JP2005091615A - Microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To collectively acquire spectral information of a relatively wide area on a sample without scanning the sample, in a microscope. <P>SOLUTION: The microscope 1 provided with an illuminating optical system 5, an objective lens 6, and a spectroscope 14 is provided with an imaging optical system 12 taking an entrance aperture 14a of the spectroscope 14 and a pupil surface 7 of the objective lens as a conjugate pair. Thus light from the entire sample in an illumination range can be collectively led to the spectroscope. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a microscope. In particular, the present invention relates to a microscope capable of acquiring spectroscopic information of a specimen.

In recent years, with the development of medicine and life sciences, there is a strong demand for microscopes for precisely studying the fine structures of living tissues and cells. Therefore, various microscopes have been proposed that can acquire spectroscopic information of a specimen and perform detailed analysis of an observation target. For example, a microscope is known in which a specimen is fluorescently stained for each specific tissue, cell, and protein, and the fluorescence can be spectroscopically observed.
For example, in Patent Document 1, illumination light is irradiated onto a sample (specimen), and a lens, a direct-viewing prism as a spectroscopic element, and a photodetector are arranged on the optical path of the transmitted light. Describes a spectroscopic scanning microscope that is arranged so that is conjugated with each other and that the sample can be scanned in a direction perpendicular to the optical axis.
Further, for example, in Patent Document 2, a laser light source capable of scanning a specimen is used as illumination means, a confocal stop is provided at a position where light from the specimen is condensed, and a grating which is a spectroscopic element is provided at the subsequent stage. A scanning optical microscope is described in which a slit is disposed at a predetermined position corresponding to the wavelength of the dispersed light to provide a photodetector that detects light of a specific wavelength.
JP-A-5-172741 (page 3, Fig. 1-4) JP-A-8-43739 (page 4-5, FIG. 1-6)

However, the conventional microscope as described above has the following problems.
In the technique described in Patent Document 1, since the entrance aperture of the specimen and the photodetector is in a conjugate positional relationship, light from one point or a minute area on the specimen is dispersed by the direct-view prism and is incident on the photodetector. . For this reason, the received light intensity is reduced, and it is further dispersed, so that there is a problem that the S / N ratio cannot be increased. In particular, the light received from the specimen has a very weak light receiving intensity when performing fluorescence observation with a low light intensity.
On the other hand, since there is a limit to the intensity of illumination light in order not to deteriorate the specimen, it is necessary to increase the incident aperture of the spectroscopic element in order to increase the amount of light received by the photodetector. In this case, the wavelength resolution is lowered, and there is a problem that precise measurement cannot be performed.
In addition, in order to acquire spectral information on the entire surface of the specimen, the specimen must be scanned, so that there is a problem that it takes time for measurement.
Further, since the scanning microscope described in Patent Document 2 is a confocal microscope, similarly to Patent Document 1, light at one point or a minute region on the specimen is dispersed and guided to a photodetector at a conjugate position. There is a problem similar to that of Patent Document 1 in that the amount of received light is small and it is necessary to scan the specimen.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a microscope that can collectively acquire spectral information of a relatively wide area on a specimen without scanning the specimen.

In order to solve the above-described problems, in the invention described in claim 1, a light source, an illumination optical system that illuminates the sample with light emitted from the light source, and an image of light from the illuminated sample are formed. The microscope includes an objective lens and a spectroscopic optical system that splits light from the specimen, and an imaging optical system that conjugates the entrance aperture of the spectroscopic optical system and the pupil of the objective lens.
According to this invention, since the pupil of the objective lens, that is, the rear focal plane of the objective lens and the entrance aperture of the spectroscopic optical system are conjugated by the imaging optical system, the light reflected from the specimen and emitted from the specimen All of the fluorescence parallel to the optical axis reaches the entrance aperture of the spectroscopic optical system. Therefore, the light from the specimen can be collectively taken into the spectroscopic optical system within the effective diameter range of the objective lens.

