorescence (light) emitted from the specimen S. ) 7 and a light detection unit 9 for detecting the fluorescence guided by the liquid light guide 7.

  The laser light source 1 emits ultrashort pulse laser light such as femtosecond pulse laser light, for example. The laser light source 1 is desirably temperature-controlled to about 15 ° C. by a temperature control device (not shown). One end of an optical fiber 3 is fixed to the laser light source 1, and the laser light emitted from the laser light source 1 is incident on the optical fiber 3.

  The microscope body 5 is covered with an exterior cover (light shielding wall) 11 made of a heat insulating material. The microscope body 5 is placed on a vibration isolation table (not shown). Further, the microscope body 5 is partitioned into three spaces of a culture space 10, an optical space 20, and a room temperature space 30 in order from the top by transparent glass (partition walls) 13 and 21.

  The culture space 10 is provided with a culture container 15 for storing the specimen S, a stage 17 on which the culture container 15 is placed, and a heater 19 for heating the inside of the culture space 10.

The culture vessel 15 is made of a transparent glass material.
The stage 17 has a through hole (not shown) penetrating in the plate thickness direction, and the culture vessel 15 can be placed so as to close the opening of the through hole.
The heater 19 can keep the temperature of the culture space 10 constant. Further, the humidity can be kept constant by a humidifier (not shown). For example, the culture space 10 is set to a temperature of 37 ° C. and a humidity of 90%.

  The optical space 20 is disposed adjacent to the culture space 10. In this optical space 20, an objective lens 23, a focus driving unit 25 that holds the objective lens 23 and can move the objective lens 23 in a direction along the optical axis in the optical space 10, and a stage 17 are supported and cultured. A stage driving unit 27 capable of moving the stage 17 in a direction intersecting the optical axis of the objective lens 23 in the space 10 and a heater (constant temperature unit) 29 for heating the optical space 20 are provided.

The objective lens 23 irradiates the sample S with the laser light emitted from the laser light source 1, while condensing the fluorescence generated in the sample S and returning it along the optical path of the laser light.
The heater 29 is configured to keep the optical space 20 at a temperature substantially equal to the temperature of the culture space 10. For example, the optical space 20 is set to 37 ° C.

  The room temperature space 30 is disposed adjacent to the optical space 20. The room temperature space 30 is not temperature-controlled and is at room temperature (for example, 20 ° C.). The outer cover 11 that forms the room temperature space 30 is connected to the other end of the optical fiber 3 and one end of the liquid light guide 7. A collimator lens 31 that converts laser light guided by the optical fiber 3 into parallel light and enters the optical space 20 is fixed to the exterior cover 11.

  In the room temperature space 30, a scanner (scanning unit) 33 such as a galvanometer mirror that two-dimensionally scans the laser light incident through the collimator lens 31 and the laser light scanned by the scanner 33 are collected. The pupil projection lens 35 for forming an intermediate image, a reflection mirror 37 for reflecting the laser light formed on the intermediate image, and the laser light reflected by the reflection mirror 37 as substantially parallel light and entering the objective lens 23. An imaging lens 39 is provided.

  Further, in the room temperature space 30, an excitation dichroic mirror (observation light branching portion) 41 that reflects and branches the fluorescence from the sample S collected by the objective lens 23 and returning the optical path of the laser light from the optical path, and an excitation dichroic mirror A condensing lens 43 that condenses the fluorescence reflected and branched by 41 and enters one end of the liquid light guide 7 is provided.

  The excitation dichroic mirror 41 is disposed between the scanner 33 and the objective lens 23, more specifically, between the imaging lens 39 and the objective lens 23. Since no other lens or the like is arranged between the objective lens 23 and the excitation dichroic mirror 41, and the excitation dichroic mirror 41 is disposed adjacent to the objective lens 23, the fluorescence from the specimen S is converted into the objective lens. The light can be branched by the excitation dichroic mirror 41 immediately after being emitted from the light source 23.

