JPH07244238A - Confocal scanning type microscope - Google Patents

Abstract

PURPOSE:To provide a confocal scanning type microscope which does not require the constitution of a confocal optical system requiring accurate alignment in the case of changing the wavelength of irradiating light. CONSTITUTION:The optical system of the confocal scanning type microscope is constituted without arranging spectroscopic parts in the confocal optical system from the minute spot of the irradiation light on a sample 900 to a diaphragm 500 for cutting stray light. The spectroscopic processing of generated light on the sample 900 is executed after the generated light passes through the confocal optical system so as to completely reduce the stray light. As a result, observation is executed by easy change, that is, by changing the wavelength of the irradiation light without changing the constitution of the confocal optical system requiring accurate adjustment.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a multi-wavelength confocal scanning microscope for observing a specimen by using irradiation light having a plurality of wavelengths and by separating the irradiation light and the observation target light by a spectroscopic means. .

[0002]

2. Description of the Related Art A confocal microscope, which illuminates only one point of a sample at a time and collects the image of only one point, effectively reaches the observation point of the image light output from the unfocused portion. In principle, it is possible to observe the image of the irradiation point of the sample to be measured very clearly because it blocks. Then, a confocal scanning microscope that scans the irradiation point two-dimensionally or three-dimensionally and collects an image of the irradiation point can clearly observe the two-dimensional or three-dimensional aspect of the sample. is there.

FIG. 5 shows an example of the configuration of a conventional confocal scanning microscope. In this device, the light beam emitted from the laser light source becomes collimated light whose beam diameter is expanded through the beam expander, and reflects the light of the wavelength of the laser light and the light of the wavelength substantially the same as the wavelength of the laser light. It is incident on the dichroic mirror. The laser light reflected by the dichroic mirror enters a two-dimensional scanner whose reflection direction is determined by the arrangement of reflective optical components, is emitted in the reflection direction set in the two-dimensional scanner, and sequentially passes through a pupil projection lens and an objective lens. To form a minute spot on the sample.

Reflected light (wavelength is the same as the irradiation laser light), fluorescence (wavelength is different from the irradiation laser light), Raman light (wavelength is the irradiation laser light The light that has entered the objective lens reverses the traveling path of the irradiation laser light and enters the dichroic mirror. The dichroic mirror reflects reflected light having the same wavelength as the irradiation laser light and transmits fluorescence or Raman light having a different wavelength from the irradiation laser light. The light transmitted through the dichroic mirror forms a spot on the diaphragm by the condenser lens. The light passing through the diaphragm is further wavelength-selected by the wavelength selection filter, and the light of the selected wavelength is incident on the photodetector and detected.

The above-mentioned photo-detecting operation is performed while scanning the reflection direction of the irradiation laser light (and further, the position of the irradiation light spot in the optical axis direction) by the two-dimensional scanner to make the sample two-dimensional (further, further). Observe three-dimensionally).

In the above-mentioned conventional example, a dichroic mirror is used as the spectroscopic means, but an apparatus having the same structure as the optical system as in the above-mentioned conventional example and using the diffracted photons as the spectroscopic means is disclosed in Japanese Unexamined Patent Publication (Kokai) No. Japanese Unexamined Patent Publication No. 5-172741 discloses a device that uses a direct-view prism.

[0007]

Since the conventional confocal scanning microscope is constructed as described above, the irradiation light of the wavelength having the highest generation efficiency of fluorescence and Raman light is observed in various samples to be observed. When it is used, there is a problem that the dichroic mirror, which is a spectroscope, and the wavelength selection filter must be replaced. In such exchange, the optical path in the optical system from the diaphragm to the sample (hereinafter called confocal optical system) that effectively blocks the image light output from the out-of-focus portion from reaching the observation point. Since it includes the exchange of the spectroscope whose optical path changes depending on the wavelength installed inside, there was a problem that favorable observation could not be performed unless the position of the diaphragm that requires precise positioning was adjusted each time. . Furthermore, since the conventional confocal scanning microscope is configured on the assumption that the wavelength of the irradiation light is constant,
There is a problem that it is not possible to deal with the case where the continuously variable wavelength is used as the irradiation light, such as observing the sample by scanning the wavelength of the irradiation light.

