Optical: systems and elements – Compound lens system – Microscope
Reexamination Certificate
2001-06-08
2002-08-13
Spyrou, Cassandra (Department: 2872)
Optical: systems and elements
Compound lens system
Microscope
C359S368000, C359S212100, C250S461100
Reexamination Certificate
active
06433929
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of acquiring an image and a scanning optical microscope for irradiating a sample emitting two or more types of fluorescences, namely, a multi-dye fluorescent sample with an excitation light as a light spot by condensing the light by an objective lens and detecting each fluorescence emitted from this sample by a photodetector through a pinhole, thereby acquiring an image of that sample.
2. Description of the Background Art
As a scanning optical microscope, there are known the following microscopes. One confocal pinhole is arranged at a position which is also conjugated by a sample. When the surface of the sample is scanned by a light spot, a plurality of rays of fluorescence are emitted from the sample. The rays of fluorescence emitted from the sample are led to the confocal pinhole. A plurality of the rays of fluorescence are subjected to optical path division by a dichroic mirror or a grating. A plurality of photodetectors corresponding to the respective rays of fluorescence are arranged to these optical paths, and a ray of fluorescence corresponding to each photodetector is detected (see Jpn. Pat. Appln. KOKAI Publication No. 8-43739).
When trying to simultaneously acquire a plurality of rays of fluorescence, a wavelength of fluorescence emitted from a fluorescent dye on a short wavelength side and a wavelength of fluorescence emitted from a fluorescent dye on a long wavelength side overlap each other. In a sample dyed using two or more kinds of fluorescent dyes, a fluorescent dye FITC is excited with an excitation wavelength of 488 nm, and emits a ray of fluorescence having a central wavelength of 520 nm. A fluorescent dye Cy
5
is excited with an excitation wavelength of 633 nm, and emits a ray of fluorescence having a central wavelength of 670 nm. These wavelengths of fluorescence overlap each other and, as shown in
FIG. 1
, a phenomenon called “fluorescent cross talk” in which a ray of fluorescence on the short wavelength side (FITC) is mixed occurs in a detector configured to detect a ray of fluorescence on the long wavelength side (Cy
5
).
In order to avoid the fluorescent cross talk, there is known a technique for detecting the respective rays of fluorescence in the time division manner (see Jpn. Pat. Appln. KOKAI Publication No. 10-206745). This technique carries out switching of each excitation wavelength for exciting a sample dyed by two or more kinds of fluorescent dyes and a optical path to each detector configured to detect each ray of fluorescence in synchronization with light scanning.
At this time, switching of the excitation wavelength and the detection optical path is performed relative to a command for acquiring an image issued by a computer in accordance with one-frame scanning or each one line or during photo acceptance of one pixel, and each ray of fluorescence is detected in the time division manner, thereby acquiring an image. Further, a product catalogue of, for example, ZEISS Co. Ltd. discloses a product such that a galvanometer mirror on a high-speed scanning side which is light scanner reciprocates for scanning and the excitation wavelength is switched by an acousto-optic device (AOTF) for selecting a wavelength in accordance with an outward route and an inward route to detect different rays of fluorescence in the respective routes, thereby avoiding the fluorescent cross talk.
The confocal effect in the scanning optical microscope depends on dimensions of a diameter of a confocal pinhole and a diameter of a light spot (diffraction ray) according to a wavelength of each ray of fluorescence whose image is formed on the confocal pinhole.
That is, although it is ideal to reduce the diameter of the confocal pinhole in order to increase the resolution, an amount of fluorescence becomes extremely small. Therefore, the light which passes through the confocal pinhole and is detected becomes weak, and acquisition of an image with the excellent SN can not be expected. Thus, the dimension of the confocal pinhole diameter is matched with that of the diffraction diameter in order to optimize the brightness and the confocal effect in the direction of an optical axis. At this time, the dimension of a diffraction diameter d can be obtained by the following expression:
d=1.22·&lgr;/NA
&lgr;=central wavelength of a ray of fluorescence to be detected
NA=NA of a fluorescent light flux incident upon the confocal pinhole
In the above expression, the diameter of the confocal pinhole is matched with the diffraction diameter d obtained by substituting a fluorescent light wavelength &lgr; to be detected and NA of the fluorescent light flux incident upon the confocal pinhole determined by an objective lens.
In the above-described prior art technique, however, since a plurality of fluorescences emitted from the sample pass through one confocal pinhole, the diameter of the confocal pinhole can matched with only the fluorescence relative to one excitation wavelength. For example, when an excitation wavelength of 488 nm is used for excitation, the FITC emits a fluorescence having a central wavelength of 520 nm. Further, when an excitation wavelength of 633 nm is used for excitation, the Cy
5
emits a fluorescence having a central wavelength of 670 nm. Therefore, assuming that NA of a fluorescence incident upon the confocal pinhole is 0.0063, the diameter of the confocal pinhole which is optimum for a fluorescence of FITC is as follows:
Confocal pinhole diameter
= 1.22 · &lgr;/NA
= 1.22 · 0.52/0.0063
= 100 &mgr;m
Moreover, the confocal pinhole diameter which is optimum for the fluorescent light of Cy
5
is as follows:
Confocal pinhole diameter
= 1.22 · &lgr;/NA
= 1.22 · 0.67/0.0063
= 130 &mgr;m
Therefore, when the confocal pinhole diameter is set to 100 &mgr;m, it is possible to obtain the confocal pinhole diameter which is optimum for the fluorescent light of FITC. However, for the fluorescent light of Cy
5
, the confocal pinhole diameter is too small, and a bright fluorescent image can not be obtained.
In addition, when the confocal pinhole diameter is set to 130 &mgr;m, the confocal pinhole diameter which is optimum for the fluorescent light of Cy
5
can be obtained. However, for the fluorescent light of FITC, the confocal pinhole diameter is too large, and the confocal effect is reduced.
In order to eliminate the above-described problems, there is disclosed a technique for optimizing the resolution and the brightness by matching the dimension of the pinhole diameter with the diffraction diameter by which the light from the sample is formed on the confocal pinhole plane (see Jpn. Utility Model Appln. KOKAI Publication No. 06-16927). By using this technique, an opening size (dimension of the pinhole diameter) of the confocal pinhole can be changed in accordance with an objective lens to be used or a wavelength to be observed.
As an adjustment mechanism for the opening size of the confocal pinhole, there is a method for performing. adjustment by arranging a plurality of pinholes on a concentric circle on a turret and rotating this turret (see Jpn. Utility Model Appln. KOKAI Publication No. 6-16927) or a method for performing adjustment by continuously moving and changing a pair of square openings each having a V shape by using a direct acting type motor (see Jpn. Pat. Appln. KOKAI Publication No. 2000-10152).
On the other hand, in a scanning optical microscope for observing fluorescences, a characteristic of a dichroic mirror for separating the illuminating lights to the sample and the fluorescences from the sample must be switched in accordance with the excitation wavelength of the sample to be observed or the fluorescent light spectral characteristic (see Jpn. Pat. Appln. KOKAI Publication No. 7-333508).
When this dichroic mirror is switched, since an image formation position on the confocal pinhole plane is shifted due to an error in a mounting angle or a difference in the parallelism of the dichroic mirror, the cente
Frishauf Holtz Goodman & Chick P.C.
Olympus Optical Co,. Ltd.
Robinson Mark A.
Spyrou Cassandra
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