  The fluorescence from the specimen S emitted from the objective lens 23 varies in the position of the light beam in accordance with the scanning of the laser light by the scanner 33, but the fluorescence immediately after emitted from the objective lens 23 has a relatively change in the position of the light beam. small. Accordingly, the fluorescence from the specimen S is branched by the excitation dichroic mirror 41 immediately after being emitted from the objective lens 23, and is incident on the liquid light guide 7 by the condenser lens 43 immediately after the branching, so that the vicinity of the objective lens 23 is obtained. It is not necessary to occupy so much space. Thereby, the temperature of the optical space 39 can be easily controlled by narrowing the optical space 30 while maintaining the detection efficiency of the fluorescence from the objective lens 23.

  The light detection unit 9 is covered with a box-shaped light shielding wall 51 having light shielding properties. The light shielding wall 51 forms a light detection space 50 separated from the culture space 10 and the optical space 20 in the light detection unit 9. Further, the light shielding wall 51 blocks light (environmental light) from the outside to the inside of the light detection space 50 and can maintain the inside of the light detection space 50 in a dry state and a sealed state. The other end of the liquid light guide 7 is connected to the light shielding wall 51.

  In the light detection space 50, a collimator lens 53 that converts the fluorescence from the specimen S guided by the liquid light guide 7 into parallel light, and the fluorescence converted into parallel light is reflected or transmitted according to the wavelength. A dichroic mirror 55 that branches into two optical paths, a barrier filter 57A that blocks laser light contained in the optical path of fluorescence reflected by the dichroic mirror 55, and a PMT (Photomultiplier Tube) that detects fluorescence that has passed through the barrier filter 57A, observation A light detection unit) 59A, a barrier filter 57B that blocks the laser light included in the optical path of the fluorescence that has passed through the dichroic mirror 55, and a PMT (observation light detection unit) 59B that detects the fluorescence that has passed through the barrier filter 57B. It has been.

  The fluorescence luminance information obtained by the PMTs 59A and 59B is sent to a PC (Personal Computer) (not shown) together with the scanning position information relating to the scanning position of the laser beam by the scanner 33. In the PC, a two-dimensional image of the specimen S is generated based on the luminance information and the scanning position information. Further, the image generated by the PC is displayed on a monitor (not shown).

  In addition, the light detection space 50 is provided with Peltier elements (cooling units) 61A and 61B that discharge the heat generated from the PMTs 59A and 59B to the outside to cool the temperature in the light detection space 50. The light detection space 50 is set to 15 ° C., for example. In addition, by maintaining the light detection space 50 in a dry state and a sealed state by the light shielding wall 51, it is possible to prevent dew condensation or the like from occurring in the PMTs 59A and 59B during cooling by the Peltier elements 61A and 61B.

The operation of the laser scanning microscope 100 configured as described above will be described.
In order to observe the specimen S with the laser scanning microscope 100 according to the present embodiment, first, the heaters 19 and 29 and the Peltier elements 61A and 61B are operated, and the culture vessel 15 containing the specimen S is placed in the culture space 10. It mounts on the stage 17 so that the opening of a through-hole may be obstruct | occluded.

  The culture space 10 is maintained at a temperature of 37 ° C. and a humidity of 90% by the operation of the heater 19. Thereby, the sample S like a living cell can be maintained in a healthy state for a long time. Further, the optical space 20 is maintained at the same 37 ° C. as the culture space 10 by the operation of the heater 29. By keeping the temperature of the optical space 20 constant at the same temperature as the culture space 10, it is possible to prevent a temperature gradient based on the temperature of the culture space 10 from being generated in the optical system and the mechanical system in the optical space 20. As a result, drift of the objective lens 23, the focus driving unit 25, and the stage driving unit 27 in the X, Y, and Z directions due to temperature changes can be prevented, and a clear image can be acquired.