The present invention has been made in order to solve the above problems, and is a confocal scanning type that does not require the configuration of a confocal optical system that requires precise alignment when changing the wavelength of irradiation light. The purpose is to provide a microscope.

[0009]

In the confocal scanning microscope of the present invention, when changing the wavelength of the irradiation light to the sample, the spectroscope that needs to be replaced or adjusted is not arranged in the confocal optical system. , It was decided to install it on the outside of it to deal with the change of the wavelength of the irradiation light without changing the configuration of the confocal optical system that requires precise placement. That is, the confocal scanning microscope of the present invention is (a)
A light source unit that outputs irradiation light that irradiates a sample to be observed, and (b) first optics that inputs the irradiation light output from the light source and changes the output direction of the irradiation light according to designation from the outside. A system, and (c) a second optical system that collects the irradiation light through the first optical system at a first point, which is one point on the sample,
(D) A reaction having a different wavelength from the reflected light or the transmitted light having the same wavelength as the irradiation light, which is output from the first point of the sample as a result of the irradiation light being irradiated to the first point, and the irradiation light. A third optical system for converging the generated light composed of the light at a second point, and (e) a second point on the plane including the second point and perpendicular to the optical axis, and the vicinity of the second point. A light transmission limiting section that selectively transmits only the generated light that has entered the region; (f) a spectroscopic section that disperses the generated light through the light transmission limiting section and sets an optical path according to the wavelength; And a photodetector that detects the generated light output from the spectroscope.

Here, the optical system arranged in the optical path from the first point to the second point includes the second point with respect to the change of the wavelength of the passing light in a predetermined wavelength range, and It is preferable that the change in the position of the point where the vertical plane intersects the optical path is within the transmission region of the light transmission limiting portion.

As the light source, a light source for outputting irradiation light of a wavelength selected according to the type of sample and a wavelength tunable laser device are provided, and the wavelength of the irradiation light is within a predetermined wavelength range when designated from the outside. It is possible to employ a light source that changes continuously. It is also possible to employ a light source that simultaneously outputs irradiation light of a plurality of wavelengths.

The first optical system is composed of a collimating optics for inputting the irradiation light through the light transmission limiter and for making the light into a substantially parallel light that is spatially spread, and a substantially parallel light output from the collimating optics. It is preferable to provide a reflection type optical scanner which inputs a certain irradiation light and changes the output direction of the irradiation light one-dimensionally or two-dimensionally by designation from the outside.

Further, it is practical that the spectroscopic unit is equipped with any one selected from a constant deviation prism, a diffraction grating, and a dichroic mirror.

The third optical system and the light transmission limiting section are arranged in the optical path between the light source section and the first optical system, and the third optical system and the light transmission limiting section are provided between the light source section and the light transmission limiting section. A fourth optical system for converging the irradiation light output from the light source unit to the second point is further provided, and the spectroscopic unit further irradiates between the light source and the fourth optical system. It may be characterized in that it is disposed in the optical path of light. In this case, the irradiation light output from the light source unit is applied to the sample through the spectroscopic unit, the fourth optical system, the light transmission limiting unit, the first optical system, and the second optical system in that order. The generated light output from the first point of the sample as a result of being irradiated with the light is transmitted through the second optical system, the first optical system, the light transmission limiter, and the fourth optical system in the order opposite to the irradiation light. And travels along the optical path of to enter the spectroscope.

Further, the photodetector section has a red component, a green component,
It is also possible to provide a filter for separating the blue component and the blue component, a red component photodetector, a green component photodetector, and a blue component photodetector, and a multichannel photodetector in which the photodetectors are arranged in the spectral direction. It is also possible to provide.

Further, a branching device for branching a part of the irradiation light traveling along the optical path in the second optical system, and a photodetector for detecting a part of the irradiation light branched by the branching device are further provided. It is possible to provide.

