JPH05172741A - Spectroscopic scanning microscope - Google Patents

Abstract

(57) [Abstract] [Purpose] To add a spectral function to a scanning optical microscope without significantly changing the configuration. [Structure] A direct-view prism 8 is provided in a detection optical system of a scanning optical microscope having an optical system of a light source 1, lenses 40 and 41, and a detector 31.
The photodetector 32 that separates and detects the light 60 and 61 split by the 0 and the direct-view prism 80 is used. [Effect] Since the direct-view prism can make the deviation angle to the center wavelength zero, the image formed by the light detected at the center is the same as that of the conventional scanning optical microscope, and the configuration of the scanning optical microscope is almost the same. does not change.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a spectroscopic scanning microscope, and more particularly to a spectroscopic scanning microscope in which a scanning optical microscope is provided with a spectroscopic function for dispersing light generated from a light source or a sample.

[0002]

2. Description of the Related Art The simplest method of adding a spectral function to a scanning optical microscope is to insert an optical filter in the optical path of a detection optical system of the scanning optical microscope. This method is actually performed in a confocal type scanning optical microscope, and Applied Optics Vol. 29, (1990) 489.
Pages 494 (Applied Optics, 29
(1990) PP489-494). Also, without significantly changing the configuration of the scanning optical microscope,
As a method for realizing the spectral function, the present inventors have invented a spectral scanning microscope in which a lens having large chromatic aberration is used in front of a pinhole of a confocal optical microscope (Japanese Patent Application No. 63-12).
667, JP-A-1-188816).

[0003]

In the spectroscopic scanning microscope in which an optical filter is inserted in the optical path of the detection optical system, the characteristics of the optical filter are fixed, so that the optical filter is adjusted according to the wavelength of fluorescence emitted from the observation sample. Need to prepare. Therefore, it is troublesome to select the optimum optical filter. Further, in order to obtain an observation image of the sample with light of a plurality of wavelengths at the same time, it is necessary to divide the light, which is difficult. A spectroscopic scanning microscope that uses a lens with large chromatic aberration before the pinhole of a confocal optical microscope can move the pinhole (detector) on the optical axis according to the wavelength.
Although the spectroscopic function can be provided without largely changing the configuration of the scanning optical microscope, it is impossible to obtain sample images of different wavelengths at the same time. In addition, it is generally considered to dispose a spectroscope on the scanning optical microscope, but the output light from the spectroscope is largely displaced from the optical axis of the scanning microscope. Inevitably, it would have to be placed in a different position, which would greatly change the configuration of the conventional scanning optical microscope. Therefore, a main object of the present invention is to realize a scanning microscope by adding a spectroscopic function to the scanning optical microscope without largely changing the configuration of the conventional scanning optical microscope. Another object of the present invention is to realize a smaller spectroscopic scanning microscope having a spectroscopic function, in which a detector can be arranged on the optical axis, and at the same time, optical images of different wavelengths can be obtained.

[0004]

In order to achieve the above object, the present invention provides a sample holding mechanism for arranging an observation object (sample) at a focus position of light from a light source in a spectroscopic scanning microscope, and the above light. An optical system that irradiates the sample as converged light, a scanning mechanism that scans the sample or the converged light, a direct-view prism that separates the transmitted light or reflected light from the sample, and a light that is split by the direct-view prism. It is configured to include a photodetector for detecting, and a display unit for displaying the irradiation position of the light on the sample and the output of the photodetector in association with each other.

[0005]

By using the direct-view prism in the light detection system of the scanning optical microscope, the optical axes of the optical system before and after the light-splitting by the direct-view prism can be kept almost the same, so that the inside of the limited barrel of the scanning optical microscope can be maintained. Since the direct-view prism and the photodetector can be arranged in the optical axis, the spectroscopic function can be easily realized without requiring a large change in the optical system of the conventional scanning optical microscope. As shown in FIG. 2, the optical system of the scanning optical microscope narrows the light emitted from the light source 1 to the observation object (sample) 2 with the lens 40 and makes it the probe light 5. The light that has passed through the observation object 2 is a lens 4
1 is collected by the photodetector 30 as detection light 60 including the information of the object 2. On the other hand, the observation object 2 is scanned by the scanning mechanism 70. The control circuit 71 controls the moving mechanism 70 and the display device 72, and displays the signal obtained at the scanning position of the object 2 on the display device 72 as an image.

In the spectroscopic scanning microscope of the present invention, since the direct-view prism is inserted in the detection optical system of the scanning optical microscope shown in FIG. 2, the deviation angle of the direct-view prism with respect to the wavelength of the light from the light source 1 is zero. Has become. When using an observation object in which fluorescence from the light source emits fluorescence that is different from the wavelength of the light from the light source, the fluorescence is slightly offset from the optical axis and is a plane perpendicular to the optical axis. The light will be condensed on the same plane as that on which the light is condensed. By detecting the light from the light source and the fluorescence with the array detector arranged on the same plane, it is possible to take an image of the observation object by the fluorescence and an image of the observation object by the light transmitted from the light source. Also, if the wavelength of the pixel position of the array detector is calibrated, it is possible to perform the spectroscopy at a certain observation place. As described above, the direct-view prism can make the deviation angle to zero with respect to the central wavelength (for example, the wavelength of the light from the light source). The conventional scanning optical microscope can be easily mounted without changing the lens barrel significantly.

[0007]

EXAMPLES Examples of the present invention will be described below. First Embodiment FIG. 1 is a block diagram showing the configuration of a first embodiment of a spectroscopic optical scanning microscope according to the present invention. The laser light emitted from the laser light source 1 is converged on the observation object 2 by the lens 40 and is emitted as the probe light 5. That is, the observation object 2 is installed at the focus position of the laser light. The observation object 2 is excited by the probe light 5 to generate fluorescence. The light transmitted through the observation object 2 is combined with the fluorescent light by the lens 4
The light is converged at 1, and is irradiated onto the detection surface of the photodetector 31 as detection light 60 via the direct-view prism 80. At that time, the laser light 60 is not deflected by the direct-view prism 80. Since the wavelength of the fluorescent light is different from the wavelength of the laser light 60, the fluorescent light 61 is deflected by the direct-view prism 80. The photodetector 31 is installed at the focal position on the optical axis. Observation object 2
Is moved by a scanning mechanism (stage) 70 on a surface perpendicular to the paper. The control circuit 71 controls the scanning mechanism 70 and the display device 72, and displays the signal obtained at the scanning position of the sample 2 on the display device 72 as an image. By separating and detecting the laser light 60 and the fluorescence 61 with the photodetector 31,
It is possible to obtain an image of the observation object 2 based on only fluorescence and an image of the observation object 2 based on only transmitted light.

The direct-view prism 80 is conventionally known, and a plurality of triangular prisms are combined to reduce the deflection of light of a specific wavelength (for example, Fraunhofer's d-line) to 0, and other wavelengths. However, the present invention is not limited to this, but a combination of 3 or 5 pieces of lead glass (flint glass) and crown glass can be used. It suffices that the deflection angle can be made zero with respect to light of a specific wavelength (center light) such as laser light from the light source 1 and that it has a function of dispersing light such as other fluorescence. As described above, in the present embodiment, since the direct-view prism 80 can make the deviation angle to the laser light from the light source 1 zero, the direct-view prism 80 and the photodetector 31 can be arranged on the optical axis.
There is no need to change the lens barrel.

Embodiment 2 FIG. 3 is a block diagram showing the configuration of the second embodiment of the spectral scanning microscope according to the present invention. In this embodiment, the transmitted light of the observation object is made into a parallel light flux, and a direct-view prism is arranged in the parallel light flux to constitute a telecentric optical system. In FIG. 3, the same functions and components as those of FIG. 1 are designated by the same reference numerals. By the arrangement of the lenses 40 and 41 and the setting of the focal length, the light transmitted through the lens 41 becomes parallel light. The direct-view prism 80 is disposed at the front focal point of the lens 42 in the transmitted light. The direct-view prism 80 includes a laser light 60 from a light source and a fluorescent light 61 excited by the laser light.
Are separated by their wavelength difference. The detector 32 has a slit arranged near the detection surface. In this embodiment, the distance between the lenses 41 and 42 can be freely set to some extent, which facilitates the design of the device with respect to the axial length of the lens barrel. The detection wavelength can be changed by arranging the detector 32 to move in the back focal plane of the lens 42. The slit may be replaced with a pinhole.

Embodiment 3 FIG. 4 is a block diagram showing the configuration of a third embodiment of the spectral scanning microscope according to the present invention. In this embodiment, the light source 11
The laser light source 11 that generates a plurality of light wavelengths of red, green, and blue is used as the above. Laser light source 1
The laser light from No. 1 is collimated by the lens 43, the pinhole 91 and the lens 44, and becomes a parallel light flux.
A semitransparent mirror 90 is arranged in the optical path of the parallel light flux. The laser light that has passed through the semi-transparent mirror 90 is condensed on the observation object 20 by the lens 45. A part of the light reflected on the observation object 20 is collimated by the lens 45, reflected by the semitransparent mirror 90, and split into the red light 62, the green light 63, and the blue light 64 by the direct-view prism 80. The arrayed detectors 31 are irradiated with the separated light of three colors by the lens 42 and detected. The respective outputs corresponding to the three color lights 62, 63 and 64 from the array detector 31 are used as RGB signals for color display of the display device 72. Reference numeral 70 denotes a scanning mechanism for scanning the observation object 20, and the moving mechanism 70 and the display device 72 are controlled by the control circuit 71. In the present embodiment, the configuration of the observation object 20 can be displayed in color by utilizing the difference in reflectance due to the difference in the wavelength of the components of the observation object 20. In the above embodiment, the laser light source 11 is a laser light source that generates a plurality of light wavelengths of red, green, and blue. However, a white light source having a wide wavelength distribution is used as a light source, and an optical filter is provided. , A slit is arranged near the detector 31 at a position according to the wavelength, and red,
You may comprise so that the signal corresponding to green and blue may be selected.

[0011]

As described above, according to the spectroscopic scanning microscope of the present invention, the structure of the conventional scanning optical microscope can be greatly increased by using the direct-view prism and the photodetector for detecting the dispersed light. There is an effect that the spectroscopic function can be easily attached to the scanning optical microscope without changing, and it is simpler than that using an ordinary spectroscope.

[Brief description of drawings]

FIG. 1 is a configuration block diagram of a first embodiment of a spectral scanning microscope according to the present invention.

FIG. 2 is a general configuration diagram of a scanning optical microscope.

FIG. 3 is a configuration block diagram of a second embodiment of the spectral scanning microscope according to the present invention.

FIG. 4 is a configuration block diagram of a third embodiment of a spectral scanning microscope according to the present invention.

[Explanation of symbols]

 1 ... Irradiation light source, 2 ... Observing object 30 ... Photodetector 31 ... Array detector 40, 41 ... Lens 5 ... Probe light 60 ... Detection light 61 ... Fluorescence 70 ... Scanning mechanism 71 ... Control circuit 72 ... Display device 80 ... Direct-view prism 90 ... Semi-transparent mirror.

Claims (6)

[Claims] 1. A mechanism for arranging a sample at a focal position of light from a light source, an optical system for converging the light to irradiate the sample, a scanning mechanism for scanning the sample or the converged light, and A direct-view prism for splitting the light of, a photodetector for detecting the light split by the direct-view prism, a display means for displaying the irradiation position of the light in the sample and the output of the photodetector in association with each other. A spectroscopic scanning microscope comprising: 2. The spectroscopic scanning microscope according to claim 1, wherein a photodetector having a movable slit or pinhole light receiving surface is used as the photodetector. 3. The spectroscopic scanning microscope according to claim 1, wherein a direct-view prism which disperses light from the sample is arranged on an optical axis of an optical system for irradiating the sample, and disperses transmitted light of the sample. The spectroscopic scanning microscope is characterized by being arranged as follows. 4. The spectroscopic scanning microscope according to claim 1, wherein a direct-view prism that disperses light from the sample is arranged to disperse reflected light reflected from the sample. Scanning microscope. 5. The spectroscopic scanning microscope according to claim 1, wherein an array detector is used as the photodetector. 6. The spectroscopic scanning microscope according to claim 5, wherein a light source for generating a plurality of light wavelengths is used as the light source, and the display means is means for displaying in color in accordance with the output from the photodetector. A spectroscopic scanning microscope characterized in that