  Further, the light detection space 50 is maintained at 15 ° C. by the operation of the Peltier elements 61A and 61B. As a result, the PMTs 59A and 59B can be cooled to prevent generation of thermal noise, and the fluorescence detection accuracy by the PMTs 59A and 59B can be improved.

  Next, laser light is generated from the laser light source 1 and guided to the microscope body 5 by the optical fiber 3. The laser light guided by the optical fiber 3 is converted into parallel light by the collimating lens 31 and scanned two-dimensionally by the scanner 33. The laser beam scanned by the scanner 33 passes through the excitation dichroic mirror 41 through the pupil projection lens 35, the reflection mirror 37, and the imaging lens 39, and is irradiated onto the sample S by the objective lens 23.

  The fluorescence generated in the specimen S by being irradiated with the laser light is collected by the objective lens 23, returns to the optical path of the laser light, is reflected by the excitation dichroic mirror 41, and is lasered between the objective lens 23 and the scanner 33. Branched from the optical path of light. Then, the fluorescence is condensed by the condenser lens 43 and enters the liquid light guide 7. As a result, the fluorescence emitted from the specimen S is guided back to the light detection unit 9 by the excitation dichroic mirror 41 and the liquid light guide 7 without returning to the scanner 33.

  In the case of one-photon excitation, the fluorescence is returned to the scanner 33 (scanning unit) to make a confocal optical system, and then the fluorescence is condensed on the confocal pinhole surface by a confocal lens. Although fluorescence is detected, in this embodiment, a confocal pinhole is not necessary because of two-photon excitation, and the above-described optical system is used.

  The fluorescence guided to the light detection unit 9 by the liquid light guide 7 is converted into parallel light by the collimator lens 53 and branched by the dichroic mirror 55 according to the wavelength. The fluorescence reflected by the dichroic mirror 55 is detected by the PMT 59A via the barrier filter 57A, and the fluorescence transmitted through the dichroic mirror 55 is detected by the PMT 59B via the barrier filter 57B.

  Next, a two-dimensional image of the specimen S is generated by a PC (not shown) based on the fluorescence luminance information obtained by the PMTs 59A and 59B and the laser beam scanning position information by the scanner 33, and displayed on a monitor. Thereby, the user can observe the sample S in a healthy state by two-photon excitation. In addition, since the ambient light is blocked by the light shielding wall 51, the PMTs 59A and 59B can eliminate the influence of the ambient light and perform stable observation.

  In this case, the PMTs 59A and 59B are thermally separated from the culture space 10 and the optical space 20 by disposing the light detection space 50 away from the culture space 10 and the optical space 20 whose temperature is set to 37 ° C. Thus, the PMTs 59A and 59B can be prevented from becoming high temperature. Further, by cooling the light detection space 50 with the Peltier elements 61A and 61B, the PMTs 59A and 59B can be cooled to suppress dark current noise, and the S / N ratio can be improved to observe the sample S.

  Further, in the optical space 20, only the objective lens 23, the focus driving unit 25, and the stage driving unit 27, which are greatly affected by drift due to temperature changes, are arranged, and the excitation dichroic mirror 41 and the PMTs 59A and 59B are arranged in other spaces. Thus, the heat capacity of the optical space 20 to be temperature-controlled can be reduced and the temperature can be kept constant with higher accuracy. Further, the microscope main body 5 can be downsized as compared with the case where the laser light source 1 and the light detection unit 9 are accommodated in the optical space 20 or the room temperature space 30. Furthermore, the footprint of the microscope body 5 is reduced, and the vibration isolation table on which the microscope body 5 is loaded can be downsized.

  Since the liquid light guide has a relatively large core diameter, the fluorescence from the sample S can be efficiently taken into the liquid light guide 7. Further, even when the pupil diameter of the objective lens 23 is large, it is not necessary to increase the reduction magnification of the condenser lens 43 that causes fluorescence to enter the liquid light guide 7 from the pupil of the objective lens 23, and the liquid light guide 7 from the objective lens 23. The distance up to can also be made relatively short. Therefore, the optical space 20 and the room temperature space 30 can be reduced in size. Furthermore, since the liquid light guide can be bent relatively easily even if the core diameter is large, the liquid light guide 7 can be routed in a narrow space.