[0017]

In the confocal scanning microscope of the present invention, first,
A predetermined output direction of the irradiation light is set in the optical system of. In this state, the irradiation light output from the light source unit is input to the first optical system and output in a predetermined direction. The irradiation light that has passed through the first optical system is condensed by the second optical system at a first point, which is one point of the sample, and a minute spot is formed at the first point. As a result of irradiation with the irradiation light, the generated light is output from the first point on the sample. Such generated light is typically fluorescence or Raman light, and includes transmitted light of irradiation light in a transmission microscope and reflected light of irradiation light in a reflection microscope.

Part of the generated light is input to the third optical system,
It is focused on the second point. A light transmission limiting portion is provided at the second point, and selectively transmits only the generated light that has entered the second point and the area in the vicinity of the second point. As a result, the light generated from the vicinity of the first point of the sample is efficiently transmitted through the light transmission limiting portion, but the light originating from a point different from the vicinity of the first point of the sample is efficiently blocked. . The generated light that has passed through the light transmission limiting unit is split by the spectroscopic unit, and the optical path according to the wavelength is set and output. In this way, the generated light separated for each wavelength is input to the photodetector, and the intensity of the generated light of a predetermined wavelength is detected.

Next, the output direction of the irradiation light different from the above is set in the first optical system. In this state, similar to the above, a minute spot of the irradiation light is formed on the sample,
The minute spot is formed at a point different from the above-mentioned first point. The generated light output from the point of the new minute spot is
In the same manner as above, the intensity of the generated light of a predetermined wavelength is detected. After that, while changing the setting of the output direction of the irradiation light to the first optical system, the intensity of the generated light of a predetermined wavelength is detected,
Observe the sample aspect.

When the wavelength of the irradiation light is changed, the spectroscopic unit and the detector can be detected without changing the configuration of the optical system (that is, the confocal optical system) in the optical path from the first point to the second point. After changing the arrangement position or setting of the components installed outside the confocal optical system, such as a section, the form of the sample is observed in the same manner as above.

[0021]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described below with reference to the accompanying drawings. In the description of the drawings, the same elements will be denoted by the same reference symbols, without redundant description.

FIG. 1 is a configuration diagram of a main part of a confocal scanning microscope according to an embodiment of the present invention. As shown in FIG. 1, the confocal scanning microscope of the present embodiment includes (a) a wavelength tunable laser device 110 which is a light source unit 100 capable of setting a wavelength of output light to be irradiation light, and (b) wavelength. A two-dimensional scanner 200 that inputs the irradiation light that is output from the variable laser device 100 and proceeds toward the sample 900, and outputs the irradiation light toward a specified direction, and (c) the irradiation light that is output from the two-dimensional scanner 200. Of the generated light output from the irradiation area on the sample 900 as a result of irradiation with the optical system 300 that forms a minute spot by condensing A condenser lens 400 that condenses the generated light that has traveled in the direction opposite to the traveling direction of the irradiation light in the order of 300 and the two-dimensional scanner 200, and (e) the condenser lens 400.
Only the light incident near the condensing point of the light generated by
A diaphragm 500 that cuts stray light, (f) a spectroscopic unit 600 that spectroscopically separates the generated light that has passed through the diaphragm 500 and sets an optical path according to the wavelength, and (g) a spectroscopic unit 600 outputs the light.
And a light detection unit 700 that detects light having a predetermined wavelength different from the wavelength of the irradiation light.

Here, the light source section 100 collects the wavelength tunable laser device 110 that emits light having a wavelength corresponding to a designation from a processing device (not shown) and the irradiation light output from the wavelength tunable laser device 110. And a condenser lens 120 that emits light.

Further, the two-dimensional scanner 200 includes a reflecting mirror and a reflecting mirror driving mechanism for orienting the reflecting surface of the reflecting mirror so that the irradiation light directed by the processing device is emitted.

Further, the optical system 300 includes a pupil projection lens 310 for inputting the irradiation light output from the two-dimensional scanner 200, which is sequentially arranged along the traveling direction of the irradiation light.
It is composed of an objective lens 320 which receives the irradiation light output from the pupil projection lens 310 and focuses the irradiation light on one point on the sample 900.

