Radiant energy – Photocells; circuits and apparatus – Optical or pre-photocell system
Reexamination Certificate
1999-09-22
2001-07-03
Allen, Stephone B. (Department: 2878)
Radiant energy
Photocells; circuits and apparatus
Optical or pre-photocell system
C356S300000
Reexamination Certificate
active
06255646
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATION
This application claims the priority of Japanese Patent Application No. 98/269561 filed on Sep. 24, 1998, which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
The present invention relates to a scanning optical microscope which disperses the fluorescence from a sample into a plurality of wavelength ranges and detects the fluorescence of each wavelength range.
The recent fluorescent observation often uses multiple dyes as well as a single dye. Since fluorescent dyeing is performed to permit cells or a specific target in an organ to be observed, each dyed portion should be detected as a clear color difference or a clear difference in fluorescent wavelength in the multiple dye observation. In this case, it is necessary to effectively remove the partial overlapping of the fluorescent wavelengths (crossover portion) in the detection. The fluorescent observation also demands a high contrast and high optical resolution. Confocal scanning laser microscopes satisfy those requirements and are becoming popular in researches in the field of biology.
Confocal scanning laser microscopes to which this invention relates and which can ensure fluorescent observation are disclosed in Jpn. Pat. Appln. Kokai Publication Nos. Hei 8-43739 and Hei 9-502269. Those microscopes use spectral resolving means like a prism or diffraction grating as fluorescence separation means for multiple dyes, and a slit for restricting the fluorescent wavelength range. This can ensure highly efficient detection of fluorescent rays from a multi-dyed sample without crossover while achieving the high contrast and high resolution of a confocal microscope.
The fluorescence from a sample is generally so weak that a photomultiplier is needed as a photosensor. Because the discoloration of a fluorescent sample becomes stronger as the excited light (laser beam) irradiated on the sample gets stronger. Therefore, an observer normally checks the balance of the discoloration of the sample and the acquired image noise and tries to make the amount of excited light as small as possible within the allowable range. For this kind of microscope, therefore, it is very important to suppress the fluorescent loss as much as possible.
We will now discuss a sample marked with two fluorescent dyes (DAPI, CY5) as one example. DAPI has an absorption wavelength in the UV range (340 to 365 nm) and an emitted fluorescent wavelength whose peak appears at approximately 450 nm. CY5 has an absorption wavelength in the red range (630 to 650 nm) and a fluorescent wavelength whose peak appears at approximately 670 nm.
The size of the spot which is formed at the position where those fluorescent rays form an image (where a confocal aperture is provided) is given by the following equation in, for example, Jpn. Pat. Appln. Kokai Publication No. Hei 9-502269.
Ø=1.22
×&lgr;/NA
where NA is the numerical aperture for emission of a lens and &lgr; is the wavelength. The comparison of the spot size of DAPI (fluorescent wavelength of 450 nm) with that of CY5 (fluorescent wavelength of 670 nm), both calculated from the above equation, show that the spot size of CY5 is about 1.5 time greater than that of DAPI.
According to the above-described prior art, therefore, the size of a confocal aperture is set in accordance with the spot size of DAPI in order to secure the confocal effect. This means that the setting of the confocal aperture is set optimized for DAPI, but is too narrow for CY5, resulting in loss of precious fluorescence. Setting the size of the confocal aperture for CY5, on the other hand, would result in an insufficient confocal effect for DAPI.
The bundle of rays that have passed the confocal aperture is resolved by the spectral resolving means (prism) and is split into wavelengths of the individual fluorescent rays using a variable slit. When a prism is used as the spectral resolving means, however, if the size of the bundle of incident rays is large, crossover of the individual wavelengths after spectral resolving occurs, the bundle of rays would not be split into the individual photosensing paths at a sufficient precision.
BRIEF SUMMARY OF THE INVENTION
Accordingly, it is a primary object of the present invention to provide a scanning optical microscope capable of leading rays of individual fluorescent wavelengths of a multi-dyed sample to the respective photosensors without reducing the confocal effect and losing the fluorescence.
It is another object of the present invention to provide a scanning optical microscope capable of leading a bundle of rays of individual fluorescent wavelengths of a multi-dyed sample to the respective photosensing paths at a high precision.
To achieve the above object, according to the main aspect of this invention, there is provided a scanning optical microscope which comprises a laser source; a scan optical system for scanning a sample with a laser beam from the laser source; a spectral resolving optical system for resolving spectra of fluorescent rays from the sample; a wavelength splitting optical system for splitting the fluorescent rays that have passed the spectral resolving optical system into rays of a plurality of different wavelengths and guiding the split rays to optical paths of the plurality of different wavelengths; a plurality of image forming optical systems, respectively provided in the optical paths of the plurality of different wavelengths, for forming images of the fluorescent rays from the sample; a plurality of confocal apertures respectively provided in the optical paths at focal points of the image forming optical systems; and a plurality of photosensors, respectively provided in the optical paths, for sensing the fluorescent rays from the sample that have passed the respective confocal apertures.
With this structure, the individual fluorescent rays from a multi-dyed sample are separated and guided to optical paths of the wavelength ranges of the respective fluorescent rays. As an image forming optical system for forming an image of the associated fluorescent ray from the sample and a confocal aperture are provided in the associated optical path, each confocal aperture can be set to the optimal aperture size for the associated wavelength range. This can provide a perfect confocal effect without any fluorescence loss.
According to one mode of the scanning optical microscope, the spectral resolving optical system includes a first optical element for resolving the spectra of the fluorescent rays from the sample; and a second optical element for transforming a bundle of rays resulting from spectral resolving by the first optical element back to a bundle of parallel rays.
As this structure allows a bundle of rays undergone spectral resolving and wavelength splitting to be emitted in parallel to the respective photosensing paths, those parallel rays all focus on the confocal points. Therefore, a scanning optical microscope can be constructed by simply arranging the confocal apertures to the respective confocal points. Since an independent confocal optical system can be provided in each path by merely arranging a single confocal aperture and a single photosensor in the optical path following the stage of separating the bundle of rays, the microscope can be constructed easily and at a low cost.
According to another mode of the scanning optical microscope, a reducing optical system for reducing a bundle of rays incident to the spectral resolving optical system is provided closer to a sample side than the spectral resolving optical system.
This structure improves the spectral resolving precision. It is preferable that the reduction ratio of this reducing optical system is at least 1/2. When the interval between the first and second optical elements is narrow, the reduction ratio is set smaller.
According to a further mode of the scanning optical microscope, the numbers of the image forming optical systems, the confocal apertures and the photosensors are equal to the number of fluorescent rays to be sensed; and the wavelength splitting optical sys
Allen Stephone B.
Frishauf, Holtz Goodman, Langer & Chick, P.C.
Olympus Optical Co,. Ltd.
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