  As described above, according to the laser scanning microscope 100 according to the present embodiment, the light detection space 50 that houses the light detection unit 9 is arranged apart from the microscope main body 5 having the culture space 10 and the optical space 20. Thus, it is possible to prevent the temperature of the PMTs 59A and 59B from becoming high and to reduce the size of the microscope body 5.

  Further, since the excitation dichroic mirror 41 (observation light branching section) for guiding the fluorescence to the light detection unit 9 is disposed between the objective lens 23 and the scanner 33 (scanning section), the fluorescence detection efficiency is maintained. However, it is possible to reduce the size.

  In the present embodiment, the PMT 59A and the PMT 59B that detect different wavelengths are exemplified as the observation light detection unit, but the observation light detection unit is not limited thereto. For example, a combination of a plurality of PMTs having different characteristics such as a high sensitivity PMT and a low sensitivity PMT that is less likely to fail due to excessive light may be used as the observation light detection unit. Furthermore, the observation light detection unit is not limited to the PMT, and another light detection device such as a photodiode may be employed.

[Second Embodiment]
Next, a laser scanning microscope according to the second embodiment of the present invention will be described.
As shown in FIG. 2, the laser scanning microscope 200 according to this embodiment includes a condenser lens 75 that collects light from the specimen S, and a liquid light that guides the light collected by the condenser lens 75. The second embodiment is different from the first embodiment in that a guide (transmission-side optical fiber) 77 and a light detection unit 79 that detects light guided by the liquid light guide 77 are further provided.
In the following, portions having the same configuration as those of the laser scanning microscope 100 according to the first embodiment are denoted by the same reference numerals and description thereof is omitted.

The condensing lens 75 condenses the laser light that has passed through the sample S and the fluorescence emitted from the sample S in the same direction as the laser light, and causes the light to enter the liquid light guide 77.
One end of the liquid light guide 77 is connected to the exterior cover 11 of the culture space 10 via a condenser lens 75.

  Similar to the light detection unit 9, the light detection unit 79 is covered with a box-shaped light shielding wall 81 having light shielding properties. The light shielding wall 81 forms a light detection space 80 that is separated from the culture space 10 and the optical space 20 in the light detection unit 79. The light shielding wall 81 blocks light (environmental light) from the outside to the inside of the light detection space 80 and can maintain the inside of the light detection space 80 in a dry state and a sealed state. The other end of the liquid light guide 77 is connected to the light shielding wall 81.

  In the light detection space 80, the laser light guided by the liquid light guide 77 and the collimator lens 83 that converts the fluorescence into parallel light and the laser light converted into the parallel light are reflected, while the fluorescence is transmitted. A dichroic mirror (transmission-side branching part) 85 for branching them, and an IR-TD (transmission-side laser light detection part, transmission detector for near-infrared laser) 87 for detecting the laser light reflected by the dichroic mirror 85; , A barrier filter 89 that blocks the laser light included in the optical path of the fluorescence that has passed through the dichroic mirror 85, and an FL-PMT (transmission-side fluorescence detector, fluorescence detector) 91 that detects the fluorescence that has passed through the barrier filter 89. And are provided. Here, since an IR (infrared) wavelength is often used for the laser light, an IR-TD (transmission-side laser light detection unit) is used for convenience of a light detection unit for detecting the laser light transmitted through the specimen S. It is said. In this embodiment, both IR-TD87 and FL-PMT91 are photomultipliers. However, the transmission side laser light detection unit and the transmission side fluorescence detection unit are not limited to these, for example, a photodiode or the like Any device can be used as long as it can detect this.

  The luminance information of the laser beam and the luminance information of the fluorescence obtained by the IR-TD87 and FL-PMT91 are sent to the PC together with the scanning position information. Then, a two-dimensional image of the specimen S is generated from the PC based on the luminance information and the scanning position information, and displayed on the monitor.