Further, the spectroscopic unit 600, the collimator lens 610 for collimating the generated light output from the diaphragm 500 into a substantially parallel light having a spatial spread, and the collimator lens 6.
The constant deviation prism 621 that inputs the generated light via 10 and changes the optical path according to the wavelength and outputs the light in a direction substantially perpendicular to the input direction, and the incident angle of the generated light is processed according to the wavelength of the irradiation light. A pulse motor 622 for rotationally driving the constant deviation angle prism 621 that sets the direction of the constant deviation angle prism 621 so that the angle is instructed by the device, and the constant deviation angle prism 6
When the generated light via 21 has the same wavelength as the irradiation light, the condensing lens 63 condenses the generated light at substantially the same point where the condensing lens 120 condenses the irradiation light.
1 and a pinhole for transmitting the input light in the vicinity of a point where the generated light having the same wavelength as the irradiation light is condensed by the condenser lens 630, and the constant deviation prism 6
The wavelength selective reflection plate 640 in which a reflection region is formed in a direction corresponding to the wavelength change direction of the optical path set according to the wavelength 21 and the generated light reflected by the wavelength selective reflection plate 640 are substantially parallelized. Collimating lens 650
And The center of rotation for rotating the constant deviation prism 621 in accordance with the wavelength of the irradiation light is the perpendicular to the incident surface passing through the incident point of the generated light having the same wavelength as the irradiation light and the perpendicular to the emitting surface passing through the emitting point. Is set at the intersection point of ## EQU1 ## to suppress the deviation of the optical axis of the generated light having the same wavelength as the irradiation light due to the wavelength of the irradiation light.

Further, the detection unit 700 reflects the red component of the generated light output from the spectroscopic unit 600 via the collimating lens and transmits the blue component and the green component, and the light transmitted through the filter 711. A filter 712 that reflects the blue component and transmits the green component, a photodetector 721 that detects the intensity of the red component reflected by the filter 711, and a photodetector that detects the intensity of the blue component reflected by the filter 712. 722 and the filter 72
Photodetector 723 that detects the intensity of the green component that has passed through 2
And Note that the photodetectors 721, 722, 723
Can detect the intensity of incident light, but a photomultiplier tube was used in this embodiment. Other than the photomultiplier tube,
A photodiode, an avalanche photodiode, or the like can be adopted.

The apparatus of this embodiment observes the sample 900 as follows. In the following description, for the sake of convenience, in an optical component or an optical system through which light passes, except for the constant deviation prism 621, a change in optical path caused by a difference in wavelength of light is ignored.

First, the processor determines the wavelength of the irradiation light,
The output of the light of that wavelength is instructed to the tunable laser 110, and the pulse motor 622 is instructed to rotate so that the incident direction of the irradiation light on the constant deviation prism 621 has the minimum deviation angle. At the same time, the processor gives an instruction that the irradiation position of the irradiation light is the initial position on the sample 900 by the two-dimensional scanner 20.
Notify 0.

After the above setting, the wavelength tunable laser device 11
The irradiation light output from 0 is condensed by the condenser lens 120 and is transmitted through the pinhole of the wavelength selective reflection plate 640. The irradiation light that has passed through the wavelength selective reflection plate 640 becomes substantially parallel light that is spatially expanded by a condenser lens (collimating lens for irradiation light) 630, and the constant deviation prism 62.
1, and is emitted in a direction substantially perpendicular to the incident direction. The irradiation light that has passed through the constant deviation prism 621 is condensed by a collimator lens (a condensing lens for irradiation light) 610, and passes through the opening of the diaphragm 500. The irradiation light that has passed through the diaphragm 500 is collimated by a condenser lens (collimation lens for irradiation light) 400, and the two-dimensional scanner 2
Enter 00. The two-dimensional scanner 200 reflects the input irradiation light in a designated direction by a reflecting mirror whose arrangement direction is set, and outputs it. The irradiation light output from the two-dimensional scanner 200 is irradiated onto the sample 900 via the pupil projection lens 310 and the objective lens 320 of the optical system 300 in order to form one minute spot.

When the irradiation light is irradiated, light (generated light) such as reflected light, fluorescence, or Raman light is output from the minute spot area on the sample 900 irradiated with the irradiation light. Of these generated light, the generated light that reaches the objective lens passes through a path opposite to the path of the irradiation light in the order of the optical system 300, the two-dimensional scanner 200, the condenser lens 400, and the diaphragm 500, and then the spectroscopic unit. Enter 600.

The generated light input to the spectroscopic unit 600 becomes substantially parallel light which is spatially spread through the collimator lens 610, and is incident on the constant deviation prism 621 in the path opposite to that of the irradiation light. In the constant deviation prism 621, the light paths of the lights having different wavelengths are different. Therefore, the constant deviation prism 6
At the time of exiting 21, the exit direction differs depending on the wavelength. Since the reflected light in the generated light has the same wavelength as that of the irradiation light, it is emitted from the constant deviation angle prism 621 via the reverse path of the irradiation light. The generated light emitted from the constant deviation prism 621 is condensed by a condenser lens. Since the reflected light takes a path opposite to that of the irradiation light, it passes through the pinhole of the wavelength selective reflection plate 640. A component of generated light having a wavelength different from the wavelength of irradiation light (such as fluorescence or Raman light)
Is incident on a position different from the pinhole when reaching the wavelength selective reflection plate 640, but such a position is subjected to reflection processing and is reflected. The generated light reflected by the wavelength selective reflection plate 640 is converted into substantially parallel light by the collimator lens 650 and input to the detection unit 700. In the detection unit 700,
The input light is decomposed into a red component, a blue component and a green component by the filter 711 and the filter 712. The red component is the photodetector 710, the blue component is the photodetector 720,
The green component is received by the photodetector 730, and a detection signal corresponding to the received light intensity is notified to the processing device. In the processing device, the information notified from the photodetection unit 700 is used as the irradiation light wavelength and the sample 9
It stores with the irradiation position of 00.

Next, the processing apparatus notifies the two-dimensional scanner 200 of an instruction to change the irradiation position of the irradiation light on the sample 900 to a new position. Thereafter, similarly to the above, the generated light (excluding the reflected light) output from the new irradiation position is detected, and the detection result is stored in the processing device together with the irradiation light wavelength and the irradiation position of the sample 900. After that, the processing device sequentially issues an instruction to scan the irradiation position on the sample 900, detects generated light (excluding reflected light) according to the irradiation position, and outputs the detection result to the irradiation light wavelength and the irradiation position of the sample 900. It is stored together with the processing device. Upon completion of scanning and collection of the detection results, the processing unit uses the stored detection results to detect the sample 9
A two-dimensional observation image of 00 is constructed and displayed.

When changing the wavelength of the irradiation light, the processor determines a new wavelength of the irradiation light, instructs the wavelength tunable laser 110 to output the light of that wavelength, and at the same time, the constant deviation prism of the irradiation light. The rotation instruction is notified to the pulse motor 622 so that the incident direction to 621 has the minimum deviation angle. At the same time, the processing device notifies the two-dimensional scanner 200 of an instruction that the irradiation position of the irradiation light is the initial position on the sample 900. Thereafter, in the same manner as described above, the sample 9 with the new irradiation light wavelength was used.
A two-dimensional observation image of 00 is constructed and displayed. In the apparatus of this embodiment, the constant deviation angle prism 621 of the irradiation light is rotated by the rotation of the constant deviation angle prism 621 according to the wavelength change of the irradiation light.
Since the aperture of the lens and the like are selected so as to be able to cope with the subsequent change of the optical axis, it is not necessary to change the position or the orientation of the parts that are not under the control of the processing device.

FIG. 2 is an explanatory view of a first modification of the spectroscopic unit in the above-mentioned embodiment, showing the structure around the spectroscopic unit.
The structural difference of this device from the above-described embodiment is that (a) the light source unit is composed only of the wavelength tunable laser 110, and (b) the spectroscopic unit 810.

As shown in FIG. 2, the spectroscopic unit 810 includes a collimating lens 811 for collimating the generated light output from the diaphragm 500 into a substantially parallel light having a spatial spread, and an acoustic signal of a frequency instructed by the processing device. Is applied to the acousto-optic element, the generated light is input through the collimating lens 811, and the optical path is changed according to the wavelength by the diffraction grating generated in the acousto-optic element (so that the irradiation light is transmitted as 0th-order light). (Arranged) acousto-optic modulator 812, a condenser lens 813 that condenses the generated light diffracted by the acousto-optic modulator 812, and a slit plate 814 that passes the generated light condensed by the condensing lens 813. , And a collimator lens 815 that substantially collimates the generated light that has passed through the slit plate 814.

In the apparatus of this modification, the sample 900 is observed as follows. As in the above embodiment, in the following description, for the sake of convenience, in optical components or optical systems through which light passes, except for the acousto-optic modulator 812, changes in optical paths caused by differences in light wavelength are ignored. I shall.

First, the processing device determines the wavelength of the irradiation light and the wavelength of the light to be measured, instructs the wavelength tunable laser 110 to output the light of that wavelength, and the light to be measured is condensed by the condenser lens 8.
An acousto-optic modulator 812 is notified of the occurrence of a diffraction grating that is a condition for diffracting light in the direction of 13. At the same time, the processing device notifies the two-dimensional scanner 200 of an instruction that the irradiation position of the irradiation light is the initial position on the sample 900.

After the above setting, the wavelength tunable laser device 11
The irradiation light output from 0 enters the acousto-optic modulator 812, is emitted without being diffracted, is condensed by the collimator lens (condensing lens for irradiation light) 811 and reaches the opening of the diaphragm 500. Thereafter, in the same manner as in the example, sample 900
The top is illuminated to form one micro-spot.

When the irradiation light is irradiated, light (generated light) such as reflected light, fluorescence, or Raman light is output from the minute spot region on the sample 900 irradiated with the irradiation light. Of these generated light, the generated light that reaches the objective lens passes through a path opposite to the path of the irradiation light in the order of the optical system 300, the two-dimensional scanner 200, the condenser lens 400, and the diaphragm 500, and then the spectroscopic unit. Input to 810.

The generated light input to the spectroscopic unit 810 becomes substantially parallel light that is spatially spread via the collimator lens 811, and enters the acousto-optic modulator 812 in the path opposite to that of the irradiation light. The acousto-optic modulator 812 diffracts light having a wavelength that meets the diffraction condition. The generated light diffracted by the acousto-optic modulator 812 is condensed by the condenser lens 813 and passes through the slit 814. The generated light that has passed through the slit 814 is converted into substantially parallel light by the collimator lens 815 and is input to the light detection unit 700. After that, the sample 900 is observed in the same manner as in the example.

FIG. 3 is an explanatory diagram of a third modification of the spectroscopic unit in the above-mentioned embodiment, showing the configuration around the spectroscopic unit.
The structural difference of this device from the above-described embodiment is the structure of the spectroscopic unit.

As shown in FIG. 3, the spectroscopic unit 820 inputs the collimating lens 821 for making the generated light output from the diaphragm 500 into substantially parallel light having a spatial spread, and the generated light via the collimating lens 821. Then, regarding the first-order diffracted light, a diffracted photon 822 that sets and outputs a different optical path for each light wavelength, a condenser lens 823 that condenses the first-order diffracted light diffracted by the diffracted photon 822, and a processing device According to the designation, a wavelength selection slit 824 for blocking the progress of the first-order diffracted light of the generated light having the same wavelength as the irradiation light according to the wavelength of the irradiation light, and a wavelength selection slit 824.
Collimating lens 8 for making the generated light transmitted through
25, and.

In the apparatus of this modification, the sample 900 is observed as follows. As in the above embodiment, in the following description, for the sake of convenience, in optical components or optical systems through which light passes, except for the acousto-optic modulator 812, changes in optical paths caused by differences in light wavelength are ignored. I shall.

First, the processing device determines the wavelength of the irradiation light and the wavelength of the light to be measured, and instructs the wavelength tunable laser 110 to output the light of that wavelength, and at the same wavelength as the irradiation light that is first-order diffracted. The wavelength selection slit 824 is notified that the first-order diffracted light of the light is blocked. At the same time, the processing device notifies the two-dimensional scanner 200 of an instruction that the irradiation position of the irradiation light is the initial position on the sample 900.

