JPH11142745A - Microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the microscope device which facilitates adjustments of an optical system and has a superior S/B. SOLUTION: The exciting light A outputted by a light source 10 is guided into a lens barrel 20 through reflecting mirrors 12 and 13, make incident on the flat surface of the front-end lens 31 of an objective lens 30, projected from the projection surface of the front-end lens 30, and totally reflected by the border surface between a cover glass 51 and a sample 60. As the light is totally reflected, a fluorescent substance present nearby the surface of the sample 60 under evanescent lighting emits fluorescent light B, which is detected by a photodetector 70 through the objective lens 30 and a reflecting mirror 21.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microscope apparatus for evanescently illuminating the vicinity of a surface of a sample, detecting a light beam generated from the vicinity of the surface, and observing the vicinity of the surface.

[0002]

2. Description of the Related Art In a case where two media having different refractive indices are in close contact with each other, if light is incident on the interface from the side of the medium having a higher refractive index at an angle of incidence equal to or greater than a predetermined angle, the light is transmitted to the interface. , And an evanescent wave is generated near the boundary surface in a medium having a low refractive index. By using this evanescent wave, only the vicinity of the boundary surface of the sample can be optically observed.

For example, in a fluorescence microscope apparatus for observing fluorescence generated by evanescently illuminating a sample containing a fluorescent substance with excitation light, only the fluorescent substance existing near the boundary surface of the sample is excited, and other fluorescent substances are excited. Since the fluorescent substance existing in the region is not excited, the spatial resolution of the fluorescence measurement in the direction perpendicular to the boundary surface is improved, and the S / B ratio is improved without generating unnecessary background light. Even weak fluorescence can be detected.

[0004]

In such a microscope apparatus for observing a sample by evanescent illumination, the sample is evanescently illuminated using a prism or the like, and is opposite to the side where the prism or the like is arranged on the sample. The luminous flux generated from the sample was observed through the objective lens arranged on the side. Therefore, if the sample is thick or opaque,
The evanescently illuminated position in the sample could not be observed with the objective lens.

In order to solve such a problem, there has been proposed a microscope apparatus which guides light while repeating total reflection on both surfaces of a thin glass plate to evanescently illuminate a sample placed on the glass surface. (Eg M.Nakache, et a
l., "Topological and modulated distribution of sur
face markers on endothelial cells ", Proc.Natl.Aca
d.Sci.USA, Vol.83, pp.2874-2878, 1986). But,
In this microscope device, it is difficult and impractical to adjust the optical system so that the sample is evanescently illuminated within the field of view of the objective lens.

Further, a light beam used for evanescent illumination passes through an objective lens, and is emitted from the most advanced lens of the objective lens (a lens located closest to the sample among a plurality of lenses in the objective lens), and thereafter, the sample surface. Microscope devices that make the sample incident on the sample so that it is totally reflected by the sample have been proposed (eg, D. Axelrod, "Total Internal Reflection Fl
uorescence Microscopy ", Methods in Cell Biology, V
ol. 30, pp. 245-270, 1989, and M. Tokunaga, et a
l., "Single Molecule Imaging of Fluorophores and E
nzymatic Reactions Achieved by Objective-Type Tota
l Internal Reflection Fluorescence Microscopy ", Bi
ochemical and Biophysical Research Communications,
Vol.235, pp.47-53, 1997). However, in the fluorescence microscope apparatus having such a configuration, since the excitation light passes through the objective lens, autofluorescence generated in the objective lens becomes a problem, and it is difficult to measure weak light.

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and it is an object of the present invention to provide a microscope apparatus which can easily adjust an optical system and has an excellent S / B ratio.

[0008]

A microscope apparatus according to the present invention is a microscope apparatus for evanescently illuminating a sample with a first light beam and observing a second light beam generated from the sample with the evanescent illumination. , A second light beam, and an illumination optical system for evanescently illuminating the sample with the first light beam via the most advanced lens of the objective lens. According to this microscope apparatus, the first light beam is evanescently illuminated on the sample via the most advanced lens of the objective lens by the illumination optical system, and the second light beam generated from the sample with the evanescent illumination by the objective lens. Is detected.

Further, the microscope apparatus according to the present invention further comprises a transparent flat plate which holds the sample in close contact with the surface and totally reflects the first light beam incident through the state-of-the-art lens on the surface. . In this case, the first light beam is emitted from the most advanced lens of the objective lens, enters the transparent flat plate, is totally reflected at the interface between the transparent flat plate and the sample, and the vicinity of the surface of the sample on the transparent flat plate is evanescent. Illuminated. Note that the term “close contact” is not only used when physical contact is made,
It also represents a case where there is a slight gap (for example, several tens of nm) at which an evanescent wave generated near the total reflection surface reaches.