  The light detection space 80 is provided with Peltier elements 93A and 93B that cool the temperature in the light detection space 80 by discharging heat generated from the IR-TD 87 and FL-PMT 91 to the outside. By the operation of the Peltier elements 93A and 93B, the light detection space 80 is set to 15 ° C., for example.

The operation of the laser scanning microscope 200 configured as described above will be described.
In order to observe the specimen S with the laser scanning microscope 200 according to the present embodiment, the heaters 19 and 29 and the Peltier elements 61A, 61B, 93A, and 93B are operated. Then, similarly to the first embodiment, a laser beam is generated from the laser light source 1 and applied to the specimen S accommodated in the culture space 10.

  As a result, the fluorescence generated in the specimen S and collected by the objective lens 23 is branched without being returned to the scanner 33 by the excitation dichroic mirror 41 and guided by the liquid light guide 7, and PMTs 59 </ b> A and 59 </ b> B of the light detection unit 9. Is detected.

  Further, the laser light transmitted through the sample S and the fluorescence emitted from the sample S to the side opposite to the objective lens 23 side are condensed by the condenser lens 75 and irradiated by the liquid light guide 77. The light is guided to the light detection unit 79.

  The laser light and fluorescence guided to the light detection unit 79 are converted into parallel light by the collimating lens 83 and then branched to respective optical paths by the dichroic mirror 85. The laser light reflected by the dichroic mirror 85 is detected by the IR-TD 87, and the fluorescence transmitted through the dichroic mirror 85 is detected by the FL-PMT 91 through the barrier filter 89.

According to the laser scanning microscope 200 according to the present embodiment, the brightness information of the fluorescence detected by the FL-PMT 91 is simultaneously observed while observing the outline of the sample S based on the brightness information of the laser light detected by the IR-TD 87. Based on the above, it is possible to observe the microstructure in the specimen S such as an organelle in the cell.
Further, if fluorescence of the same wavelength is detected by the PMTs 59A and 59B and the FL-PMT 91 of the light detection unit 9 and the obtained luminance information is added, brighter fluorescence observation becomes possible.

Each of the above embodiments can be modified as follows.
In each of the above embodiments, the entire objective lens 23 is arranged in the optical space 20, but for example, as shown in FIGS. 3 and 4, the tip of the objective lens 23 is inserted into the culture space 10. It is good. It is assumed that at least a part (base) of the objective lens 23 is accommodated in the optical space 20.

  In this case, the glass 13 may have a through hole 13a into which the objective lens 23 can be inserted. Further, the objective lens 23 may be held movably in the optical axis direction by the focus driving unit 25 while the tip of the objective lens 23 is inserted into the culture space 10 through the through hole 13a of the glass 13. . Also, as shown in FIG. 3, the O-ring 95 closes the gap between the glass 13 and the objective lens 23 without hindering the movement of the objective lens 23 in the optical axis direction, or it can be expanded and contracted as shown in FIG. The gap between the glass 31 and the objective lens 23 may be closed by the bellows 97 made of the above member.

At this time, a gap (for example, the front lens 23a and the lens frame 23b) of the portion protruding into the culture space 10 of the objective lens 23 is prevented so that the high-humidity gas in the culture space 10 does not enter the objective lens 23. The gap between the lens frames 23b when the lens frame 23b has a multiple structure is sealed.
In this way, even the objective lens 23 having a short WD (distance from the tip of the objective lens 23 to the sample S) can be used. The bright and high-resolution objective lens 23 having a large NA (numerical aperture) generally has a short WD. However, with the above-described structure, a bright and clear image can be obtained.

  As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the specific structure is not restricted to this embodiment, The design change etc. of the range which does not deviate from the summary of this invention are included. For example, the present invention is not limited to those applied to the above-described embodiments and modifications, but may be applied to embodiments in which these embodiments and modifications are appropriately combined, and is not particularly limited. .

