U.S. patent number RE38,860 [Application Number 10/613,664] was granted by the patent office on 2005-11-01 for scanning optical microscope.
This patent grant is currently assigned to Olympus Optical Co., Ltd.. Invention is credited to Yoshihiro Shimada.
United States Patent |
RE38,860 |
Shimada |
November 1, 2005 |
Scanning optical microscope
Abstract
A scanning optical microscope comprising 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.
Inventors: |
Shimada; Yoshihiro (Sagamihara,
JP) |
Assignee: |
Olympus Optical Co., Ltd.
(Tokyo, JP)
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Family
ID: |
17474098 |
Appl.
No.: |
10/613,664 |
Filed: |
July 2, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
401203 |
Sep 22, 1999 |
06255646 |
Jul 3, 2001 |
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Foreign Application Priority Data
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Sep 24, 1998 [JP] |
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10-269561 |
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Current U.S.
Class: |
250/234;
356/300 |
Current CPC
Class: |
G02B
21/0076 (20130101); G02B 21/16 (20130101); G01J
3/021 (20130101); G01J 3/14 (20130101); G01J
3/4406 (20130101); G01N 21/64 (20130101); G01N
21/6458 (20130101) |
Current International
Class: |
G01J
3/28 (20060101); G01J 003/28 () |
Field of
Search: |
;250/234-236,201.3,458.1
;356/300,310,319,320,326 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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53-135660 |
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Nov 1978 |
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JP |
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4-350816 |
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Dec 1992 |
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JP |
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5-509417 |
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Dec 1993 |
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JP |
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8-043739 |
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Feb 1996 |
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JP |
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9-502269 |
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Mar 1997 |
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JP |
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Primary Examiner: Allen; Stephone B.
Attorney, Agent or Firm: Frishauf, Holtz, Goodman &
Chick, PC
Claims
What is claimed is:
1. A scanning optical microscope comprising: a laser source; a scan
optical system for scanning a sample with a laser beam from said
laser source; a spectral resolving optical system for resolving
spectra of fluorescent rays from said sample; a wavelength
splitting optical system for splitting said fluorescent rays that
have passed said spectral resolving optical system into rays of a
plurality of different wavelengths and guiding said split rays to
optical paths of said plurality of different wavelengths; a
plurality of image forming optical systems, respectively provided
in said optical paths of said plurality of different wavelengths,
for forming images of said fluorescent rays from said sample; a
plurality of confocal apertures respectively provided in said
optical paths at focal points of said image forming optical
systems; and a plurality of photosensors, respectively provided in
said optical paths, for sensing said fluorescent rays from said
sample that have passed the respective confocal apertures.
2. The scanning optical microscope according to claim 1, wherein
said spectral resolving optical system includes: a first optical
element for resolving said spectra of said fluorescent rays from
said sample; and a second optical element for transforming a bundle
of rays resulting from spectral resolving by said first optical
element back to a bundle of parallel rays.
3. The scanning optical microscope according to claim 1, further
comprising a reducing optical system, provided closer to a sample
side than said spectral resolving optical system, for reducing a
bundle of rays incident to said spectral resolving optical
system.
4. The scanning optical microscope according to claim 1, wherein
the numbers of said image forming optical systems, said confocal
apertures and said photosensors are equal to the number of
fluorescent rays to be sensed; and said wavelength splitting
optical system has wavelength splitting optical elements smaller in
number by one than said number of said photosensors.
5. The scanning optical microscope according to claim 4, further
comprising an optical-element positioning drive mechanism for
positioning said wavelength splitting optical elements.
6. The scanning optical microscope according to claim 5, wherein
said optical-element positioning drive mechanism positions said
wavelength splitting optical elements in a direction perpendicular
to an incident optical axis..Iadd.
7. A scanning optical microscope which scans a sample with a laser
beam from a laser source, said scanning optical microscope
comprising: a spectral resolving unit which resolves fluorescent
rays from the sample into successive spectral components; a
wavelength splitter which splits the successive spectral components
resolved by the spectral resolving unit into rays of a plurality of
different wavelengths; and a plurality of side-on type
photomultipliers, respectively provided in optical paths of the
rays of different wavelengths split by the wavelength splitter, for
sensing the rays, wherein axial centers of the plurality of side-on
type photomultipliers come approximately within planes to be
spectrally-resolved by the spectral resolving
unit..Iaddend..Iadd.
8. A scanning optical microscope which scans a sample with a laser
beam from a laser source, said scanning optical microscope
comprising: a spectral resolving unit which resolves spectra of
fluorescent rays from the sample; a wavelength splitter which
splits the fluorescent rays resolved by the spectral resolving unit
into rays of a plurality of different wavelengths; a plurality of
side-on type photomultipliers, respectively provided in optical
paths of the rays of different wavelengths split by the wavelength
splitter, for sensing the rays, wherein axial centers of the
plurality of side-on type photomultipliers come approximately
within planes to be spectrally-resolved by the spectral resolving
unit; a plurality of image forming optical systems, respectively
provided at fronts of the plurality of side-on type
photomultipliers in each of the optical paths of the rays of
different wavelengths split by the wavelength splitter, for forming
images of the rays; and a plurality of confocal apertures
respectively provided at focal points of said plurality of image
forming optical systems..Iaddend..Iadd.
9. The scanning optical microscope of claim 8, wherein said
spectral resolving unit comprises an optical unit which emits a
resolved bundle of rays in parallel..Iaddend..Iadd.
10. A scanning optical microscope which scans a sample with a laser
beam from a laser source, said scanning optical microscope
comprising: a spectral resolving unit which resolves spectra of
fluorescent rays from the sample; a wavelength splitter which
splits the fluorescent rays resolved by the spectral resolving unit
into rays of a plurality of different wavelengths; a plurality of
side-on type photomultipliers, respectively provided in optical
paths of the rays of different wavelengths split by the wavelength
splitter, for sensing the rays, wherein axial centers of the
plurality of side-on type photomultipliers come approximately
within planes to be spectrally-resolved by the spectral resolving
unit; a beam splitter for splitting the fluorescent rays from the
sample and the laser beam; and a reducing optical system, provided
between the beam splitter and said spectral resolving unit, for
reducing the flourescent rays..Iaddend.
Description
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.
where NA is the numerical aperture for emission of a lens and
.lambda. 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
system has wavelength splitting optical elements smaller in number
by one than the number of the photosensors.
This setting can provide a microscope having a desired number of
channels.
According to a modification of the third mode, the scanning optical
microscope further comprises an optical-element positioning drive
mechanism for positioning the wavelength splitting optical elements
in a direction perpendicular to an incident optical axis.
This structure can facilitate microadjustment of the optical
elements to ensure high-precision ray sensing.
Additional objects and advantages of the invention will be set
forth in the description which follows, and in part will be obvious
from the description, or may be learned by practice of the
invention. The objects and advantages of the invention may be
realized and obtained by means of the instrumentalities and
combinations particularly pointed out hereinafter.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING
The accompanying drawings, which are incorporated in and constitute
a part of the specification, illustrate presently preferred
embodiments of the invention, and together with the general
description given above and the detailed description of the
preferred embodiments given below, serve to explain the principles
of the invention.
FIG. 1 is a structural diagram of a scanning optical microscope
according to a first embodiment of this invention;
FIG. 2 is a structural diagram of the scanning optical microscope
according to the first embodiment;
FIG. 3 is a diagram showing sensitivity distribution data of a
side-on type photomultiplier according to the first embodiment;
FIG. 4 is a structural diagram showing of a modification of the
first embodiment;
FIG. 5 is a structural diagram showing of another modification of
the first embodiment;
FIG. 6 is a diagram illustrating the structure of a scanning
optical microscope according to a second embodiment of this
invention; and
FIG. 7 is a diagram illustrating the structure of the scanning
optical microscope according to the second embodiment of this
invention.
DETAILED DESCRIPTION OF THE INVENTION
Preferred embodiments of this invention will be described below
with reference to the accompanying drawings.
First Embodiment
A first embodiment of this invention will now be discussed
referring to FIGS. 1 through 5.
FIG. 1 shows the general structure of a scanning optical microscope
according to this first embodiment.
FIG. 1 shows a typical laser source 1 which is comprised of an
argon-krypton gas laser. The laser beam that has been emitted from
this laser source 1 sequentially passes a beam expander 2, a
wavelength-selection filter 3, a beam splitter 4, an X-Y scan
optical system 5, a pupil projection lens 6, an image-forming lens
7 and an objective lens 8 and reaches a sample 9.
The laser source 1 emits argon rays of mainly 351 nm and 488 nm and
krypton rays of 568 nm and 647 nm. The beam expander 2 is set in
such a way that the size of the laser beam nearly satisfies the
pupil size of the objective lens 8. The beam splitter 4 reflects
about 20% of the arrived light and passes about 80% of that
light.
The wavelength-selection filter 3 selectively passes rays of a
wavelength of 351 nm, 488 nm, 568 nm or 647 nm. For example, the
sample 9 is dyed with four dyes, DAPI, FITC, Texas Red and CY5;
DAPI is excited by the argon rays of 351 nm, FITC by the argon rays
of 488 nm, Texas Red by the krypton rays of 568 nm and CY5 by the
krypton rays of 647 nm.
When excited, DAPI emits fluorescent rays which have a peak at
approximately 450 nm. Likewise, FITC emits fluorescent rays having
a peak at approximately 530 nm, Texas Red emits fluorescent rays
having a peak at approximately 610 nm, and CY5 emits fluorescent
rays having a peak at approximately 670 nm. Those fluorescent rays
pass through the beam splitter 4 after passing the objective lens
8, the image-forming lens 7, the pupil projection lens 6 and the
X-Y scan optical system 5.
A bundle of rays 20 that have passed the beam splitter 4 travels
through a reducing optical system 10 which reduces the bundle of
rays, thus forming a bundle of parallel rays 21. This bundle of
parallel rays 21 passes a prism 11a for spectral resolving, and a
bundle of rays with the resolved spectra is formed into a bundle of
parallel rays 22 by a prism 11b which is optically identical to the
prism 11a. The bundle of parallel rays 22 then reaches a first
prism mirror 12a. The first prism mirror 12a is so designed as to
be movable in a direction perpendicular to the incident axis by a
first mirror drive mechanism 17a, and is set in such a manner that
the bundle or rays is separated into right and left optical paths
with about 570 nm as the boundary. Consequently, the fluorescent
rays of DAPI and FITC are separated as a bundle of rays 23a, while
the fluorescent rays of Texas Red and CY5 are separated as a bundle
of rays 23b.
The bundle of parallel rays 23a enters a second prism mirror 12b.
This second prism mirror 12b is likewise so designed as to be
movable in a direction perpendicular to the incident axis by a
second mirror drive mechanism 17b, and is set in such a manner that
the bundle of rays is separated into right and left optical paths
with about 490 nm as the boundary. As a result, the fluorescent
rays of DAPI are separated as a bundle of rays 24a, and the
fluorescent rays of FITC are separated as a bundle of rays 24b.
The other bundle of parallel rays 23b likewise enters a third prism
mirror 12c. This third prism mirror 12c is also so designed as to
be movable in a direction perpendicular to the incident axis by a
third mirror drive mechanism 17c, and is set in such a manner that
the bundle of rays is separated into right and left optical paths
with about 650 nm as the boundary. Consequently, the fluorescent
rays of Texas Red are separated as a bundle of rays 24c, and the
fluorescent rays of CY5 are separated as a bundle of rays 24d.
The individual bundles of rays 24a, 24b, 24c and 24d pass through
slits 13a, 13b, 13c and 13d, which have variable widths and are
each movable in a direction perpendicular to the optical axis, so
that the return rays of the excited rays from the sample and
partial overlapping portions of the fluorescent wavelengths
(crossover portions of the fluorescent rays) are restricted by
those slits 13a-13d. The individual bundles of fluorescent rays
24a, 24b, 24c and 24d that have passed the slits 13a, 13b, 13c and
13d reach confocal lenses 14a, 14b, 14c and 14d respectively. As
the bundles of fluorescent rays 24a, 24b, 24c and 24d are emitted
as bundles of parallel rays, they focus on confocal apertures 15a,
15b, 15c and 15d located at their focal points, and pass through
the confocal apertures 15a-15d to be sensed by photomultipliers
16a, 16b, 16c and 16d.
The aperture sizes of the confocal apertures 15a-15d are set to the
ones that are calculated by the following equation.
where NA is the numerical aperture for emission of each of the
confocal lenses 14a, 14b, 14c and 14d and .lambda. is the
fluorescent wavelength.
With this structure where the confocal lenses 14a, 14b, 14c and 14d
and the confocal apertures 15a, 15b, 15c and 15d are respectively
arranged in the individual fluorescent optical paths for DAPI,
FITC, Texas Red and CY5, for example, the aperture sizes .O
slashed. of the confocal apertures 15a, 15b, 15c and 15d can be set
optimally for the respective fluorescent rays. It is thus possible
to provide the best confocal effect without losing the
fluorescence.
As the above structure has the reducing optical system 10 provided
immediately before the first prism 11a, ray sensing can be
implemented with a higher precision.
If the beam size of the bundle of rays 21 incident to the first
prism 11a is large, the spectra resolved by the first prism 11a may
overlap, disabling the optimal dispersion for each fluorescent
frequency by the prism mirror 12a. This embodiment does not however
suffer such a problem because the reducing optical system 10 which
reduces the beam size of the incident bundle of rays to at least
1/2 is provided immediately before the first prism 11a.
The photomultipliers 16a, 16b, 16c and 16d are of a side-on type
and have their axial centers coming approximately within planes to
be spectral-resolved by the prism 11a. The side-on type
photomultipliers, which are generally highly sensitive, are cheaper
than head-on type photomultipliers, and are often used in scanning
confocal laser microscopes. However, the side-on type
photomultipliers are inferior to the head-on type photomultipliers
in a large difference between the axial and vertical sensitivities,
though there is not much difference in the axial sensitivity
distribution. As a reference, the sensitivity distribution data of
the side-on type photomultiplier is shown in FIG. 3.
Since the axial centers of the side-on type photomultipliers come
approximately within the planes to be spectral-resolved by the
prism 11a in this embodiment, the sensitivity distribution of the
side-on type photomultipliers is hardly significant.
Although the foregoing description has discussed a way of guiding
the individual fluorescent rays to the respective sensing paths in
a case where the sample 9 is dyed with flour dyes, this invention
can also cope with a case where the sample 9 is dyed with a single
dye without any difficulty. When the sample 9 is dyed with a single
dye of FITC, crossover with the other fluorescent rays need not be
considered, so all the fluorescent rays to be acquired have only to
be guided to a single photosensor.
In this case, positions of the prism mirrors 12a and 12b should be
adjusted as shown in FIG. 2 so that FITC can be acquired
completely. In FIG. 2, like or same reference numerals are given to
those components which are the same as the corresponding components
in FIG. 1. As the structure below the reducing optical system 10 is
the same as the one shown in FIG. 1, it is not illustrated in FIG.
2.
If rays of 600 nm or lower are acquired completely, for example,
fluorescent rays of FITC can be gotten completely, so that the
positions of the prism mirrors 12a and 12b are so set as to be able
to get rays of 600 nm or lower. The slit 13b has only to be set to
cut the excited light of 488 nm and get light of 600 nm or lower.
This invention can also easily be adapted to cases of a double-dyed
sample and a triple-dyed sample.
Although a pair of prisms 11a and 11b which are optical identical
are used as the spectral resolving optical system in the first
embodiment, a pair of diffraction gratings or a pair of holograms
which are optical identical to each other may be used as well. As
the bundle of rays undergone spectral resolving should be emitted
as a bundle of parallel rays, different optical elements like a
prism and a diffraction grating may be combined.
FIG. 4 shows an optical system which is a combination of a prism
and a diffraction grating. In this case, since the diffraction
directions of the prism and diffraction grating are opposite to
each other, the axis of the outgoing bundle of rays forms, for
example 90 degrees to the axis of the incident bundle of rays if
one wants to acquire a bundle of parallel rays for each
fluorescence.
Because the fluorescent rays can be split into a plurality of
wavelengths by prism mirrors according to the above-described first
embodiment, the number of fluorescence sensing paths can be set as
desired. Although this example has a four-channel structure, it can
easily be modified into a two-channel structure, 3-channel
structure, 5-channel structure and so forth. FIG. 5 shows a case of
the 5-channel structure. In this example, the bundle of rays
separated by the third prism mirror 12c is further separated by a
fourth prism mirror 12d into two bundles of rays which respectively
pass slits 13d and 13e, confocal lenses 14d and 14e and confocal
apertures 15d and 15e to be respectively detected by
photomultipliers 16d and 16e. This structure can provide five
fluorescence sensing paths.
Since the axial centers of the side-on photomultipliers come
approximately within the planes to be spectral-resolved by the
prism 11a, the sensitivity distribution is hardly significant. The
use of such cheap side-on type photomultipliers can realize an
inexpensive scanning optical microscope.
Second Embodiment
FIGS. 6 and 7 show the structure of a scanning optical microscope
according to a second embodiment of this invention. In FIGS. 6 and
7, like or same reference numerals are given to corresponding or
identical components. As the structure below the reducing optical
system 10 in FIGS. 6 and 7 is the same as the one in FIG. 1, its
illustration is omitted.
A laser source 1 shown in FIG. 6 is a light source for an
argon-krypton gas. The laser beam that has been emitted from this
laser source 1 sequentially passes a beam expander 2, a
wavelength-selection filter 3, a beam splitter 4, an X-Y scan
optical system 5, a pupil projection lens 6, an image-forming lens
7 and an objective lens 8 and reaches a sample 9.
The laser source 1 emits argon rays of mainly 488 nm and krypton
rays of 568 nm and 647 nm. The beam expander 2 is set in such a way
that the size of the laser beam nearly satisfies the pupil size of
the objective lens 8. The beam splitter 4 reflects about 20% of the
arrived light and passes about 80% of that light.
The wavelength-selection filter 3 selectively passes rays of a
wavelength of 488 nm, 568 nm or 647 nm. For example, the sample 9
is triple-dyed with FITC, Texas Red and CY5; FITC is excited by the
argon rays of 488 nm. Texas Red by the krypton rays of 568 nm and
CY5 by the krypton rays of 647 nm.
When excited, FITC emits fluorescent rays which have a peak at
approximately 530 nm. Likewise, Texas Red emits fluorescent rays
having a peak at approximately 610 nm, and CY5 emits fluorescent
rays having a peak at approximately 670 nm. Those fluorescent rays
pass through the beam splitter 4 after passing the objective lens
8, the image-forming lens 7, the pupil projection lens 6 and the
X-Y scan optical system 5.
As shown in FIG. 6, a bundle of rays 20 that have passed the beam
splitter 4 travels through a reducing optical system 10 which
reduces the bundle of rays, thus forming a bundle of parallel rays
21. This bundle of parallel rays 21 passes a prism 11 for spectral
resolving, and the resultant bundle of rays enters a lens 30. The
lens 30 is arranged in such a way that its focal point coincides
with an incident point 11x of the bundle of rays to the prism 11.
Therefore, the individual spectral-resolved bundles of rays from
the lens 30 are emitted as bundles of parallel rays within the
spectral-resolved planes. The spectral-resolved bundles of rays are
separated by mirrors 31a, 31b and 31c to go to the respective
fluorescence sensing paths. The mirrors 31b and 31c are movable in
a direction of 45.degree. with respect to the incident light
axis.
Specifically, an end face 31cx of the mirror 31c is set to a
position where excited rays of approximately 647 nm bit, and an end
face 31bx of the mirror 31b is set to a position where excited rays
of approximately 568 nm bit. This structure allows the fluorescent
rays of FITC to be separated as a bundle of rays 33a, the
fluorescent rays of Texas Red to be separated as a bundle of rays
33b, and the fluorescent rays to CY5 to be separated as a bundle of
rays 33c. Although the positions of the mirrors 31a, 31b and 31c
are slightly shifted from the focal point of the lens 30, ray
separation to the individual fluorescence sensing paths can be
executed with a sufficiently high precision because the size of the
bundle of rays is reduced by the reducing optical system 10.
The individual bundles of rays 33a, 33b and 33c pass through slits
13a, 13b and 13c, which have variable widths and are each movable
in a direction perpendicular to the optical axis, so that the
return rays of the excited rays from the sample and partial
overlapping portions of the fluorescent wavelengths (crossover
portions of the fluorescent rays) are removed by those slits
13a-13c. The slits 13a, 13b and 13c are located at the focal point
of the lens 30. As the individual bundles of rays after spectral
resolving form spots at the positions of the respective slits, the
slits can restrict the wavelengths with a very high precision. The
positions of the slits 13a to 13c and the mirrors 31a to 31c may be
set in such a way that the focal point of the lens 30 comes to an
intermediate position thereof.
The individual bundles of rays that have passes the slits pass
lenses 34a, 34b, 34c and prism 35a, 35b and 35c. The lenses 34a,
34b, 34c are identical to the lens 30 and the prisms 35a, 35b and
35c are identical to the prism 11. The spectral-resolved bundles of
rays are combined in the individual optical paths to respectively
become bundles of rays 40a, 40b and 40c, which enter confocal
lenses 14a, 14b and 14c. The bundles of rays 40a, 40b and 40c then
pass confocal apertures 15a, 15b and 15c, arranged at their focal
points, and are sensed by photomultipliers 16a, 16b and 16c that
are so arranged that their axes become perpendicular to the
sheet.
As the spectra of the bundles of rays 40a, 40b and 40c are
completely combined, unlike in the first embodiment, the bundles of
rays 40a, 40b and 40c do not spread toward the spectral resolving
direction after passing the confocal apertures 15a, 15b and 15c.
Therefore, no problem would arise even if the photomultipliers 16a,
16b and 16c are arranged in such a way that their axes become
perpendicular to the sheet.
The aperture sizes of the confocal apertures are set to the ones
that are calculated by the following equation.
where NA is the numerical aperture for emission of each of the
confocal lenses 14a, 14b, 14c and 14d and .lambda. is the
fluorescent wavelength.
Like the first embodiment, therefore, this embodiment can provide
the best confocal effect without losing the fluorescent rays of
FITC, Texas Red and CY5. Although the illustrated example has a
3-channel structure, the number of channels is not limited to three
but can be set arbitrarily according to the purpose.
Although the foregoing description has discussed a way of guiding
the individual fluorescent rays to the respective sensing paths in
a case where the sample 9 is dyed with three dyes, this invention
can also cope with a case where the sample 9 is dyed with a single
dye without any difficulty. When the sample 9 is dyed with a single
dye of FITC, crossover with the other fluorescent rays need not be
considered, so all the fluorescent rays to be acquired have only to
be guided to a single photosensor.
In this case, the position of the mirror 31c should be adjusted as
shown in FIG. 7 so that FITC can be acquired completely. If rays of
600 nm or lower are acquired completely, for example, fluorescent
rays of FITC can be obtained completely, so that the position of
the mirror 31c is so set as to be able to get rays of 600 nm or
lower. The slit 13c has only to be set to cut the excited light of
488 nm and get light of 600 nm or lower. This invention can also
easily be adapted to cases of a double-dyed sample and a
triple-dyed sample.
Although a prism is used as the spectral resolving means in the
second embodiment, a diffraction grating or a hologram may be used
as well.
Since the spectral-resolved bundle of rays is formed as a spot by
the image-forming lens 30 in the second embodiment, it is possible
to restrict the wavelengths of fluorescent rays with very high
precision. As the focal point of the lens 30 is set to match with
the incident position 11x of the bundle of rays 21 to the prism 11,
the spectral-resolved bundle of rays emitted from the lens 30
become parallel in the spectral-resolved plane. This can allow the
use of the lenses 34a, 34b and 34c each identical to the lens 30
and the prisms 35a, 35b and 35c each identical to the prism 11, so
that the microscope can be constructed easily and at a low
cost.
It should be apparent to those skilled in the art that the present
invention is not limited to the above-described embodiments, but
may be embodied in many other specific forms without departing from
the spirit or scope of the invention.
Additional advantages and modifications will readily occur to those
skilled in the art. Therefore, the invention in its broader aspects
is not limited to the specific details and representative
embodiments shown and described herein. Accordingly, various
modifications may be made without departing from the spirit or
scope of the general inventive concept as defined by the appended
claims and their equivalents.
* * * * *