According to a second aspect of the present invention, in the microscope according to the first aspect, the illumination optical system is configured such that the illumination range on the specimen can be varied.
According to the present invention, by changing the illumination range, light in a region on the illuminated specimen reaches the entrance aperture of the spectroscopic optical system without scanning the specimen or the optical system, and spectral information is acquired. be able to.

According to a third aspect of the present invention, in the microscope according to the first or second aspect, the imaging optical system is an optical system that projects a reduced pupil of the objective lens.
According to the present invention, since the pupil of the objective lens is reduced and projected, the light passing through the pupil of the objective lens can be efficiently collected, and light quantity loss due to the entrance aperture of the spectroscopic optical system can be prevented. Therefore, it is possible to perform spectroscopic observation with high light utilization efficiency.

  According to the microscope of the present invention, all of the reflected light from the specimen and the fluorescence emitted from the specimen that is parallel to the optical axis reach the entrance aperture of the spectroscopic optical system. Thus, spectral information of a relatively wide area can be acquired collectively, and bright spectral observation can be performed quickly.

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In all the drawings, even if the embodiments are different, the same or corresponding members are denoted by the same reference numerals, and redundant description is omitted.
FIG. 1 is a configuration block diagram for explaining a schematic configuration of a microscope according to an embodiment of the present invention. 2A and 2B are schematic explanatory diagrams for explaining a configuration example of a spectroscopic optical system that can be used in the microscope according to the embodiment of the present invention.
These are schematic views, and for example, even a plurality of lenses and lens groups are represented as one member in the drawing for convenience.

As shown in FIG. 1, the microscope 1 according to the embodiment of the present invention is an episcopic microscope, and the schematic configuration thereof includes an illumination optical system 5, an objective lens 6, an eyepiece lens 15, an imaging optical system 12, and a spectroscopic system. Instrument 14 (spectral optical system).
The illumination optical system 5 illuminates the object surface 8 on which the specimen is arranged through the objective lens 6 and the light source 2 such as a mercury lamp, the field stop 3 for adjusting the field of view, and the light transmitted through the field stop 3. The lens 4 is provided. As the illumination method using the lens 4, for example, a well-known illumination method such as Kohler illumination can be adopted. The half mirror 9 is for guiding the light beam emitted from the condenser lens 4 onto the optical axis of the objective lens 6.
Although not shown, the illumination optical system 5 includes appropriate optical elements such as a filter element and a polarizing plate element, for example, as required for the observation application of the microscope 1.

In the present embodiment, the field stop 3 can naturally change the size of the aperture in accordance with the magnification of the objective lens 6, for example, but the size of the aperture can be freely changed in order to change the illumination range around the optical axis. It can be varied.
Further, the aperture center is decentered with respect to the optical axis so that the illumination range on the specimen placed on the object plane 8 can be varied. Further, the aperture shape of the field stop 3 may be variable to an appropriate shape other than a circle.

In the case of this embodiment, the objective lens 6 is for imaging light from the specimen, and the pupil plane 7 of the objective lens is formed on the rear focal plane. The objective lens 6 is configured so that the magnification can be varied by switching and arranging a plurality of lenses, for example, by a turret method, or by using the objective lens 6 as a zoom lens.
The eyepiece 15 is for enlarging the image of the specimen formed by the objective lens 6. In FIG. 1, reference numeral 50 indicates a pupil.

  The imaging optical system 12 includes an imaging lens 10 provided on the optical path between the half mirror 9 and the eyepiece lens 15, and a half mirror 13 that branches a part of the light beam transmitted through the imaging lens 10 to the outside of the optical axis. And the lens 11 provided on the optical path of the light beam branched by the half mirror 13, and the pupil plane 7 of the objective lens and the entrance aperture 14a of the spectroscope 14 are conjugate and projected so as to be reduced. Placed in.

The spectroscope 14 is an optical system for acquiring spectral information of an incident light beam.
As the optical system of the spectroscope 14, various known spectroscopic optical systems can be adopted depending on the purpose of spectroscopic observation. Here, those examples will be briefly described.
FIG. 2A shows an example in which the spectrometer 14 is a so-called Czerny-Turner spectrometer.
In this example, an incident aperture 14a, a concave mirror 16, a diffraction grating 17, a concave mirror 18, and a line sensor 19 are arranged along the optical path.
The divergent light that has passed through the incident aperture 14a is collected by the concave mirror 16 to be substantially parallel light, and is diffracted by the diffraction grating 17 in a direction corresponding to the wavelength of the incident light. Then, the concave mirror 18 forms an image of those light beams. A line sensor 19 for detecting the light intensity at the imaging position of each wavelength light is disposed on the imaging surface so that the spectral intensity distribution can be measured.
In this example, it is preferable to increase the number of diffraction gratings 17 so that the diffraction efficiency in the detection wavelength band is not lowered. In this case, the dispersion increases, so that even if the slit width of the incident aperture 14a is wide, the spectral components on the imaging surface are not easily mixed, and there is an advantage that the spectral can be efficiently performed.

FIG. 2B shows an example in which the spectroscope 14 is a spectroscope using a prism.
In this example, the incident aperture 14a, the lens 20, the prism 21, the lens 22, and the line sensor 19 are arranged along the optical path.
The divergent light that has passed through the incident aperture 14a is collected by the lens 20 to be substantially parallel light, and is refracted by the prism 21 in a direction corresponding to the wavelength of the incident light. The lens 22 forms an image of these light beams. A line sensor 19 is disposed on the image plane.
In the above two examples, the line sensor 19 is used as the spectral distribution measuring means. However, an area sensor or a sensor in which a slit scans the photodetector may be used.

The action of the microscope 1 of the present embodiment will be described focusing on the action at the time of spectroscopic observation.
The specimen on the object plane 8 is illuminated by the illumination optical system 5. At that time, by setting the field stop 3 to have a substantially circular opening at the center of the optical axis, it is possible to illuminate the sample as a whole within a range corresponding to the numerical aperture of the objective lens 6. Then, reflected light or fluorescence from the specimen exiting substantially parallel to the optical axis is condensed by the objective lens 6 near the center of the pupil plane 7 of the objective lens.
The light beam that has passed through the vicinity of the center of the pupil plane 7 of the objective lens passes through the half mirror 9 while being diverged, is collected by the imaging lens 10, and partly reflected off the optical axis by the half mirror 13. Then, the light is condensed at the position of the entrance opening 14 a by the lens 11.
Here, since the pupil plane 7 of the objective lens and the entrance aperture 14a are conjugated, information over the entire illumination range of the specimen is condensed on the entrance aperture 14a. Further, since the magnification of the imaging optical system 12 is an optical system for reducing and projecting the pupil plane 7 of the objective lens, by setting the reduction magnification to an appropriate value, an image in the range of the pupil diameter can be entered. The diameter can be reduced to a diameter smaller than the opening width. As a result, the entire pupil of the objective lens is incident on the incident aperture 14a without loss of light quantity.
The spectral distribution of the light beam incident on the incident aperture 14a is measured by the spectroscope 14, and the spectral information of the sample can be acquired.

  As described above, according to the microscope 1 of the present embodiment, the light beam passing through the pupil plane 7 of the objective lens can be guided to the entrance aperture 14a as a whole, so that efficient spectroscopic observation with no light loss is performed. There is an advantage that can be. Further, since the entire spectroscopic information of the sample image can be acquired collectively by the spectroscope 14 without scanning the sample, there is an advantage that the spectral information can be acquired quickly.

Further, according to the microscope 1, the field stop 3 can be reduced in diameter so that the illumination range can be reduced around the optical axis.
In addition, the field stop 3 can be reduced in diameter or the aperture shape can be varied to narrow the illumination range. Further, by decentering the optical axis center, the size and position of the illumination range on the specimen can be reduced. Can be varied. For example, the illumination range can be limited to the range of the region W in FIG. 1, and only the reflected light of the region W on the specimen reaches the entrance opening 14a along the optical path as illustrated. In this way, there is an advantage that spectral information of a desired region on the specimen can be acquired.
For example, during fluorescence observation, when a fluorescent protein with a fluorescent wavelength close to a specific region of a specimen is distributed and cannot be accurately identified with the naked eye, the specific region is illuminated and a precise spectral distribution is measured. It becomes possible. At this time, since the wavelength light in the other region that causes noise can be excluded in advance, there is an advantage that measurement with a large dynamic range can be performed.

In the present embodiment, the microscope 1 has been described as an epi-illumination microscope, but it goes without saying that the same effect can be obtained even if it is a transmission microscope. It is very easy to provide a transmissive illumination optical system in place of the illumination optical system 5 in FIG.
In addition, since the incident aperture 14a has a slit shape, it is preferable that the light beam incident on the incident aperture 14a has a flat shape parallel to the slit in order to improve the light utilization efficiency while ensuring the spectral resolution. Therefore, the imaging optical system 12 may include an optical element such as a cylindrical lens.

As a modification of the present embodiment, an example in which the illumination optical system 5 is changed can be given. The following briefly describes the differences from the above embodiment.
FIG. 3 is a configuration block diagram for explaining a schematic configuration of a microscope according to a first modification of the embodiment of the present invention. FIG. 4 is a block diagram illustrating a schematic configuration of a microscope according to a second modification of the embodiment of the present invention.

The microscope 100 according to the first modification of the present embodiment is a laser microscope, and is particularly suitable for a laser microscope of two-photon excitation or multiphoton excitation (hereinafter collectively referred to as multiphoton excitation). Is. As shown in FIG. 3, an illumination optical system 24 is provided instead of the illumination optical system 5 described above.
The illumination optical system 24 includes a laser 25 (light source), a scanner 26, and lenses 27 and 28.
The laser 25 is a light source for performing multiphoton excitation.
The scanner 26 is a mechanism for scanning the spot of the laser 25 on the specimen, and is composed of an optical scanning element such as a galvanometer mirror.
The lenses 27 and 28 are scanning optical systems for forming an image of a light beam emitted from the laser 25 on the object plane 8 with a minute spot diameter and scanning at an appropriate speed.

Multi-photon excitation is an excitation method in which a light beam having a wavelength n times the excitation light wavelength is irradiated as excitation light for exciting fluorescent molecules, for example. Such long-wavelength excitation light can cause photoexcitation similar to that in which one photon of the original excitation wavelength light is absorbed by absorbing n photons with respect to the fluorescent molecule. Therefore, it is known that there is an advantage that fluorescence observation can be performed using long wavelength light with little damage to the specimen.
However, in the case of 1-photon excitation, a large number of photo-excitations occur in the light transmission range, whereas in n-photon excitation, the probability that n photons are absorbed simultaneously (in a very short time) by a specific fluorescent molecule is extremely small. Therefore, photoexcitation occurs substantially only at a focal position with a high energy density. Therefore, there is a problem that the light amount is smaller than that of the one-photon excitation, and the S / N ratio becomes small when performing spectroscopic observation.

  However, according to the microscope 100 of the present modification, the light beam passing through the pupil plane can be condensed within the width of the entrance aperture 14a, so that even if the light from the specimen is very small, the spectrometer 14 can be efficiently used. This has the advantage that a relatively high S / N ratio can be obtained. Therefore, the microscope 100 is very suitable as a laser microscope for two-photon excitation or multiphoton excitation.

The microscope 101 of the second modified example of the present embodiment is a microscope capable of total reflection fluorescence observation, and includes an illumination optical system 29 instead of the illumination optical system 5 as shown in FIG. 6 is provided with an oil immersion objective lens 33 (objective lens).
The illumination optical system 29 irradiates the specimen 8b disposed on the cover glass 8a with illumination light totally reflected from the cover glass 8a side, and the specimen 8b in the vicinity of the cover glass 8a surface by the evanescent light that oozes out to the specimen 8b side. Is used for lighting. Such an illumination optical system can be provided in either a transmission type or an epi-illumination microscope, but FIG. 4 shows an epi-illumination configuration.
The configuration of the illumination optical system 29 is the same as that of a known evanescent illumination optical system, and includes a laser 25 (light source), a lens 30, a field stop 31, a lens 32, and an oil immersion objective lens 33.

The light beam emitted from the laser 25 is incident on the lens 30 in an eccentric manner to be a parallel light beam that passes through the focal point of the lens 30. The field stop 31 is for restricting the size of the light beam transmitted through the focal position to restrict the illumination range.
The light beam that has passed through the field stop 31 is collected by the lens 32, reflected by the half mirror 9, and decentered into the oil immersion objective lens 33. Here, the lenses 30 and 32 constitute an optical system in which the emission point of the laser 25 and the pupil plane 7 of the objective lens which is the rear focal plane of the oil immersion objective lens 33 are conjugated.
The oil immersion objective lens 33 is an optical element that diffuses from the pupil plane 7 of the objective lens while decentering it and makes it a parallel light flux, skews it toward the center of the optical axis and totally reflects it on the inner surface side of the cover glass 8a. . Reference numeral 33a is oil.

According to such a microscope 101, the light from the specimen 8 b that has been evanescently illuminated by the illumination optical system 29 is condensed on the pupil plane 7 of the objective lens by the oil immersion objective lens 33 and is incident on the imaging optical system 12. Then, the light enters the spectroscope 14 through the incident aperture 14a.
Since evanescent illumination uses evanescent light that oozes out to a range of about several hundreds of nanometers from the total reflection surface, the light from the specimen 8b is extremely weak light. Therefore, for example, when the amount of light at one point or a minute region of the specimen is guided to the spectrometer, the light becomes much weaker, but in this modification, the light from the entire specimen can be incident on the spectrometer 14. There is an advantage that spectroscopic measurement with a high S / N ratio can be performed.

In the above description, in order to perform efficient spectroscopic observation, the entrance aperture 14a and the pupil plane 7 of the objective lens are both conjugate to each other. The lens arrangement of the imaging optical system 12 may be variable so that the above can be performed. That is, by switching the lens arrangement, the state where the entrance aperture 14a is conjugated with the pupil plane 7 of the objective lens and the state where it is conjugated with the object plane 8 may be switched. In that case, it is preferable to arrange | position the pinhole slit for making it confocal before the entrance opening 14a.
If constituted in this way, there is an advantage that it can be set as the microscope which has the function of the conventional spectroscopic observation.

It is a block diagram for explaining a schematic configuration of a microscope according to an embodiment of the present invention. It is a schematic explanatory drawing for demonstrating the structural example of the spectroscopic optical system which can be used for the microscope which concerns on embodiment of this invention. It is a block diagram for demonstrating schematic structure of the microscope of the 1st modification of embodiment of this invention. It is a block diagram for demonstrating schematic structure of the microscope of the 2nd modification of embodiment of this invention.

Explanation of symbols

1, 100, 101 Microscope 2 Light source 3 Field stop 5, 24, 29 Illumination optical system 6 Objective lens 7 Objective lens pupil plane 8 Object plane 8b Sample 12 Imaging optical system 14 Spectrometer (spectral optical system)
14a Entrance aperture 15 Eyepiece 17 Diffraction grating 19 Line sensor 21 Prism 25 Laser (light source)
26 Scanner 33 Oil immersion objective (objective lens)

Claims (3)

A light source; an illumination optical system that illuminates the specimen with light emitted from the light source; an objective lens that forms an image of light from the illuminated specimen; and a spectroscopic optical system that separates the light from the specimen A microscope,
A microscope comprising an imaging optical system in which an entrance aperture of the spectroscopic optical system and a pupil of the objective lens are conjugate.   The microscope according to claim 1, wherein the illumination optical system is configured to change an illumination range on the specimen.   The microscope according to claim 1, wherein the imaging optical system is an optical system that projects a reduced pupil of the objective lens.

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