  For example, in each of the above-described embodiments, description has been made on the use of fluorescence observation. However, even when SHG (Second Harmonic Generation, second harmonic) or THG (Third Harmonic Generation, third harmonic) emitted from the specimen S is observed. Good. Furthermore, the laser scanning microscopes 100 and 200 according to each of the above embodiments are applied to various observation methods using a nonlinear effect that generates light from a specimen in proportion to the square of the intensity of light or a power of more than that. it can. In addition, the laser scanning microscopes 100 and 200 according to the above embodiments may perform multiphoton excitation observation such as three-photon excitation instead of two-photon excitation.

1 Laser light source
3 Optical fiber (Laser light guiding fiber)
7 Liquid light guide (observation light guide fiber)
DESCRIPTION OF SYMBOLS 10 Culture space 20 Optical space 23 Objective lens 29 Heater (constant temperature part)
33 Scanner (scanning unit)
41 Excited dichroic mirror (observation beam splitter)
51 light shielding wall 59A, 59B PMT (observation light detection part)
61A, 61B Peltier element (cooling part)
77 Liquid Light Guide (Transmission-side optical fiber)
80 Photodetection space 85 Dichroic mirror (transmission side branch)
87 IR-TD (Transmission side laser beam detector)
91 FL-PMT (transmission-side fluorescence detection unit)
100,200 Laser scanning microscope

Claims (10)

A scanning section for two-dimensionally scanning laser light;
An objective lens for condensing the light from the sample while irradiating the sample with laser light scanned by the scanning unit;
An observation light branching unit for branching light from the sample collected by the objective lens from the optical path of the laser light between the objective lens and the scanning unit;
An observation light guiding fiber for guiding the light branched by the observation light branching unit;
An observation light detection unit for detecting light guided by the observation light guide fiber;
A culture space capable of accommodating the specimen and maintaining an internal temperature and humidity and an optical space accommodating the objective lens are disposed adjacent to each other,
A laser scanning microscope in which a light detection space for accommodating the observation light detection unit is disposed apart from the culture space and the optical space. A partition having a through-hole into which the objective lens can be inserted, and separating the culture space and the optical space;
A seal portion capable of closing a gap between the objective lens and the partition wall while allowing movement of the objective lens inserted in the through-hole of the partition wall in the optical axis direction;
The laser scanning microscope according to claim 1, wherein a part of the objective lens is inserted into the culture space through the through hole.   The laser scanning microscope according to claim 1, further comprising a light shielding wall that surrounds the light detection space and blocks light from outside to inside of the light detection space.   The laser scanning microscope according to any one of claims 1 to 3, wherein the optical space has a thermostat that constants the temperature of the optical space substantially equal to the temperature of the culture space. A light source;
The laser scanning microscope according to any one of claims 1 to 4, further comprising a laser light guide fiber that guides the laser light emitted from the light source to the scanning unit.   The laser scanning microscope according to claim 1, wherein the observation light branching unit is disposed adjacent to the objective lens.   The laser scanning microscope according to any one of claims 1 to 6, wherein the light detection space includes a cooling unit that cools a temperature in the light detection space.   The laser scanning microscope according to any one of claims 1 to 7, wherein the observation light guiding fiber is a liquid light guide. A transmission side optical fiber that guides the laser light transmitted through the specimen and the fluorescence emitted from the specimen in the same direction as the laser light;
A transmission side branching section for branching the laser light guided by the transmission side optical fiber and the fluorescence;
A transmission side laser beam detection unit for detecting the laser beam branched in the transmission side branch unit;
The laser scanning microscope according to any one of claims 1 to 8, further comprising a transmission-side fluorescence detection unit that detects fluorescence branched in the transmission-side branching unit.   The laser scanning microscope according to any one of claims 1 to 9, wherein the laser light has at least one of a characteristic of multiphoton absorption by the specimen and a characteristic of generating harmonics from the specimen.

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