After the above setting, the wavelength tunable laser device 11
The irradiation light output from 0 enters the diffraction grating 822,
Next-order diffracted light is condensed by a collimator lens (condenser lens for irradiation light) 821 and reaches the aperture of the diaphragm 500.
After that, in the same manner as in the example, the sample 900 is irradiated and one minute spot is formed.

When the irradiation light is irradiated, light (generated light) such as reflected light, fluorescence, or Raman light is output from the minute spot area on the sample 900 irradiated with the irradiation light. Of these generated light, the generated light that reaches the objective lens passes through a path opposite to the path of the irradiation light in the order of the optical system 300, the two-dimensional scanner 200, the condenser lens 400, and the diaphragm 500, and then the spectroscopic unit. Input to 820.

The generated light input to the spectroscopic unit 820 becomes substantially parallel light that is spatially spread via the collimator lens 821, and enters the diffraction grating 822 in the path opposite to the irradiation light,
Be diffracted. The generated light, which is first-order diffracted by the diffraction grating 822, is condensed by the condenser lens 823, passes through the wavelength selection slit 824 except for the light having the same wavelength as the irradiation light. The generated light that has passed through the wavelength selection slit 824 is made into substantially parallel light by the collimator lens 825 and is input to the light detection unit 700. After that, the sample 900 is observed in the same manner as in the example.

The present invention is not limited to the above example, but can be modified. For example, in the photodetector, it is possible to employ a condenser lens for condensing incident generated light and a multichannel photodetector including photodetectors arranged in the spectral direction (see FIG. 4). It is possible to measure the wavelength distribution of the generated light. Further, in the above example, a lens is used for condensing and collimating, but a reflecting optical device such as a concave mirror can be used, and in this case, deviation of the optical axis due to wavelength can be reduced. Further, even if a dichroic mirror is adopted as the spectroscope of the spectroscopic unit, the spectroscopic unit can be similarly configured.

Further, by providing a half mirror in the confocal optical system and detecting the intensity of the irradiation light reflected by this half mirror, it is possible to monitor the supply of the irradiation light to the confocal optical system. Is. If the arrangement of the light sources is adjusted so that the intensity of the monitor light is maximized, efficient observation can be performed. It should be noted that it is preferable that such monitoring be performed prior to the observation and that the half mirror be removed from the confocal optical system during the observation.

Further, as the light source of the irradiation light, it is possible to adopt a light source which simultaneously outputs irradiation light of a plurality of wavelengths.

Furthermore, in addition to the two-dimensional scanner of the above example,
If the pupil projection lens, the objective lens, the sample, or the like is movable in the optical axis direction and a minute spot of the irradiation light is scanned in the thickness direction of the sample, the three-dimensional aspect of the sample can be observed.

[0053]

As described above in detail, the confocal scanning microscope of the present invention reaches the condensing position of the generated light in which the component for removing the stray light from the minute spot irradiated with the irradiation light on the sample is arranged. In the optical path of the generated light (confocal optical system), since optical parts such as a spectroscope whose optical path changes greatly depending on the wavelength are not installed, when changing the wavelength of the irradiation light,
It is possible to provide a confocal scanning microscope that can easily change the wavelength of irradiation light without changing the configuration of the confocal optical system that requires precise alignment.

[Brief description of drawings]

FIG. 1 is a configuration diagram of a main part of a confocal scanning microscope according to an embodiment of the present invention.

FIG. 2 is an explanatory diagram of a first modified example of the embodiment.

FIG. 3 is an explanatory diagram of a second modification of the embodiment.

FIG. 4 is an explanatory diagram of a modified example of the light source unit of the embodiment.

FIG. 5 is a configuration diagram of a main part of a conventional confocal scanning microscope.

[Explanation of symbols]

100 ... Light source part, 110 ... Laser light source, 120 ... Condensing lens, 130 ... Beam expander, 200 ... Two-dimensional scanner, 300 ... Optical system, 310 ... Pupil projection lens, 32
0 ... Objective lens, 400 ... Condensing / collimating lens, 5
00 ... diaphragm, 600, 810, 820 ... spectroscopic section, 61
0, 811, 821 ... Collimating / condensing lens, 621
... Constant deviation prism, 812 ... Acousto-optic modulator, 822 ...
Diffraction grating, 630, 813, 823 ... Condensing lens, 64
0 ... Wavelength selection reflection plate, 814 ... Slit, 824 ... Wavelength selection slit, 650, 815, 825 ... Collimating lens, 700 ... Photodetector, 711, 712 ... Wavelength filter, 721, 722, 723 ... Photodetector, 900 …sample.

Claims (10)

[Claims] 1. A light source section for outputting irradiation light for irradiating a sample to be observed, and irradiation light output from the light source for inputting, and changing an output direction of the irradiation light according to designation from the outside. 1. an optical system, a second optical system for converging the irradiation light through the first optical system to a first point which is one point on the sample, and the irradiation light is irradiated to the first point. As a result, the generated light composed of reflected light or transmitted light having the same wavelength as the irradiation light and reaction light having a different wavelength from the irradiation light, which is output from the first point of the sample, is generated at the second point. And a third optical system for condensing the light selectively to only the generated light incident on the second point on the plane including the second point and perpendicular to the optical axis and the area in the vicinity of the second point. A light transmission limiting unit that transmits the light and the generated light that has passed through the light transmission limiting unit are dispersed, and an optical path is set according to the wavelength. That the spectroscopic unit and the confocal scanning microscope, characterized in that it comprises a light detector for detecting the output by the generated light from the spectroscope. 2. An optical system arranged in an optical path from the first point to the second point includes a light including the second point with respect to a change of a wavelength of passing light in a predetermined wavelength range. The confocal scanning microscope according to claim 1, wherein a change in position of a point where a plane perpendicular to the axis and the optical path intersect is within a transmission region of the light transmission limiting portion. 3. The confocal scanning microscope according to claim 1, wherein the light source section outputs irradiation light having a wavelength selected according to the type of the sample. 4. The light source comprises a tunable laser device,
The confocal scanning microscope according to claim 1, wherein the wavelength of the irradiation light continuously changes in a predetermined wavelength range by designation from the outside. 5. The first optical system receives the irradiation light from the light transmission limiter and collimates the light into a substantially parallel light which is spatially spread, and a collimator optics output from the collimator optics. Input the irradiation light that is parallel light, and one-dimensional or two-dimensional according to the designation from the outside.
The confocal scanning microscope according to claim 1, further comprising: a reflection type optical scanner that three-dimensionally changes an output direction of the irradiation light. 6. The confocal scanning microscope according to claim 1, wherein the spectroscopic unit includes any one selected from a constant deviation prism, a diffraction grating, and a dichroic mirror. 7. The third optical system and the light transmission limiting section are arranged in an optical path between the light source section and the first optical system, and the light source section and the light transmission limiting section are provided. And a fourth optical system for converging the irradiation light output from the light source unit to the second point, and the spectroscopic unit is provided between the light source and the fourth optical system. The irradiation light output from the light source unit is disposed in the optical path of the irradiation light, and the irradiation light is emitted from the light splitting unit, the fourth optical system, the light transmission limiting unit, the first optical system,
And the generated light output from the first point in the sample as a result of being irradiated with irradiation light through the second optical system, the second optical system, and the second optical system. 2. The confocal point according to claim 1, wherein the first optical system, the light transmission restrictor, and the fourth optical system are sequentially input to the spectroscope by advancing an optical path opposite to the irradiation light. Scanning microscope. 8. The light detecting section comprises a red component, a green component,
The multi-wavelength confocal scanning microscope according to claim 1, further comprising a filter for separating the blue component and the blue component, a red component photodetector, a green component photodetector, and a blue component photodetector. 9. The confocal scanning microscope according to claim 1, wherein the photodetection unit includes a multichannel photodetector in which photodetectors are arranged in a spectral direction. 10. A branching device for branching a part of the irradiation light traveling along the optical path in the second optical system, and a photodetector for detecting a part of the irradiation light branched by the branching device, The confocal scanning microscope according to claim 1, further comprising: