JP3599754B2 - Confocal scanning optical microscope - Google Patents

Description

[0001]
[Industrial applications]
The present invention relates to a confocal scanning optical microscope, and more particularly, to a confocal scanning optical microscope capable of efficiently detecting reflected light and fluorescence from a sample.
[0002]
[Prior art]
Conventionally, there is a confocal scanning optical microscope configured as follows. The light from the light source is focused on a sample (specimen) with an objective lens to form a light spot, and the sample is two-dimensionally scanned with the light spot, and the reflected light or fluorescence from the sample is determined by the focal position of the objective lens. The light is extracted by passing through a confocal stop disposed at a position conjugate with the above, and the extracted light is photoelectrically converted to obtain image information of the sample.
[0003]
Hereinafter, this type of conventional microscope will be described with reference to FIGS. FIG. 8 is a diagram showing a first example of the related art, which is mainly used for observing materials, semiconductors, and the like. That is, the laser beam emitted from the light source 1 is reflected by the polarization beam splitter 2 and is two-dimensionally scanned in XY by the two-dimensional optical deflector 3.
[0004]
The two-dimensionally scanned laser beam passes through the λ / 4 plate 4 and becomes circularly polarized light. The reflected light of the laser beam applied to the sample 6 by the objective lens 5 passes through the objective lens 5 and the λ / 4 plate 4 again, and becomes linearly polarized light orthogonal to the vibration direction emitted from the light source 1. This linearly polarized light passes through the polarization beam splitter 2 and the confocal stop 7 and is guided to the detector 8.
[0005]
FIG. 9 is a diagram showing a second conventional example, which is mainly used for fluorescence detection of an organism such as a cell or a tissue. FIG. 9 is different from FIG. 8 in that a dichroic mirror 9 is used instead of the polarization beam splitter 2 in FIG. 8 and the fluorescence from the sample 6 is detected through an absorption filter 10, but the other points are different. It is the same as FIG.
[0006]
[Problems to be solved by the invention]
The conventional scanning optical microscope described above has different types of optical systems depending on the application. For this reason, for example, in the biological field, when performing cell adhesion and fluorescence observation of the entire morphology, or when performing fluorescence observation of the layer structure of bone and osteoclasts / osteoblasts, for example, in the semiconductor field, contamination on wafers and chips In order to identify the form of the nation and whether it is derived from an organic or inorganic substance, two different types of microscopes as shown in FIGS. 8 and 9 were required. In reality, it is extremely difficult to observe the exact same place of the specimen with different microscopes.
[0007]
The present invention has been made based on the above circumstances, and provides a confocal scanning optical microscope capable of simultaneously or individually observing reflected light and fluorescence from a sample with one optical system. With the goal.
[0008]
[Means for Solving the Problems]
In order to achieve the above object, the invention corresponding to claim 1 is a condensing optical system that condenses light from a light source on a sample, and a first photodetector that detects reflected light from the sample, A second photodetector for detecting fluorescence from the sample; and a first and second photodetector for directing light from the light source toward the sample and for detecting a part of reflected light from the sample and fluorescence. A first wavelength-dependent light splitting element directed toward the first wavelength-dependent light splitting element, and a part of the reflected light and the fluorescence guided from the first wavelength-dependent light splitting element. 10 is a confocal scanning optical microscope including a second wavelength-dependent light splitting element that leads to a second photodetector .
[0009]
[Action]
According to the first aspect of the present invention, the reflected light and the fluorescence from the sample are guided to the first and second photodetectors by at least two wavelength-dependent light splitting elements. The system allows the reflected light and the fluorescence from the sample to be observed simultaneously or individually.
[0010]
【Example】
Hereinafter, embodiments of the present invention will be described with reference to the drawings. First, a first embodiment of the present invention will be described with reference to FIGS. FIG. 1 is a schematic configuration diagram showing a first embodiment of the present invention, in which a light source 1, an objective lens 5 for condensing light from the light source 1 into a minute spot on a sample 6, and A light detector 8 for detecting reflected light (left side in the figure) for detecting reflected light, a light detector 8 for detecting fluorescence from the sample 6 (upper side in the figure), and each light detector 8 , 8 at least two wavelength-dependent light splitting elements such as dichroic mirrors 11 and 12 and a scanning optical system (not shown) for relatively two-dimensionally scanning the spot on the sample 6 and the sample 6. ) And a position optically conjugate with the in-focus position of the objective lens 5 and in front of the photodetectors 8, 8 with the aperture center aligned with the optical axis of the light incident on the photodetectors 8 And a confocal stop 7 whose aperture diameter can be varied.
[0011]
The dichroic mirrors 11 and 12 are mirrors that reflect only light in a specific wavelength region of visible light using light interference by a thin film and transmit light in the remaining wavelength region. The one having a spectral characteristic corresponding to is used. For example, when the laser wavelength of the light source 1 is 488 nm, the dichroic mirror 11 has a spectral characteristic shown in FIG. 2, and the dichroic mirror 12 has a spectral characteristic shown in FIG. Use Of course, by adjusting the spectral characteristics of the dichroic mirrors 11 and 12 to the wavelength of the light source, the same can be achieved even if the wavelength of the light source 1 is different.
[0012]
Hereinafter, the operation of the first embodiment configured as described above will be described. The laser beam emitted from the light source 1 is reflected by the dichroic mirror 11, is two-dimensionally scanned XY by the two-dimensional optical deflector 3, and is irradiated on the sample 6 by the objective lens 5. The reflected light irradiating the sample 6 with the laser beam again passes through the objective lens 5 and the two-dimensional optical deflector, and becomes linearly polarized light orthogonal to the vibration direction emitted from the light source 1. This linearly polarized light is reflected by the dichroic mirror 12 and guided to the photodetector 8 for detecting reflected light. Thereby, reflected light from the sample 6 can be detected.
Light passing through the dichroic mirror 12 is input to a photodetector 8 for detecting fluorescence of living organisms such as cells and tissues, and fluorescence from the sample 6 is detected.
With this configuration, the reflected light and the fluorescence can be detected at the same time, so that it is possible to observe the form and the distribution (localization) of the fluorescence with high precision without changing the tailoring of the microscope. .
[0014]
FIG. 4 is a view showing a second embodiment of the present invention, in which a bandpass filter 13 and an optical filter 14 are added to the embodiment of FIG. Specifically, a band-pass filter 13 is provided between a photodetector (left side in the figure) 8 for detecting reflected light and a dichroic mirror 12, and an optical filter 14 is provided for a photodetector for detecting fluorescence (upper side in the figure). ) 8 and the dichroic mirror 12.
[0015]
As the bandpass filter 13, a narrow bandpass filter corresponding to the laser wavelength of the light source 1 is used. For example, when the laser wavelength of the light source 1 is 488 nm, the bandpass filter 13 has a spectral characteristic as shown in FIG. Become.
The optical filter 14 uses one of a narrow band, a high band, a long pass filter, and the like in accordance with the fluorescent staining.
According to the second embodiment configured as described above, it is particularly effective to detect only the reflected light except for the fluorescent crossover.
[0016]
FIG. 6 is a view showing a third embodiment of the present invention. In this embodiment, a dichroic mirror 15 is added to the embodiment of FIG. 4, and an optical filter 14 and a photodetector 8 are further added. In this case, the dichroic mirror 15 whose spectral characteristics have been adjusted in accordance with the double staining of fluorescence is used. According to the third embodiment, one is used to observe reflected light, and the other two is used to detect FITL and rhodamine etc. at the same time because they are used as fluorescent probes.
[0017]
FIG. 7 is a view showing a fourth embodiment of the present invention, in which a dichroic cube 16 is provided instead of the dichroic mirrors 12 and 15 in the embodiment of FIG. This makes the configuration simpler than that of FIG.
[0018]
The present invention is not limited to the above-described embodiment, and may be, for example, as follows. Instead of the dichroic mirrors 11 and 12 used in the embodiment, a prism having a similar function may be used. In addition to this, a wavelength-dependent light splitting whose spectral characteristics can be obtained according to the wavelength of the light from the light source 1 is used. Any element may be used. In addition, any of the narrow-band bandpass filters 13 and the spectral characteristics shown in FIGS. 4, 6, and 7 may be used as long as they can be adjusted to the wavelength of the light source 1.
[0019]
【The invention's effect】
As described above, according to the present invention, a confocal scanning optical microscope capable of efficiently detecting reflected light and fluorescence from a sample can be provided.
[Brief description of the drawings]
FIG. 1 is a diagram showing a schematic configuration of a first embodiment of a confocal scanning optical microscope according to the present invention.
FIG. 2 is a diagram showing spectral characteristics of the dichroic mirror 11 of FIG.
FIG. 3 is a diagram showing spectral characteristics of the dichroic mirror 12 of FIG.
FIG. 4 is a diagram showing a schematic configuration of a second embodiment of a confocal scanning optical microscope according to the present invention.
FIG. 5 is a diagram showing spectral characteristics of the narrow bandpass filter of FIG. 4;
FIG. 6 is a diagram showing a schematic configuration of a third embodiment of the confocal scanning optical microscope according to the present invention.
FIG. 7 is a diagram showing a schematic configuration of a fourth embodiment of the confocal scanning optical microscope according to the present invention.
FIG. 8 is a diagram showing a schematic configuration of a first example of a conventional confocal scanning optical microscope.
FIG. 9 is a diagram showing a schematic configuration of a second example of a conventional confocal scanning optical microscope.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Light source, 3 ... Two-dimensional optical deflector, 4 ... λ / 4 plate, 5 ... Objective lens, 6 ... Sample (sample), 7 ... Confocal stop, 8 ... Photodetector, 11, 12 ... Dichroic filter, 13: narrow band pass filter, 14: optical filter, 15: dichroic filter, 16 ..., 14: CCD television camera, 15: CMD control circuit, 16: dichroic filter.

Claims (2)

A focusing optical system for focusing light from the light source on the sample,
A first light detector for detecting reflected light from the sample;
A second light detector for detecting fluorescence from the sample;
A first wavelength-dependent light splitting element that guides light from the light source toward the sample and guides a part of reflected light from the sample and fluorescence toward the first and second photodetectors;
Second wavelength-dependent light that separates part of the reflected light and the fluorescence guided from the first wavelength-dependent light splitting element and guides the fluorescence to the first photodetector and the second photodetector. A splitting element;
A confocal scanning optical microscope, characterized in that the light reflected from the sample and the fluorescence from the sample can be detected. The first wavelength-dependent light splitting element has a characteristic that transmittance changes between a wavelength range of the fluorescence and a wavelength range of light from the light source, and the wavelength of light from the light source is transmitted through the light source. 2. A confocal scan according to claim 1, wherein a portion of the light from the light source is directed toward the first and second photodetectors by being included in a region where the rate transitions. Optical microscope.

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