In the microscope apparatus according to the present invention, the sample is held in close contact with the outer surface of the frontmost lens of the objective lens, and the first light beam is totally reflected at the boundary surface between the frontmost lens and the sample. It is characterized by the following. In this case, the first light flux is totally reflected by the outer surface of the frontmost lens of the objective lens, and the vicinity of the surface of the sample on the frontmost lens is evanescently illuminated.

The microscope apparatus according to the present invention is further characterized by further comprising a reflected light guiding means for guiding the reflected light of the first light beam to the outside of the objective lens. In this case, the first light flux is not reflected and scattered in the objective lens, and the generation of autofluorescence is suppressed when the microscope device is used as a fluorescence microscope device.

In the microscope apparatus according to the present invention, the most advanced lens of the objective lens is mainly composed of quartz. Further, in the microscope apparatus according to the present invention, the transparent flat plate is mainly composed of quartz. In any of these cases, when this microscope apparatus is used as a fluorescence microscope apparatus, the generation of autofluorescence is suppressed.

Further, the microscope apparatus according to the present invention comprises a photodetector for detecting an image of the second light beam passing through the objective lens, and an image analyzer for image-analyzing the image of the second light beam detected by the photodetector. And a unit. In this case, the image of the second light flux formed by the objective lens is
The image is detected by the photodetector and analyzed by the image analysis unit.

[0014]

Embodiments of the present invention will be described below in detail 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. Further, a fluorescence microscope apparatus for irradiating the sample with excitation light (first light flux) and observing fluorescence (second light flux) will be described below, but the present invention is not limited to this.

FIG. 1 is a configuration diagram of a microscope device according to the present embodiment, and FIG. 2 is an enlarged configuration diagram of a peripheral portion of an objective lens of the microscope device. The light source 10 outputs excitation light A that excites a fluorescent substance existing in the sample 60. For example, an argon laser light source that outputs laser light having a wavelength of 488 nm as the excitation light A is suitably used as the light source 10. An irradiation time control unit 11 is arranged near the emission port of the light source 10. This irradiation time control unit 11
Performs a shutter opening / closing operation by the control unit 72. The irradiation range adjusting optical system 14 is an optical system for adjusting the range in which the evanescent wave generated by the total reflection of the excitation light A is irradiated on the sample 60.

Each of the reflecting mirrors 12 and 13 sequentially reflects the excitation light A output from the irradiation time control unit 11 and guides the excitation light A into the housing 32. Each of these reflecting mirrors 12 and 13 can be adjusted in position and orientation, and the excitation light A for the foremost lens 31 of the objective lens 30 in the housing 32 is adjusted.
And / or one or both of the incident position and the incident angle can be adjusted.

A stage 40 is provided above the objective lens 30, and a cover glass (transparent flat plate) on which the sample 60 is placed.
51 is placed on the stage 40. This cover glass 5
1 is preferably made of quartz that transmits the excitation light A and the fluorescence B and does not generate autofluorescence as a main component. Further, the space between the cover glass 51 and the most advanced lens 31 of the objective lens 30 is preferably filled with glycerol 50 in order to effectively perform the evanescent illumination of the sample 60 with the excitation light A. The sample 60 is enclosed by a cover glass 52 via a spacer 53. Sample 6
Numeral 0 denotes, for example, a nerve cell (NG-108) stained with 5- (N-hexadecanoyl) aminofluorescein, which is encapsulated alive in a salt solution filled in a space sandwiched between cover glasses 51 and 52. Is done.

A part of the side surface of the foremost lens 31 of the objective lens 30 provided in the mirror body 20 is polished and flattened.
The flat surface 31A has an anti-reflection film formed thereon. Further, it is preferable that the most advanced lens 31 has quartz as a main component that transmits the excitation light A and the fluorescent light B and does not generate autofluorescence. This state-of-the-art lens 31
The excitation light A reflected by the reflecting mirror 13 and introduced into the mirror body 20 is incident on the flat surface 31A and is emitted from the emission surface 31B.

Excitation light A emitted from the exit surface 31B of the state-of-the-art lens 31 is incident on the cover glass 51 via the glycerol 50, and is totally reflected at the interface between the cover glass 51 and the sample 60. An evanescent wave is generated near the boundary. Then, fluorescence B is generated from the fluorescent substance existing near the boundary surface of the sample 60 illuminated by evanescent illumination. The most advanced lens 31 of the objective lens 30 is
Of the generated fluorescence B, the fluorescence B that has reached via the cover glass 51 and the glycerol 50 is incident. Then, the reflecting mirror 21 reflects the fluorescent light B passing through the objective lens 30 and guides the fluorescent light B to the outside of the mirror body 20.

The photodetector 70 has an image pickup surface at an image forming position of the fluorescent light B guided to the outside of the mirror body 20 to detect an image of the fluorescent light B. The image of the detected fluorescence B is analyzed. The control unit 72 controls the output of the excitation light A in the light source 10, controls the shutter opening / closing operation in the irradiation time control unit 11, and also controls the image analysis in the image analysis unit 71.

This microscope apparatus operates as follows.
The excitation light A output from the light source 10 and the irradiation time control unit 11 controlled by the control unit 72 is guided into the mirror body 20 via the irradiation range adjusting optical system 14 and the reflecting mirrors 12 and 13, and Flat surface 31A of state-of-the-art lens 31
At a predetermined incident angle. State-of-the-art lens 31
Is emitted from the emission surface 31B, and enters the cover glass 51 via the glycerol 50. Then, the excitation light A is totally reflected at the interface between the cover glass 51 and the sample 60 and evanescently illuminates the vicinity of the interface of the sample 60.

Fluorescence B generated when a fluorescent substance existing near the boundary surface of the sample 60 is excited by evanescent illumination
Passes through a cover glass 51 and glycerol 50,
The light enters the most advanced lens 31 of the objective lens 30. Then, the fluorescent light B is guided to the outside of the mirror body 20 through the objective lens 30 and the reflecting mirror 21 to form an image on the detection surface of the photodetector 70, and the image is detected by the photodetector 70 and The image is analyzed by the analysis unit 71.

In the microscope apparatus according to this embodiment configured as described above, the sample cell (the cell formed by the cover glass 51, the cover glass 52, and the spacer 53) is moved or replaced on the stage 40. Even so, if the optical thickness of the cover glass 51 is constant, the illumination optical system (the optical system including the reflecting mirror 12, the reflecting mirror 13, and the state-of-the-art lens 31) and the detecting optical system (the objective lens 30) It is not necessary to adjust the optical system including the reflecting mirror 21 and the photodetector 70). In addition, since the excitation light A passes through only the frontmost lens 31 and the cover glass 51 before reaching the surface of the sample 60, the generation of autofluorescence is small, and the material of one or both of the frontmost lens 31 and the cover glass 51 is small. By using quartz, the generation of auto-fluorescence can be further reduced.

Next, the configuration of the objective lens will be described in more detail. FIGS. 3 and 4 are cross-sectional views of the objective lens of the microscope apparatus according to the present embodiment, and show cross-sectional views taken along a plane including the optical axis. The objective lens 30 is composed of eleven lenses including the most advanced lens 31. The state-of-the-art lens 31 has a substantially hemispherical shape, a shape in which both ends are cut perpendicular to the plane of the hemisphere, and one of the cut surfaces at both ends is a flat surface 31A on which the excitation light A is incident. The other is a flat surface from which the totally reflected excitation light is emitted.

FIG. 3 shows a case in which a sample cell is placed via a glycerol 50 on the foremost lens 31 of the objective lens 30 as in the case shown in FIGS. On the other hand, FIG. 4 shows a case where the sample 60 is placed directly on the most advanced lens 31. In any case, the excitation light A needs to be totally reflected on the surface of the sample 60 on the optical axis of the objective lens 30. Therefore, in the cases of FIGS. Face 31
The incident position or incident angle of the excitation light A on A is changed by the reflecting mirrors 12 and 13.

Depending on the incident position and the incident angle of the excitation light A on the most advanced lens 31, the excitation light A is
It is necessary to enter the front end lens 31 after entering the lens housing 32 through the entrance hole (which may be a transparent medium) provided in a part of the lens 2. In this case,
An exit hole (which may be a transparent medium) is provided in the lens housing 32 at a position symmetrical to the entrance hole, and the excitation light A totally reflected on the surface of the sample 60 passes through the frontmost lens 31 and exits The light is guided to the outside of the objective lens 30 through the hole (reflected light guide means). By doing so, the excitation light A
Is not reflected or scattered inside the lens housing 32, so that the generation of auto-fluorescence can be suppressed also in this regard.

FIG. 5 shows the most advanced lens 3 of the objective lens 30.
FIG. FIG. 5B is a diagram viewed from the optical axis direction, and FIGS. 5A and 5C are diagrams viewed from two directions perpendicular to the optical axis and perpendicular to each other.
An antireflection film is formed on the flat surface 31A.

[0028]

As described above in detail, according to the present invention, the first light beam is evanescently illuminated on the sample by the illumination optical system via the most advanced lens of the objective lens, and the evanescent light is emitted by the objective lens. Since the configuration is such that the second light beam generated from the sample due to the illumination is detected, the optical system does not need to be adjusted even when the sample is moved or replaced, and the handling is easy. In addition, since the sample is evanescently illuminated, the spatial resolution of measurement in a direction perpendicular to the boundary surface is high.

A transparent plate for holding the sample in close contact with the surface is provided, and the first light beam incident on the transparent plate via the state-of-the-art lens is totally reflected on the surface. In the case where the sample is held in close contact with the outer surface of the sample and the first light flux is totally reflected at the interface between the state-of-the-art lens and the sample, it is particularly suitable for evanescent illumination of the vicinity of the surface of the sample.

In the case where the apparatus further comprises reflected light guiding means for guiding the reflected light of the first light beam to the outside of the objective lens, the first light
Is not reflected or scattered in the objective lens,
When this microscope device is used as a fluorescence microscope device, generation of autofluorescence is suppressed. In addition, when the most advanced lens of the objective lens is mainly composed of quartz, and when the transparent flat plate is mainly composed of quartz,
When this microscope apparatus is used as a fluorescence microscope apparatus, the generation of autofluorescence is suppressed, and a sample with an excellent S / N ratio can be observed. Further, since the sample can be irradiated with the first light flux having a wavelength of 340 nm or less, it is possible to excite and observe a protein which is not fluorescently labeled.
The photodegradable reagent contained in the sample can be photolyzed.

In a case where the apparatus further includes a photodetector for detecting an image of the second light beam having passed through the objective lens, and an image analyzing unit for performing image analysis on the image of the second light beam detected by the photodetector. The image of the second light flux formed by the objective lens is detected by the photodetector and image-analyzed by the image analysis unit, thereby facilitating the analysis of the vicinity of the surface of the evanescently illuminated sample.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of a microscope apparatus according to an embodiment.

FIG. 2 is an enlarged configuration diagram of a peripheral portion of an objective lens of the microscope device according to the present embodiment.

FIG. 3 is a sectional view of an objective lens of the microscope device according to the embodiment.

FIG. 4 is a cross-sectional view of the objective lens of the microscope device according to the present embodiment.

FIG. 5 is an explanatory diagram of the most advanced lens of the objective lens of the microscope device according to the present embodiment.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 10 ... Light source, 11 ... Irradiation time control part, 12, 13 ... Reflecting mirror, 14 ... Irradiation range adjustment optical system, 20 ... Mirror, 21 ... Reflecting mirror, 30 ... Objective lens, 31 ... State-of-the-art lens, 32 ...
Lens housing, 40 ... stage, 50 ... glycerol,
51, 52: cover glass, 53: spacer, 60: sample, 70: photodetector, 71: image analysis unit, 72: control unit, A: excitation light, B: fluorescence.

Claims (7)

[Claims] 1. A microscope apparatus for evanescently illuminating a sample with a first light beam and observing a second light beam generated from the sample with the evanescent illumination, wherein the objective lens inputs the second light beam. A microscope apparatus comprising: an illumination optical system configured to evanescently illuminate the first light flux on the sample via a most advanced lens of the objective lens. 2. The apparatus according to claim 1, further comprising: a transparent flat plate that holds the sample in close contact with a surface and totally reflects the first light beam incident through the front-end lens on the surface. Microscope equipment. 3. The method according to claim 1, wherein the sample is held in close contact with an outer surface of the frontmost lens of the objective lens, and the first light flux is totally reflected at a boundary surface between the frontmost lens and the sample. The microscope device according to claim 1, wherein 4. The microscope apparatus according to claim 1, further comprising a reflected light guiding means for guiding the reflected light of the first light beam to outside the objective lens. 5. The microscope apparatus according to claim 1, wherein the most advanced lens of the objective lens is mainly composed of quartz. 6. The microscope apparatus according to claim 2, wherein the transparent flat plate has quartz as a main component. 7. A photodetector that detects an image of the second light beam that has passed through the objective lens, and an image analysis unit that performs image analysis on the image of the second light beam detected by the photodetector. The microscope apparatus according to claim 1, further comprising: