U.S. patent application number 09/877767 was filed with the patent office on 2002-03-28 for scanning optical microscope and method of acquiring image.
This patent application is currently assigned to Olympus Optical Co., Ltd.. Invention is credited to Sasaki, Hiroshi.
Application Number | 20020036824 09/877767 |
Document ID | / |
Family ID | 26593749 |
Filed Date | 2002-03-28 |
United States Patent
Application |
20020036824 |
Kind Code |
A1 |
Sasaki, Hiroshi |
March 28, 2002 |
Scanning optical microscope and method of acquiring image
Abstract
A scanning optical microscope comprises a light source
configured to selectively output to a sample dyed with two or more
types of fluorescent dyes an excitation light having an excitation
wavelength according to the each fluorescent dye, scanner
configured to scan the excitation light outputted from the light
source, an objective lens configured to condense the excitation
light scanned by the scanner on the sample, a detector configured
to detect a fluorescence of the fluorescent light dye according to
the excitation light by the excitation light condensed by the
objective lens, one confocal pinhole whose pinhole diameter
arranged in front of the detector is adjustable, and controller
configured to adjust the pinhole diameter of the confocal pinhole
to a diameter suitable for the fluorescence emitted from the sample
by using the excitation light in synchronization with switching of
the excitation lights from the light source when acquiring one
image by detecting each fluorescence according to each excitation
light in the time division manner through the confocal pinhole by
switching the excitation lights with which the sample is irradiated
in synchronization with scanning by the scanner.
Inventors: |
Sasaki, Hiroshi; (Tokyo,
JP) |
Correspondence
Address: |
FRISHAUF, HOLTZ, GOODMAN &
LANGER & CHICK, PC
767 THIRD AVENUE
25TH FLOOR
NEW YORK
NY
10017-2023
US
|
Assignee: |
Olympus Optical Co., Ltd.
43-2, Hatagaya 2-chome, Shibuya-ku
Tokyo
JP
|
Family ID: |
26593749 |
Appl. No.: |
09/877767 |
Filed: |
June 8, 2001 |
Current U.S.
Class: |
359/385 ;
359/368; 359/738 |
Current CPC
Class: |
G02B 21/002 20130101;
G02B 21/16 20130101 |
Class at
Publication: |
359/385 ;
359/368; 359/738 |
International
Class: |
G02B 021/00; G02B
021/06; G02B 009/00; G02B 009/08 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 12, 2000 |
JP |
2000-175630 |
Sep 18, 2000 |
JP |
2000-282695 |
Claims
What is claimed is:
1. A scanning optical microscope comprising: a light source
configured to selectively output to a sample dyed with two or more
types of fluorescent dyes an excitation light having an excitation
wavelength according to each of said fluorescent dyes; scanner
configured to scan said excitation light outputted from said light
source; an objective lens configured to condense said excitation
light scanned by said scanner on said sample; a detector configured
to detect a fluorescence from said fluorescent dye according to
said excitation light by using said excitation light condensed by
said objective lens; one confocal pinhole whose pinhole diameter
arranged in front of said detector is adjustable; and controller
configured to adjust said pinhole diameter of said confocal pinhole
to a diameter suitable for said fluorescence emitted from said
sample by said excitation light in synchronization with switching
of said excitation light from said light source, when acquiring one
image by detecting each fluorescence according to said each
excitation light in the time division manner though said confocal
pinhole by switching said excitation light with which said sample
is irradiated in synchronization with scanning by scanner.
2. The scanning optical microscope according to claim 1, wherein
said confocal pinhole is a minute device group having a plurality
of minute devices and said plurality of said minute devices are
controlled by said minute device group controller.
3. The scanning optical microscope according to claim 1, wherein
said confocal pinhole includes a minute device group configured by
arranging a plurality of minute deflecting mirrors in a
two-dimensional matrix form, further comprising minute device group
controller configured to control an angle of each minute deflecting
mirror within said diffraction diameter so as to reflect said light
spot in an arrangement direction of said photodetector, and to
control an angle of said each minute deflecting mirror outside said
diffraction diameter to an angle different from said angle of said
each minute deflecting mirror within said diffraction diameter.
4. The scanning optical microscope according to claim 1, wherein
said controller switches said excitation light in synchronization
with scanning by said light scanner in accordance with each one
line with respect to said sample.
5. The scanning optical microscope according to claim 4, wherein
said confocal pinhole is a minute device group having a plurality
of minute devices, and said plurality of said minute devices are
controlled by said minute device group controller.
6. The scanning optical microscope according to claim 4, wherein
said confocal pinhole includes a minute device group configured by
arranging a plurality of minute deflecting mirrors in a
two-dimensional matrix form, further comprising a minute device
controller configured to control an angle of said each minute
deflecting mirror within said diffraction diameter so as to reflect
said light spot in an arrangement direction of said photodetector,
and to control an angle of said each minute deflecting mirror
outside said diffraction diameter to an angle different from said
each angle of said each minute deflecting mirror in said
diffraction diameter.
7. The scanning optical microscope according to claim 1, wherein
said controller switches said excitation lights in synchronization
with scanning by said light scanner in accordance with each one
frame.
8. The scanning optical microscope according to claim 7, wherein
said confocal pinhole is a minute device group having a plurality
of minute devices, and said plurality of said minute devices are
controlled by said minute device controller.
9. The scanning optical microscope according to claim 7, wherein
said confocal pinhole includes a minute device group configured by
arranging a plurality of minute deflecting mirrors in a
two-dimensional matrix form, further comprising minute device group
controller configured to control an angle of said each minute
deflecting mirror within said diffraction diameter so as to reflect
said light spot in an arrangement direction of said photodetector,
and to control an angle of said each minute deflecting mirror
outside said diffraction diameter to an angle different from said
angle of said each minute deflecting mirror in said diffraction
diameter.
10. The scanning optical microscope according to claim 1, wherein
said controller switches said excitation lights in units of pixel
during scanning with respect to said sample by said light
scanner.
11. The scanning optical microscope according to claim 10, wherein
said confocal pinhole is a minute device group having a plurality
of minute devices, and said plurality of said minute devices are
controlled by said minute device controller.
12. The scanning optical microscope according to claim 10, wherein
said confocal pinhole includes a minute device group configured by
arranging a plurality of minute deflecting mirrors in a
two-dimensional matrix form, further comprising minute device group
controller to control an angle of said each minute deflecting
mirror within said diffraction diameter so as to reflect said light
spot in an arrangement direction of said photodetector, and to
control an angle of said each minute deflecting mirror outside said
diffraction diameter to an angle different from said angle of said
each minute deflecting mirror within said diffraction diameter.
13. The scanning optical microscope according to claim 1, wherein
said detector is configured by one detector.
14. The scanning optical microscope according to claim 13, further
comprising a barrier filter which is fixed in front of said
detector, blocks two or more types of excitation lights for
exciting said two or more types of fluorescences, and transmits
therethrough said two or more types of fluorescences emitted by
said sample by said excitation lights.
15. The scanning optical microscope according to claim 13, further
comprising: a first barrier filter for blocking a first excitation
light of said excitation lights and transmitting therethrough said
fluorescence emitted from said sample by said first excitation
light; a second barrier filter for blocking a second excitation
light of said excitation lights and transmitting therethrough said
fluorescence emitted from said sample by said second fluorescence;
and means for switching said first barrier filter and said second
barrier filter between said confocal pinhole and said detector in
synchronization with change of said excitation lights.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is based upon and claims the benefit of
priority from the prior Japanese Patent Application No. 2000-175630
and 2000-282695, filed Jun. 12, 2000 and Sep. 18, 2000,
respectively, the entire contents of which are incorporated herein
by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] 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.
[0004] 2. Description of the Background Art
[0005] 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).
[0006] 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 Cy5 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 (Cy5).
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.multidot..lambda./NA
[0011] .lambda.=central wavelength of a ray of fluorescence to be
detected
[0012] NA=NA of a fluorescent light flux incident upon the confocal
pinhole
[0013] In the above expression, the diameter of the confocal
pinhole is matched with the diffraction diameter d obtained by
substituting a fluorescent light wavelength .lambda. to be detected
and NA of the fluorescent light flux incident upon the confocal
pinhole determined by an objective lens.
[0014] 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 Cy5 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: 1 Confocal pinhole diameter =
1.22 / NA = 1.22 0.52 / 0.0063 = 100 m
[0015] Moreover, the confocal pinhole diameter which is optimum for
the fluorescent light of Cy5 is as follows: 2 Confocal pinhole
diameter = 1.22 / NA = 1.22 0.67 / 0.0063 = 130 m
[0016] Therefore, when the confocal pinhole diameter is set to 100
.mu.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 Cy5, the confocal pinhole diameter is too
small, and a bright fluorescent image can not be obtained.
[0017] In addition, when the confocal pinhole diameter is set to
130 .mu.m, the confocal pinhole diameter which is optimum for the
fluorescent light of Cy5 can be obtained. However, for the
fluorescent light of FITC, the confocal pinhole diameter is too
large, and the confocal effect is reduced.
[0018] 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. Pat. Appln. KOKAI
Publication No. 6-16927). 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.
[0019] 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. Pat. 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).
[0020] 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).
[0021] 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 center of the confocal pinhole and the
image formation position must be corrected by moving the optical
axis or the confocal pinhole position.
[0022] As the correction method, there is, for example, a
crisscross moving stage system using two motors for moving the
confocal pinhole itself within the plane (see Jpn. Pat. Appln.
KOKAI Publication No. 7-333508) or a system for matching the image
formation position with the center of the confocal pinhole by
rotating two parallel plane glasses by a motor and then moving the
optical axis (see Jpn. Pat. Appln. KOKAI Publication No.
8-271792).
BRIEF SUMMARY OF THE INVENTION
[0023] It is an object of the present invention to provide a method
of acquiring an image and a scanning optical microscope by which
the cross talk does not occur in all fluorescences and an optimum
confocal effect can be obtained in accordance with each
fluorescence to be detected when detecting a plurality of
fluorescences.
[0024] It is another object of the present invention to provide a
scanning optical microscope by which mechanical abrasion in a
driving section does not occur and a speed of diameter correction
by means for effectively restricting the diffraction diameter can
be increased.
[0025] The present invention is characterized in that, when an
excitation light having an excitation wavelength according to each
fluorescent dye is switched and emitted with respect to a sample
dyed with two or more kinds of fluorescent dyes in synchronization
with light scanner and each fluorescence according to each
excitation light is detected through one confocal pinhole in the
time division manner to obtain one image, a pinhole diameter of the
confocal pinhole is adjusted to a diameter suitable for the
fluorescence emitted from the sample by the excitation light in
accordance with the wavelength of the excitation light.
[0026] Specifically, the structure of the present invention is
described as follows. It is to be noted that the "pinhole" means a
transmission type pinhole such that the fluorescence passes through
the pinhole as well as a reflection type pinhole for reflecting the
fluorescence such as a mirror having a pinhole shape.
[0027] According to the present invention, there is provided a
scanning optical microscope comprising: a light source configured
to selectively output an excitation light having an excitation
wavelength according to each fluorescent dye to a sample dyed with
two or more kinds of fluorescent dyes; scanner configured to scan
the excitation light outputted from the light source; an objective
lens configured to condense the excitation light scanned by the
scanner onto the sample; a detector configured to detect a
fluorescence having the fluorescent dye according to the excitation
light by using the excitation light condensed by the objective
lens; one confocal pinhole capable of adjusting a diameter of a
pinhole arranged in front of the detector; controller configured to
adjust a pinhole diameter of the confocal pinhole to a diameter
suitable for the fluorescent rays emitted from the sample by the
excitation light in synchronization with switching of the
excitation light from the light source when each fluorescence
according to each excitation light is detected in the time division
manner through the confocal pinhole to acquire one image by
changing over the excitation light with which the sample is
irradiated in synchronization with scanning of the scanner.
Preferred embodiments according to the present invention are as
follows. It is to be noted that each of the following embodiments
may be solely applied or combined and applied.
[0028] (1) The controller switches the excitation light in
synchronization with scanning performed for each line relative to
the sample by the light scanner.
[0029] (2) The controller switches the excitation light in
synchronization with scanning performed for each frame relative to
the sample by the light scanner.
[0030] (3) The controller switches the excitation light in units of
pixel during scanning relative to the sample by the light
scanner.
[0031] (4) The detector is configured by one detector.
[0032] (5) There is further included a barrier filter which is
fixed in front of the detector, blocks two or more types of
excitation lights for exciting the two or more types of
fluorescences and transmits therethrough two or more types of
fluorescences emitted from the sample.
[0033] (6) There are further included: a first barrier filter which
blocks a first excitation light of the excitation lights and
transmits therethrough fluorescences emitted from the sample by the
first excitation light; a second barrier filter which blocks a
second excitation light of the excitation lights and transmits
therethrough fluorescences emitted from the sample by the second
excitation light; and means for switching the first barrier filter
and the second barrier filter between the confocal pinhole and the
detector in synchronization with change of the excitation
lights.
[0034] (7) The confocal pinhole is a minute device group having a
plurality of minute devices, and a plurality of the minute devices
are controlled by minute device controller.
[0035] (8) The confocal pinhole includes a minute device group
configured by arranging a plurality of minute deflecting mirrors in
the form of a two-dimensional matrix, and further includes minute
device controller configured to control an angle of each minute
deflecting mirror in the diffraction diameter in such a manner that
the light spot is reflected in an arrangement direction of the
photodetector and controlling an angle of each minute deflecting
mirror outside the diffraction diameter to an angle different from
that of each minute deflecting mirror in the diffraction
diameter.
[0036] (9) In (7) or (8), the minute device group controller has a
function for varying an area of each minute device for controlling
to lead the light spot to the photodetector in accordance with the
dimension of the diffraction diameter imaged to the minute device
group.
[0037] (10) In (7) or (8), the minute device group controller has a
function for correcting a central position of each minute device
for controlling to lead the light spot to the photodetector in
accordance with the displacement of the light spot imaged to the
minute device group.
[0038] (11) In (10), there is provided a function for correcting
the displacement of the light spot in the minute device group
generated by switching of at least one optical device arranged
between the sample and the minute device group.
[0039] (12) In (7) or (8), there are further included: a light
source capable of selectively outputting to a sample dyed with two
more types of fluorescent dyes an excitation light having an
excitation wavelength according to each fluorescent dye; scanner
configured to scan an excitation light outputted from the light
source; and an objective lens configured to condense the excitation
light scanned by the scanner, wherein the minute device group
controller adjusts each minute device of the minute device group
for leading a light from the sample to the photodetector to a
diffraction diameter of a light spot imaged to the minute device
group through the confocal lens in synchronization with switching
of the excitation light from the light source when each
fluorescence according to each excitation light is detected in the
time division manner through one minute device group to acquire one
image by changing over the excited lights with which the sample is
irradiated in synchronization with scanning of the light
scanner.
[0040] (13) In (12), switching of excitation lights by the minute
device group controller is synchronized with scanning in accordance
with each one line or scanning in an outward route and an inward
route by the light scanner, respectively.
[0041] (14) In (12), switching of the excitation lights by the
minute device group controller is synchronized with scanning in
accordance with one frame by the light scanner.
[0042] (15) In (12), switching of the excitation lights by the
minute device group controller is synchronized with scanning in
accordance with one pixel by the light scanner.
[0043] Since the pinhole diameter is adjusted in accordance with a
wavelength of a fluorescence emitted from the sample, there occurs
no cross talk in all fluorescences when detecting a plurality of
fluorescences, and an optimum confocal effect can be obtained in
accordance with each fluorescence to be detected.
[0044] Further, since the minute deflecting mirror is adopted as
the pinhole, mechanical abrasion of the driving section is not
generated, and it is possible to speed up the diameter correction
or the position correction of means for restricting the effective
range of the diffraction diameter.
[0045] 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 DRAWINGS
[0046] The accompanying drawings, which are incorporated in and
configure 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.
[0047] FIG. 1 is a view showing cross talk of a fluorescence from a
sample dyed with each fluorescent reagent;
[0048] FIG. 2 is a block diagram showing a first embodiment of a
scanning optical microscope according to the present invention;
[0049] FIG. 3 is an image acquisition control flowchart in the
first embodiment of the scanning optical microscope according to
the present invention;
[0050] FIG. 4 is a block diagram showing a second embodiment of a
scanning optical microscope according to the present invention;
[0051] FIG. 5 is an image acquisition control flowchart according
to the second embodiment of the scanning optical microscope
according to the present invention;
[0052] FIG. 6 is an image acquisition control flowchart according
to a third embodiment of a scanning optical microscope according to
the present invention;
[0053] FIG. 7 is a block diagram showing a fourth embodiment of a
scanning optical microscope according to the present invention;
[0054] FIG. 8 is a block diagram of a minute deflecting mirror
array in the fourth embodiment of the scanning optical microscope
according to the present invention;
[0055] FIG. 9 is a view showing the angle control of each minute
deflecting mirror in a light area in which fluorescences are imaged
in the minute deflecting mirror array in the fourth embodiment of
the scanning optical microscope according to the present
invention;
[0056] FIG. 10 is a side view showing paths of fluorescences
incident upon each minute deflecting mirror in a plane on which
fluorescences are reflected in the fourth embodiment of the
scanning optical microscope according to the present invention;
[0057] FIG. 11 is a view showing the angle control of each minute
deflecting mirror in a light area in which fluorescences are imaged
in a minute deflecting mirror array in a fifth embodiment of a
scanning optical microscope according to the present invention;
[0058] FIG. 12 is a block diagram showing a sixth embodiment of a
scanning optical microscope according to the present invention;
[0059] FIG. 13 is a block diagram showing a seventh embodiment of a
scanning optical microscope according to the present invention;
[0060] FIG. 14 is a view showing the angle control of each minute
deflecting mirror with respect to correction of the displacement of
a light spot on a minute deflecting mirror array in the seventh
embodiment of the scanning optical microscope according to the
present invention; and
[0061] FIG. 15 is a partial block diagram showing the case where a
confocal lens and a minute deflecting mirror array are provided at
the rear of a switching spectral dichroic mirror in the scanning
optical microscope according to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0062] Preferred embodiments according to the present invention
will now be described with reference to the accompanying
drawings.
[0063] FIG. 2 is a block diagram showing a scanning optical
microscope according to a first embodiment. A laser unit 1 outputs
each excitation laser beam having an excitation wavelength thereof
to a sample 2 (multi-dye fluorescent sample) dyed with two or more
types of fluorescent dyes. The laser unit 1 includes: an Ar laser
device 3 for emitting an excitation laser beam having an excitation
wavelength 488 nm; an HeNe-G laser device 4 for emitting an
excitation laser beam having an excitation wavelength of 543 nm; an
HeNe-R laser device 5 for emitting an excitation laser beam having
an excitation wavelength of 633 nm; a mirror 6; a dichroic mirror 7
for combining two exiting laser beams having wavelengths of 488 nm
and 543 nm; a dichroic mirror 8 for combining excitation laser
beams having wavelengths of 488 nm, 543 nm and 633 nm; and an
acousto-optic device (AOTF) 9 for selecting an excitation laser
beam having an arbitrary wavelength among the respective
wavelengths of 488 nm, 543 nm and 633 nm.
[0064] The excitation laser beam outputted from the laser unit 1,
i.e., the excitation laser beam having an excitation wavelength
selected by the AOTF 9 is led to a scanning unit 11 through a
single-mode fiber 10. A collimator lens 12 is arranged at an
emission end of the single-mode fiber 10. The collimator lens 12
forms the excitation laser beam outgoing from the single-mode fiber
10 into a parallel light.
[0065] An excitation dichroic mirror 13 is arranged on a optical
path of the excitation laser beam formed into the parallel light by
the collimator lens 12. The excitation dichroic mirror 13 has a
characteristic for reflecting the respective excitation laser beams
having the excitation wavelengths of 488 nm, 543 nm and 633 nm and
a characteristic for transmitting therethrough the wavelengths of
the fluorescences emitted from the sample 2.
[0066] X/Y galvanometer mirrors 14a and 14b are arranged on a
reflection optical path of the excitation dichroic mirror 13. The
X/Y galvanometer mirrors 14a and 14b have a function for scanning
the excitation laser beams having the respective excitation
wavelengths of 488 nm, 543 nm and 633 nm in the two-dimensional
direction of an X direction and a Y direction on the sample 2 (the
X direction may be referred to as a horizontal direction and the Y
direction may be referred to as a vertical direction hereinafter).
Additionally, in the present specification, as will be described in
detail, it is determined that the X galvanometer mirror 14a of the
X/Y galvanometer mirrors 14a and 14b carries out high-speed
scanning and the Y galvanometer mirror 14b performs low-speed
scanning. That is, the X galvanometer mirror 14a conducts
reciprocating scanning for one line at a high speed, and the Y
galvanometer mirror 14b is driven in such a manner that scanning
line is shifted by one line upon completion of scanning of one
line, for example. It is to be noted that scanning by the X
galvanometer mirror 14a in a first direction of the reciprocating
scanning is referred to as "outward route scanning" and scanning by
the same in a second direction opposed to the first direction is
referred to as "inward route scanning".
[0067] A pupil projection lens 15, a mirror 16, an image formation
lens 17 and an objective lens 18 for forming a light spot on the
sample 2 are arranged on the scanning optical path from the X/Y
galvanometer mirrors 14a and 14b to the sample 2.
[0068] The fluorescence emitted from the sample 2 proceeds in the
reverse direction of the illuminating optical path (namely, the
direction from the objective lens 18 to the image formation lens
17, the mirror 16, the pupil projection lens 15, and the X/Y
galvanometer mirrors 14a and 14b) and passes through the excitation
dichroic mirror 13 to enter a confocal lens 19.
[0069] A confocal pinhole 20 is arranged at an image formation
position of the confocal lens 19. The confocal pinhole 20 has a
structure the inner diameter of which can be adjusted without
restraint (complete control of opening and closing). The confocal
pinhole 20 is driven by a diameter drive mechanism 21 consisting
of, e.g., a motor mechanism. The diameter drive mechanism 21
performs control to adjust the diameter of the confocal pinhole 20
to an optimum pinhole diameter in accordance with a wavelength of
each fluorescence emitted from the sample 2 when the sample 2 is
irradiated with each excitation laser beam having an excitation
wavelength of, e.g., 488 nm, 543 nm, or 633 nm (control concerning
opening/closing. This will be simply referred to as "control"
hereinafter).
[0070] A spectral dichroic mirror 22 is arranged on the optical
path of the fluorescence (detected light) having passed through the
confocal pinhole 20. The spectral dichroic mirror 22 has a
characteristic for separating, for example, a fluorescence having a
wavelength shorter than a wavelength of 570 nm (fluorescence
acquired by excitation of the excitation wavelength 488 nm) and a
fluorescence having a wavelength longer than a wavelength of 570 nm
(fluorescence acquired by excitation of the excitation wavelength
of 543 nm or 633 nm).
[0071] A first photodetector 24 is arranged on the reflection
optical path (fluorescence having a wavelength shorter than the
wavelength 570 nm) of the spectral dichroic mirror 22 through a
barrier filter 23 for setting an area of a fluorescence having a
desired wavelength by cutting the reflected light of the laser
beam. A second photodetector 26 is arranged on a transmission
optical path (fluorescence having a wavelength longer than the
wavelength 570 nm) of the spectral dichroic mirror 22 through a
barrier filter 25 for setting an area of a fluorescence having a
desired wavelength by cutting the reflected light of the laser
beam.
[0072] Upon receiving an execution command from a computer 28, a
control section 27 executes the following function. (1) A function
for selecting the Ar laser device 3, the HeNe-G laser device 4 or
the HeNe-R laser device 5 from the laser unit 1. (2) A function for
driving the X/Y galvanometer mirrors 14a and 14b for scanning. (3)
A function for controlling a pinhole diameter of the confocal
pinhole 20. (4) A function for distinguishing a signal by a
fluorescent dye F1 fetched from the first photodetector 24 and a
signal by a fluorescent dye Cy5 fetched from the second
photodetector 26 by using different colors. (5) A function for
displaying one multi-dye fluorescent image on, e.g., a monitor.
[0073] A computer 28 has a function for causing an observer to give
an observation start command to a control section 27.
[0074] Description will now be given as to an image acquisition
method by using a scanning optical microscope having the structure
mentioned above.
[0075] Here, a method for observing two types of fluorescences in
the time division manner. Specifically, a fluorescence according to
a fluorescent dye FITC is detected in the outward route scanning
and a fluorescence according to a fluorescent dye Cy5 is detected
in the inward route scanning by the X galvanometer mirror 14a.
[0076] The outline of this observation method is as described
below. A fluorescence according to the fluorescent dye FITC at each
pixel position on one line is detected by a first photodetector 24
in the outward route scanning and a fluorescence according to the
fluorescent dye Cy5 at each pixel position on the same line as the
one line in the outward route is detected by a second photodetector
26 in the inward route scanning by using the X galvanometer mirror
14a. Here, scanning for one line is completed. Upon completing
scanning for one line, the light spot is then shifted by one
scanning line in the vertical direction on the sample 2 by the Y
galvanometer mirror 14b, and scanning for one line is similarly
carried out as described above. The above-mentioned operation is
repeated until detection of a detection range is terminated.
[0077] The specific operation when executing the above observation
method from the computer 28 will now be described with reference to
FIG. 3.
[0078] When an execution command is issued from the computer 28 to
the control section 27, the control section 27 first receives the
execution command from the computer (step S1). The control section
27 makes judgment upon whether scanning using the X/Y galvanometer
mirrors 14a and 14b is carried out in the outward route or the
inward route (step S2). If it is the outward route scanning, the
control section 27 issues a command for selecting an excitation
laser beam of the Ar laser device 3 to the acousto-optic device 9
of the laser unit 1 (step S3).
[0079] Here, the acousto-optic device 9 selects the excitation
laser beam having the excitation wavelength of 488 nm outputted
from the Ar laser device 3 among the Ar laser device 3, the HeNe-G
laser device 4 and the HeNe-R laser device 5 and leads the selected
laser beam to the single-mode fiber 10.
[0080] At the same time, the control section 27 issues to the
diameter drive mechanism 27 a command for controlling a pinhole
diameter of the confocal pinhole 20 to 100 .mu.m which is an
optimum diameter for the fluorescence having a central wavelength
of 520 nm emitted from the fluorescent dye FITC when the sample 2
is irradiated with the excitation wavelength 488 nm (step S4). As a
result, the pinhole diameter of the confocal pinhole 20 is
controlled to 100 .mu.m diameter which is optimum for the central
wavelength 520 nm of the fluorescence of the fluorescent dye
FITC.
[0081] The excitation laser beam having the excitation wavelength
of 488 nm is led to the scanning unit 11 through the single-mode
fiber 10. The excitation laser beam is then formed into a parallel
beam by the collimator lens 12, reflected by the excitation
dichroic mirror 13, and scanned by the X/Y galvanometer mirrors 14a
and 14b. Further, the excitation laser beam is transmitted through
the pupil projection lens 15, reflected downwards by the mirror 16,
and imaged as a light spot on the sample 2 through the image
formation lens 17, the objective lens 18 and others. At this time,
the light spot is used for scanning in the outward route in the
horizontal direction on the sample 2.
[0082] The fluorescence having the central wavelength of 520 nm by
the fluorescent dye FITC generated when the sample 2 is scanned by
using the light spot of the laser beam proceeds in a direction
opposite to the illumination optical path, namely, proceeds from
the objective lens 18 to the image formation lens 17, the mirror
16, the pupil projection lens 15, and X/Y galvanometer mirrors 14a
and 14b and enters the confocal lens 19 through the excitation
dichroic mirror 13. The fluorescence is condensed by the confocal
lens 19 and imaged on the confocal pinhole 20.
[0083] At this moment, the diameter of the confocal pinhole 20 is
controlled to the diameter of 100 .mu.m which is optimum for the
central wavelength 520 nm of the fluorescence of the fluorescent
dye FITC. Further, the fluorescence of the fluorescent dye FITC
transmitted through the confocal pinhole 20 is reflected by the
spectral dichroic mirror 22. Its unnecessary laser reflected light
is cut by the barrier filter 23 and only the fluorescence of the
FITC enters the first photodetector 24.
[0084] The control section 27 fetches a signal from the first
photodetector 24. At this time, the control section 27 prevents the
second photodetector 26 from electrically measuring the detected
light so as not to detect a leaked light which is transmitted
through the spectral dichroic mirror 22 (step S5).
[0085] Furthermore, the control section 27 fetches a signal from
the first photodetector 24 in accordance with each pixel during the
outward route scanning in the horizontal direction by the X
galvanometer mirror 14a.
[0086] When the outward route scanning by the X galvanometer mirror
14a is finished and the inward route scanning is started, the
control section 27 issues a command for selecting the excitation
laser beam of the HeNe-R laser device 5 to the acousto-optic device
9 of the laser unit 1, selects and outputs the excitation laser
beam having the excitation wavelength of 633 nm from the HeNe-R
laser device 5 so that the selected excitation laser beam is led to
the single-mode fiber 10 (step S6).
[0087] At the same time, the control section 27 issues to the
diameter drive mechanism 21 a command for controlling the pinhole
diameter of the confocal pinhole 20 to 130 .mu.m which is an
optimum diameter of the fluorescence having the central wavelength
of 670 nm emitted from the fluorescent dye Cy5 when the sample 2 is
irradiated with the excitation wavelength of 633 nm (step S7). As a
result, the pinhole diameter of the confocal pinhole 20 is
controlled to the diameter of 130 .mu.m which is optimum for the
central wavelength 670 nm of the fluorescence of the fluorescent
dye Cy5.
[0088] The excitation laser beam having the excitation wavelength
of 633 nm is led to the scanning unit 11 through the single-mode
fiber 10. The excitation laser beam is then formed into a parallel
light by the collimator lens 12, reflected by the excitation
dichroic mirror 13, and scanned by the X/Y galvanometer mirrors 14a
and 14b. This excitation laser beam is further transmitted through
the pupil projection lens 15, reflected downwards by the mirror 16,
and imaged as a light spot on the sample 2 through the image
formation lens 17, the objective lens 18 and others. At this time,
the light spot is used for scanning in the inward route in the
horizontal direction on the sample 2.
[0089] The fluorescence having the central wavelength of 670 nm by
the fluorescent dye Cy5 generated when scanned on the sample 2 in
this manner proceeds in a direction opposite to the illumination
optical path, namely, proceeds from the objective lens 18 to the
image formation lens 17, the mirror 16, the pupil projection lens
15, and the X/Y galvanometer mirrors 14a and 14b, is transmitted
through the excitation dichroic mirror 13 and enters the confocal
lens 19. Moreover, the fluorescence is condensed by the confocal
lens 19 and imaged on the confocal pinhole 20.
[0090] At this time, the diameter of the confocal pinhole 20 is
controlled to the diameter of 130 .mu.m which is optimum for the
central wavelength of 670 nm of the fluorescence of the fluorescent
dye Cy5, and the fluorescence of the fluorescent dye Cy5
transmitted through the confocal pinhole 20 is transmitted through
the spectral dichroic mirror 22. Its unnecessary laser reflected
light is cut by the barrier filter 25, and only the fluorescence of
Cy5 enters the second photodetector 26.
[0091] The control section 27 fetches a signal from the second
photodetector 26. At this moment, the control section 27 prevents
the first photodetector 24 from electrically measuring the detected
light so as not to detect a leaked light reflected on the spectral
dichroic mirror 22 (step S8).
[0092] This control section 27 fetches a signal from the second
photodetector 26 in accordance with each pixel during the inward
route scanning in the horizontal direction by the X galvanometer
mirror 14a.
[0093] Then, when the outward and inward route scanning by the X
galvanometer mirror 14a is completed, the control section 27 makes
judgment upon whether the horizontal scanning line by the X
galvanometer mirror 14a has reached a last line (step S9).
[0094] If the horizontal scanning line has reached the last line in
the step S9, the processing is terminated. However, if it is
determined that the horizontal scanning line has not reached the
last line in the step S9, the light spot on the sample 2 is shifted
by one scanning line in the vertical direction by the Y
galvanometer mirror 14b (step S10).
[0095] Thereafter, the operation from the step S2 to the step S10
is repeated till the detection operation in the detection range is
terminated.
[0096] Subsequently, the control section 27 separates the signal
according to the fluorescent dye FITC fetched from the first
photodetector 24 and the signal according to the fluorescent dye
Cy5 fetched from the second photodetector 26 by using different
colors and displays them as one multi-dye fluorescent light image
on, e.g., a monitor.
[0097] As described above, in the first embodiment, the laser
waveform of 488 nm is selected in the outward route scanning by the
X galvanometer mirror 14a, and the diameter of the confocal pinhole
is controlled to 100 .mu.m which is optimum for the fluorescence
according to the fluorescent light dye FITC. Further, the laser
wavelength of 633 nm is selected in the inward route scanning, and
the diameter of the confocal pinhole is controlled to 130 .mu.m
which is optimum for the fluorescence according to the fluorescent
dye Cy5. That is, when the respective fluorescences according to
the two types of fluorescent dyes FITC and Cy5 are detected for one
image in the time division manner, the pinhole diameter of the
confocal pinhole 20 can be controlled to the dimension which is
optimum for each fluorescent light wavelength. Therefore, there
occurs no cross talk in the two types of fluorescences, and the
optimum confocal effects can be obtained in accordance with each
fluorescence to be detected.
[0098] Although the unit of time division for acquiring the
respective fluorescences of the fluorescent dyes FITC and Cy5 is
the unit of line in the first embodiment, scanning may be carried
out for two frames in one image acquisition for example, and time
division may be performed in accordance with each frame. In this
case, in the first frame scanning, the laser wavelength, the
confocal pinhole diameter and setting of the photodetector are
switched and controlled so as to acquire the fluorescence of the
FITC, and the fluorescence of the FITC is obtained by the first
photodetector 24. Then, in the next frame scanning, the laser
wavelength, the confocal pinhole diameter and setting of the
photodetector are controlled so as to acquire the fluorescence of
the Cy5, and the fluorescence of the Cy5 is obtained by the second
photodetector 26.
[0099] During scanning of one pixel, the two fluorescences may be
subjected to time division and detected. In this case, when
detecting one pixel, the laser waveform, the confocal pinhole
diameter, and setting of the photodetector are first controlled so
as to acquire the fluorescence of the fluorescent dye FITC, and the
fluorescence of the FITC is obtained by the first photodetector 24.
Then, the laser wavelength, the confocal pinhole diameter, and
setting of the photodetector are switched so as to acquire the
fluorescence of the Cy5, and the fluorescence of the Cy5 is
obtained by the second photodetector 26. Additionally, in all
pixels, the fluorescences of the fluorescent dyes FITC and Cy5 are
detected. In this modification in which the two fluorescences are
switched and acquired in one pixel, the time lag produced when
acquiring the two fluorescences can be greatly reduced as compared
with the time division detection for each frame scanning or the
time division detection in the outward route and the inward route
using the X galvanometer mirror 14a.
[0100] In the above-described first embodiment, although detection
is carried out in both the outward route scanning and the inward
route scanning. However, scanning may be carried out twice on the
same line only in one direction so that the fluorescence of the
FITC is acquired in the first outward route scanning and the
fluorescence of the Cy5 is obtained in the second outward route
scanning.
[0101] A second embodiment according to the present invention will
now be described with reference to the accompanying drawings. It is
to be noted that same reference numerals as those in FIG. 2 denote
like or corresponding parts, thereby omitting the detailed
description.
[0102] FIG. 4 is a block diagram showing a scanning optical
microscope according to the second embodiment of the present
invention. The scanning optical microscope according to the second
embodiment omits the spectral dichroic mirror 22, the barrier
filter 23 and the first photodetector 24 in the first embodiment,
and uses only one photodetector (second photodetector in the first
embodiment) 26 in order to acquire the two fluorescences. It is to
be noted that a barrier filter 31 substitutes for the barrier
filter 25.
[0103] The barrier filter 31 has a characteristic for cutting both
the two excitation wavelengths 488 nm and 633 nm and causing two
fluorescent light wavelength regions corresponding to the
fluorescent dyes FITC and Cy5 to pass. Further, the barrier filter
31 may be configured so as to switch two types of barrier filters,
i.e., a barrier filter for FITC having a characteristic for cutting
the excitation wavelength 488 nm and causing the fluorescent light
wavelength of the fluorescent dye FITC to pass, and a barrier
filter for Cy5 having a characteristic for cutting the excitation
wavelength 633 nm and causing the fluorescent light wavelength of
the fluorescent dye Cy5 to pass by an electric motor-driven type
mechanism in synchronization with switching of the excitation
wavelengths for exciting the respective fluorescences.
[0104] An image acquisition method by using the scanning optical
microscope configured as described above will now be described with
reference to an image acquisition control flowchart shown in FIG.
5.
[0105] The control after issue of an execution command from the
computer 28 to the control section 27 till the pinhole diameter in
the outward route scanning in the X galvanometer mirror 14a is
controlled to 100 .mu.m which is optimum for the fluorescence
having the central wavelength 520 nm (step S11 to Step S13) is the
same as the control from the step S2 to the step S4 in FIG. 3,
thereby omitting the explanation.
[0106] The control section 27 fetches a signal from the
photodetector 26 as a signal of the fluorescence of the fluorescent
dye FITC and accumulates it in a non-illustrated memory section
(step S14). The control section 27 fetches a signal from the
photodetector 26 in accordance with each pixel during the outward
route scanning in the horizontal direction by the X galvanometer
mirror 14a.
[0107] The control till the pinhole diameter in the inward route
scanning by the X galvanometer mirror 14a is controlled to 130
.mu.m which is optimum for the fluorescence having the central
wavelength 670 nm (step S11, step S15 and step S16) is the same as
those in the step S2, the step S6 and the step S7 in FIG. 3,
respectively, thereby omitting the explanation.
[0108] The control section 27 fetches a signal from the
photodetector 26 as a signal of the fluorescent light of the
fluorescent dye Cy5 and accumulates it in a non-illustrated memory
section (step S17). This control section 27 fetches a signal from
the photodetector 26 in accordance with each pixel during the
inward scanning in the horizontal direction by the X galvanometer
mirror 14a.
[0109] Upon completing the outward and inward route scanning by the
X galvanometer mirror 14a, the control section 27 makes judgment
upon whether the horizontal scanning line by the X galvanometer
mirror 14a has reached a last line (step S18).
[0110] If the horizontal scanning line has reached the last line in
the step S18, the processing is terminated. However, if it is
determined that the horizontal scanning line has not reached the
last line in the step S18, the light spot on the sample 2 is
scanned for one pixel in the vertical direction by the Y
galvanometer mirror 14b, and the scanning line is shifted by one
line (step S19).
[0111] Thereafter, the operation from the step S11 to the step S19
is repeated till the detection operation in the detection range is
completed. Then, the control section 27 separates the signal by the
fluorescent dye FITC and the signal by the fluorescent dye Cy5 by
using different colors, and displays them as one multi-dye
fluorescent image on, e.g., a monitor.
[0112] As described above, according to the second embodiment, in
addition to the effects in the first embodiment, it is possible to
cut back one photodetector.
[0113] A third embodiment according to the present invention will
now be described with reference to the accompanying drawings. Since
the structure of the third embodiment is the same as that of the
second embodiment, illustration and description are omitted.
[0114] In the third embodiment, description will be given as to a
method for acquiring three fluorescences during scanning of one
pixel in the time division manner. Incidentally, in the third
embodiment, detection of the fluorescence having the central
wavelength 590 nm by excitation using the excitation laser beam
having the excitation wavelength 543 nm with respect to the sample
2 dyed with a fluorescent dye PI will be explained in addition to
the first and second embodiments. At this time, the pinhole
diameter of the confocal pinhole 20 which is optimum for the
fluorescence with respect to the fluorescent dye PI is as follows:
3 Confocal pinhole diameter = 1.22 / NA = 1.22 0.59 / 0.0063 = 114
m
[0115] It is to be noted that NA of the fluorescence incident upon
the confocal pinhole 20 is determined as NA=0.0063 as similar to
the case of the fluorescent dyes FITC and Cy5 described in the
first and second embodiments.
[0116] In this case, the control section 27 has the following
function. That is, in case of acquiring an image of the sample 2
triple-dyed with the fluorescent dyes FITC, PI and Cy5, the
excitation laser beam of the Ar laser device 37 the excitation
laser beam of the HeNe-G laser device 4, and the excitation laser
beam of the HeNe-R laser device 5 are sequentially selected from
the laser unit 1. In synchronization with selection of these
excitation laser beams, the pinhole diameter of the confocal
pinhole 20 is controlled to the pinhole diameters 100 .mu.m, 114
.mu.m and 130 .mu.m which are optimum for the fluorescent
wavelengths according to the respective fluorescent dyes. At the
same time, signals from the detector 26 are fetched as signals by
the fluorescent dye FITC, the fluorescent dye PI and the
fluorescent dye Cy5 in synchronization with selection of these
excitation laser beams and accumulated in non-illustrated memory
sections according to the fluorescent dye FITC, the fluorescent dye
PI and the fluorescent dye Cy5. These operations are effected for
all pixels while performing scanning using the X/Y galvanometer
mirrors 14a and 14b.Further, the respective signals by the
fluorescent dyes FITC, PI and Cy5 accumulated in accordance with
each pixel are distinguished by using different colors and
displayed as one multi-dye fluorescent image on, e.g., a
monitor.
[0117] The barrier filter 31 arranged in front of the photodetector
26 has a characteristic for cutting all of three excitation
wavelengths 488 nm, 543 nm and 633 nm and causing three fluorescent
light wavelength regions corresponding to the fluorescent dyes
FITC, PI and Cy5 to pass. The barrier filter 31 may be configured
so as to switch three types of barrier filters, i.e., a barrier
filter for FITC having a characteristic for cutting the excitation
wavelength 488 nm and causing the fluorescent light wavelength of
the fluorescent dye FITC to pass, a barrier filter for PI having a
characteristic for cutting the excitation wavelength 543 nm and
causing the fluorescent light wavelength of the fluorescent dye PI
to pass, and a barrier filter for Cy5 having a characteristic for
cutting the excitation wavelength 633 nm and causing the
fluorescent light wavelength of the fluorescent dye Cy5 to pass by
an electric motor-driven type mechanism in synchronization with the
detection time of each fluorescence.
[0118] An image acquisition method by using the scanning optical
microscope having the above-described structure will now be
described with reference to an image acquisition control flowchart
shown in FIG. 6. Incidentally, measurement is carried out with
respect to the three fluorescent dyes FITC, PI and Cy5 in the order
of FITC, PI and Cy5 in the following explanation.
[0119] When an execution command is issued from the computer 28 to
the control section 27, the control section 27 drives and moves the
X/Y galvanometer mirrors 14a and 14b so that the spot light is
imaged on one point on the sample 2 according to first one pixel.
The control section 27 then fixes the X/Y galvanometer mirrors 14a
and 14b (step S20).
[0120] The control section 27 determines the fluorescence to be
measured (step S21). Here, measurement is conducted with respect to
the three types of the fluorescent dyes FITC, PI and Cy5 in the
order of FITC, PI and Cy5. Therefore, a selection command is issued
to the acousto-optic device 9 of the laser unit 1 in order to
select the excitation laser beam of the Ar laser device 3, and the
excitation laser beam having the excitation wavelength 488 nm is
outputted from the Ar laser device 3 (step S22).
[0121] At the same time, the control section 27 issues to the
diameter drive mechanism 21 a command for controlling the pinhole
diameter of the confocal pinhole 20 to 100 .mu.m which is an
optimum diameter which can be calculated from the central
wavelength 520 nm of the fluorescence of the fluorescent dye FITC
when the sample 2 is irradiated with the excitation laser beam
having the excitation wavelength 488 nm (step S23). As a result,
the pinhole diameter of the confocal pinhole 20 can be controlled
to the diameter of 100 .mu.m which is optimum for the central
wavelength 520 nm of the fluorescence of the fluorescent dye
FITC.
[0122] The excitation laser beam having the excitation wavelength
488 nm is led to the scanning unit 11 through the single-mode fiber
10, and imaged as a light spot on the sample 2 through the
collimator lens 12, the excitation dichroic mirror 13, the X/Y
galvanometer mirrors 14a and 14b, the pupil projection lens 15, the
mirror 16, the image formation lens 17, the objective lens 18 and
others.
[0123] The fluorescence having the central wavelength 520 nm
according to the fluorescent dyes FITC from the sample 2 proceeds
in a direction opposite to the illumination optical path, enters
the confocal lens 19, and imaged on the confocal pinhole 20.
[0124] At this time, since the confocal pinhole 20 is controlled to
the diameter of 100 .mu.m which is optimum for the fluorescence
having the central wavelength 520 nm according to the fluorescent
dye FITC, the fluorescence passes through the confocal pinhole 20
and enters the photodetector 26 through the barrier filter 31.
[0125] The control section 27 fetches a signal from the
photodetector 26 as a signal of the fluorescence of the fluorescent
dye FITC and accumulates it in a non-illustrated memory section
according to the fluorescent dye FITC (step S24).
[0126] Subsequently, the control section 27 makes judgment upon
whether detection of the respective fluorescences of the three
types of fluorescent dyes FITC, PI and Cy5 is completed with
respect to one pixel. If detection of all of the three types of the
fluorescences is not completed, the processing returns to the step
S21 and the fluorescent dye to be subsequently subjected to
fluorescent light measurement, namely, PI or Cy 5 in this example
is determined (step S25).
[0127] As a result of this determination, the control section 27
shifts from the step S21 to the step S27. The control section 27
issues a selection command to the acousto-optic device 9 of the
laser unit 1 on the same pixel and causes this device to select the
excitation laser beam of the HeNe-G laser device 4 so that the
excitation laser beam having the excitation wavelength 543 nm is
outputted from the HeNe-G laser device 4 (step S27).
[0128] At the same time, the control section 27 issues to the
diameter drive mechanism 21 a command for controlling the pinhole
diameter of the confocal pinhole 20 to 114 .mu.m which is an
optimum diameter for the central wavelength 590 nm according to the
fluorescent dye PI when the sample 2 is irradiated with the
excitation laser beam having the excitation wavelength 543 nm (step
S28). As a result, the pinhole diameter of the confocal pinhole 20
is controlled to 114 .mu.m which is optimum for the central
wavelength 590 nm of the fluorescence of the fluorescent dye
PI.
[0129] The excitation laser beam having the excitation wavelength
543 nm is led to the scanning unit 11 through the single-mode fiber
10, and imaged as a light spot on the sample 2 through the
collimator lens 12, the excitation dichroic mirror 13, the X/Y
galvanometer mirrors 14a and 14b, the pupil projection lens 15, the
mirror 16, the image formation lens 17, the objective lens 18 and
others.
[0130] The fluorescence having the central wavelength 590 nm
according to the fluorescent dye PI from the sample 2 proceeds in a
direction opposite to the illumination optical path, enters the
confocal lens 19, and is imaged on the confocal pinhole 20.
[0131] At this time, since the confocal pinhole 20 is controlled to
the diameter of 114 .mu.m which is optimum for the fluorescence
having the central wavelength 590 nm according to the fluorescent
dye PI, the fluorescence passes through this confocal pinhole 20
and enters the photodetector 26 through the barrier filter 31.
[0132] The processing advances to the step S24, and the control
section 27 fetches a signal from the photodetector 26 as a signal
of a fluorescence of the fluorescent dye PI and accumulates it in a
non-illustrated memory section according to the fluorescent dye
PI.
[0133] Again in the step S25, the control section 27 then makes
judgment upon whether detection of the respective fluorescences of
the three types of the fluorescent dyes FITC, PI and Cy5 is
completed with respect to one pixel. If detection of all of the
three types of the fluorescences is not completed, the processing
returns to the step S21 and determines the fluorescent dye to be
subsequently subjected to the fluorescent light measurement,
namely, Cy5 in this example.
[0134] As a result of this determination, the control section 27
shifts from the step S21 to the step S29. The control section 27
issues a selection command to the acousto-optic device 9 of the
laser unit 1 on the same pixel and causes this device to select the
excitation laser beam of the HeNe-R laser device 5 so that the
excitation laser beam having the excitation wavelength 633 nm is
outputted from the HeNe-R laser device 5 (step S29).
[0135] At the same time, the control section 27 issues to the
diameter drive mechanism 21 a command for controlling the pinhole
diameter of the confocal pinhole 20 to 130 .mu.m which is a
diameter optimum for the fluorescence having the central wavelength
670 nm according to the fluorescent dye Cy5 when the sample 2 is
irradiated with the excitation laser beam having the excitation
wavelength 633 nm (step S30). As a result, the pinhole diameter of
the confocal pinhole 20 can be controlled to the diameter of 130
.mu.m which is optimum for the central wavelength 670 nm of the
fluorescence of the fluorescent dye Cy5.
[0136] The excitation laser beam having the excitation wavelength
633 nm is led to the scanning unit 11 through the single-mode fiber
10, and imaged as a light spot on the sample 2 through the
collimator lens 12, the excitation dichroic mirror 13, the X/Y
galvanometer mirrors 14a and 14b, the pupil projection lens 15, the
mirror 16, the image formation lens 17, the objective lens 18 and
others.
[0137] The fluorescence having the central wavelength 670 nm
according to the fluorescent dye Cy5 from the sample 2 proceeds in
a direction opposite to the illumination optical path, enters the
confocal lens 19 and is imaged on the confocal pinhole 20.
[0138] At this moment, since the confocal pinhole 20 is controlled
to the diameter of 130 .mu.m which is optimum for the fluorescent
light having the central wavelength 670 nm according to the
fluorescent dye Cy5, the fluorescence passes through the confocal
pinhole 20 and enters the photodetector 26 through the barrier
filter 31.
[0139] At the step S24, the control section 27 fetches a signal
from the photodetector 26 as a signal of the fluorescent light of
the fluorescent dye Cy5 and accumulates it in a non-illustrated
memory section according to the fluorescent dye Cy5.
[0140] Then, again at the step S25, the control section 27 makes
judgment upon whether detection of the respective fluorescences of
the three types of fluorescent dyes FITC, PI and Cy5 is completed
with respect to one pixel. If detection of all of the three types
of fluorescences is completed, the processing proceeds to the next
step S26, and judgment is made upon whether the pixel scanned by
the X galvanometer mirror 14a or the X/Y galvanometer mirrors 14a
and 14b has reached a last pixel.
[0141] If it is determined that the scanned pixel has reached the
last pixel in the last line at the step S26, the processing is
completed. However, if the scanned pixel has not reached the last
pixel in the last line, the processing advances to the next step
S27, the spot light is fixedly emitted by the X galvanometer mirror
14a or the X/Y galvanometer mirrors 14a and 14b in accordance with
next each one pixel.
[0142] Thereafter, as similar to the above description, the spot
light is fixedly emitted by the X/Y galvanometer mirrors 14a and
14b in accordance with each one pixel, and the diameter of the
confocal pinhole is controlled to 100 .mu.m in order to detect the
fluorescence according to the fluorescent dye FITC. Then, the
diameter of the confocal pinhole is controlled to 114 .mu.m in
order to detect the fluorescence according to the fluorescent dye
PI, and the diameter of the confocal pinhole is subsequently
controlled to 130 .mu.m in order to detect the fluorescence
according to the fluorescent dye Cy5. Further, the operation for
accumulating the signals according to the fluorescent dyes FITC, PI
and Cy5 in each non-illustrated memory section is carried out with
respect to all the pixels.
[0143] Then, the control section 27 distinguishes the signal
according to the fluorescent dye FITC, the signal according to the
fluorescent dye PI and the signal according to the fluorescent dye
Cy5 by using different colors, and displays them as one multi-dye
fluorescent image on, e.g., a monitor.
[0144] As described above, according to the third embodiment, the
pinhole diameter of the confocal pinhole 20 which is optimum for
each of the fluorescent light wavelengths of the three types of
fluorescent dyes FITC, PI and Cy5 can be set by using one
photodetector 26, and the signal for each of the three types of
fluorescent light wavelengths can be acquired in the time division
manner. Moreover, there occurs no cross talk in all of the three
types of fluorescences, and the optimum confocal effect can be
obtained.
[0145] According to the third embodiment, for example, the three
types of fluorescent dyes FITC, PI and Cy5 are sequentially
switched and their fluorescences are acquired. However, switching
of these fluorescent dyes FITC, PI and Cy5 is not restricted to a
particular order, and they may be randomly switched. In addition,
the present invention is not restricted to the three types of
fluorescent dyes FITC, PI and Cy5, and any other fluorescent dye
may be used.
[0146] Although switching of the respective excitation wavelengths
is carried out in units of the reciprocating scanning of the
excitation laser beam (units of each line), units of a frame or
units of one pixel in the first to third embodiments, the present
invention is not restricted thereto, and the excitation wavelengths
can be switched with any timing as long as switching is
synchronized with scanning of the excitation laser beam.
[0147] In the first to third embodiments, the pinhole diameter of
the confocal pinhole 20 is switched (changed) in accordance with
each wavelength of the fluorescence in order to acquire a confocal
image which is optimum for each fluorescence. Besides, it is
possible to adopt a structure for, e.g., reflecting the
fluorescence emitted from the sample by a mirror instead of passing
the fluorescence. Such an embodiment will now be described
hereinafter.
[0148] FIG. 7 is a block diagram showing a scanning optical
microscope of a fourth embodiment according to the present
invention. In FIG. 7, like reference numerals denote parts similar
to those in FIG. 1, thereby omitting the detailed explanation. In
this embodiment, a minute deflecting mirror array 30 is arranged in
place of the confocal pinhole 20 in FIG. 2.
[0149] That is, in the fourth embodiment, the minute deflecting
mirror array 30 as a minute device group is arranged at an image
formation position of the confocal lens 19, i.e., a position
conjugated with the sample 2 through the confocal lens 19. This
minute deflecting mirror array 30 effectively restricts a
diffraction diameter and has the same function as the confocal
pinhole 20 in the first and second embodiments. This minute
deflecting mirror array 30 has a structure in which a plurality of
minute deflecting mirrors 31 are arranged in the two-dimensional
matrix form as shown in FIG. 8, and an angle of each minute
deflecting mirror 31 can be varied without restraints.
[0150] An angle of the minute deflecting mirror 31 is controlled
by, e.g., on/off switching of an electromagnet or deformation of an
electrostriction device. This minute deflecting mirror array 30 is
manufactured by a semiconductor process using, e.g., a
semiconductor material. As to the dimension of each minute
deflecting mirror 31, this mirror is formed into a square of
approximately 10 .mu.m.times.10 .mu.m. In this minute deflecting
mirror array 30, a filling ratio of the minute deflecting mirrors
31 aligned in the matrix form is not less than 90%.
[0151] In this minute deflecting mirror array 30, an angle of each
minute deflecting mirror 31 in a light area of the fluorescent
light spot is controlled in such a manner that the fluorescent
light spot from the sample 2 formed by image formation of the
confocal lens 19 is reflected on the optical path 33 on the
arrangement direction side of the first and second photodetectors
24 and 26. Incidentally, in this minute deflecting mirror array 30,
each minute deflecting mirror 31 outside the light area of the
fluorescent light spot is controlled to an angle different from
that of each minute deflecting mirror 31 in the light area of the
fluorescent light spot, and its reflection direction is, for
example, a optical path 32.
[0152] On the optical path 33 of the fluorescence reflected by the
minute deflecting mirror array 30, a reflecting mirror 35 is
arranged, and a spectral dichroic mirror 22 is arranged on the
reflection optical path of this reflecting mirror 35. This spectral
dichroic mirror 22 has a characteristic for separating a
fluorescence having a wavelength shorter than a wavelength 570 nm
(fluorescence acquired by excitation of the excitation wavelength
488 nm) and a fluorescence having a wavelength longer than a
wavelength 570 nm (fluorescence acquired by excitation of the
excitation wavelength 543 nm or 633 nm), for example. This
structure following this dichroic mirror 22 is similar to the first
embodiment, omitting the explanation.
[0153] The control section 27 has a function of: selecting the Ar
laser device 3, HeNe-G laser device 4 or HeNe-R laser device 5 from
the laser unit 1; driving the X/Y galvanometer mirrors 14a and 14b
for scanning; distinguishing a signal outputted from the first
photodetector 24 which has fetched the fluorescence from, e.g., the
fluorescent dye FITC and a signal outputted from the second
photodetector 26 which has fetched the fluorescence from, e.g., the
fluorescent dye Cy5 by using different colors; and displaying them
as one multi-dye fluorescent image on, e.g., one monitor.
[0154] Further, the control section 27 controls an angle of each
minute deflecting mirror 31 in the light area of the fluorescent
light spot in the minute deflecting mirror array 30 so as to
reflect the fluorescence from the sample 2 on the optical path 33
parallel to the arrangement direction of the first and second
photodetectors 24 and 26. Furthermore, the control section 27
controls an angle of each minute deflecting mirror 31 so as to
reflect an angle of each minute deflecting mirror 31 outside the
light area of the fluorescent light spot on the optical path 32
different from the arrangement direction of the first and second
photodetectors 24 and 26.
[0155] Description will now be given as to the effect of the
scanning optical microscope having the above-described structure.
It is to be noted that only differences from the first embodiment
will be explained in the following description.
[0156] When the sample 2 is scanned by selecting the Ar laser
device 3, the fluorescence having the central wavelength 520 nm
according to the fluorescent dye FITC is generated, and this
fluorescence proceeds in a path similar to that in the first
embodiment and enters the confocal lens 19. Then, the fluorescence
is condensed by the confocal lens 19 and imaged as a light spot on
the minute deflecting mirror array 30.
[0157] The control section 27 selects the Ar laser device 3, and
controls an angle of each minute deflecting mirror 31 in the light
area of the fluorescent light spot imaged in the minute deflecting
mirror array 30 so as to reflect the fluorescence from the sample 2
on the optical path 33 which is the arrangement direction of the
first and second photodetectors 24 and 26. The control section 27
controls an angle of each minute deflecting mirror 31 so as to
reflect an angle of each minute deflecting mirror 31 outside the
light area of the fluorescent light spot on the optical path 32
different from the arrangement direction of the first and second
photodetectors 24 and 26.
[0158] Here, it is assumed that NA of the fluorescence condensed
from the confocal lens 19 onto the minute deflecting mirror array
30 is 0.0063, and the fluorescent dye FITC is excited by the laser
beam having the excitation wavelength 488 nm and generates the
fluorescence having the fluorescent light wavelength 520 nm. Then,
the dimension (diffraction diameter) .phi.D of the light spot on
the minute deflecting mirror array 30 can be obtained by
calculating the following formula: 4 D = 1.22 / NA = 1.22 0.52 /
0.0063 100 m
[0159] Therefore, the control section 27 controls an angle of each
minute deflecting mirror 31 within the diffraction diameter
.phi.D.apprxeq.100 .mu.m (within an area Q1 in FIG. 9) of the
fluorescent light spot to be imaged which is obtained by
calculating the above formula on the minute deflecting mirror array
30 as shown in FIG. 9. Moreover, the control section 27 controls
each minute deflecting mirror 31 in such a manner that the
fluorescence from the sample 2 reflected on these minute deflecting
mirrors 31 proceeds on the optical path 33 in the arrangement
direction of the first or second photodetector 24 or 26. It is to
be noted that "a" denotes each minute deflecting mirror 31 within
the area Q1 in FIG. 9.
[0160] Besides, the control section 27 controls an angle of each
minute deflecting mirror 31 outside the area Q1 in the minute
deflecting mirror array 30 to an angle different from that of each
minute deflecting mirror 31 within the area Q1, and controls each
minute deflecting mirror 31 in such a manner that the light
reflected on these minute deflecting mirrors 31 proceeds on the
optical path 32 deviated from the arrangement direction of the
first or second photodetector 24 or 26.
[0161] FIG. 10 is a side cross-sectional view showing the path of
the fluorescent spot light imaged on each minute deflecting mirror
31 within a plane on which the fluorescence is reflected. Angles of
the respective minute deflecting mirrors 31-1 to 31-11 in the
diffraction diameter (in the area Q1) .phi.D (.apprxeq.100 .mu.m)
of the light spot are controlled in a direction along which the
fluorescent light spot proceeds on the optical path 33, and angles
of the respective minute deflecting mirrors 31-12 to 31-15 outside
the diffraction diameter .phi.D of the light spot are controlled in
a direction along which the light proceeds on the optical path
32.
[0162] By setting the angle of each minute deflecting mirror 31 in
the minute deflecting mirror array 30 having the above-described
arrangement, the fluorescent light spot from a focusing surface of
the sample 2 is reflected on the respective minute deflecting
mirrors 31-1 to 31-11 within the diffraction diameter .phi.D of the
light spot and proceeds on the optical path 33. Moreover, it is
reflected on the reflecting mirror 35 and enters the spectral
dichroic mirror 22. As a result, the minute deflecting mirror array
30 serves as a reflecting type confocal pinhole.
[0163] Besides, the light reflected on the respective minute
deflecting mirrors 31-12 to 31-15 other than the minute deflecting
mirrors 31-1 to 31-11, namely, the light from a surface deviated
from the focusing surface of the sample 2 (de-focusing surface)
does not enter the first or second photodetector 24 or 26.
[0164] The fluorescence of the fluorescent dye FITC which has
entered the spectral dichroic mirror 22 is reflected here and its
unnecessary laser reflected light is cut by the barrier filter 23.
Thus, only the fluorescence of the FITC enters the first
photodetector 24.
[0165] The control section 27 fetches a signal from the first
photodetector 24 and finally acquires a fluorescent image of the
sample 2.
[0166] On the other hand, when increase in the confocal pinhole
diameter is desired in favor of the brightness even though the
confocal effect is sacrificed to some extent, for example, when it
is desired to set the confocal pinhole diameter to 200 .mu.m which
is twice as large as the diffraction diameter .phi.D (.apprxeq.100
.mu.m), the angle of each minute deflecting mirror 31 within the
area Q2 is controlled as shown in FIG. 9 so that the fluorescence
from the sample 2 reflected on these minute deflecting mirrors 31
can proceed on the optical path 33 in the arrangement direction of
the first or second photodetector 24 or 26. That is, the angles of
the respective minute deflecting mirrors "a" or "e" are controlled
in a direction along which the fluorescence proceeds on the optical
path 33.
[0167] By setting the angle of each minute deflecting mirror 31 in
such a minute deflecting mirror array 30, the fluorescent light
spot from the focusing surface of the sample 2 is reflected on each
minute deflecting mirror 31 within Q2 (=200 .mu.m) and proceeds on
the optical path 33. In addition, this fluorescent light spot is
reflected on the reflecting mirror 35 and enters the spectral
dichroic mirror 22. As a result, the minute deflecting mirror array
30 serves as a reflecting type confocal pinhole.
[0168] Besides, the light reflected on each minute deflecting
mirror 31 outside Q2 (=200 .mu.m) proceeds on the optical path 32
and does not enter the first or second photodetector 24 or 26.
[0169] The fluorescence of the fluorescent dye FITC which has
entered the spectral dichroic mirror 22 is reflected here, and its
unnecessary laser reflected light is cut by the barrier filter 23.
Thus, only the fluorescence of the FITC enters the first
photodetector 24.
[0170] The control section 27 fetches a signal from the first
photodetector 24 and finally acquires a fluorescent image of the
sample 2.
[0171] In the fourth embodiment, the minute deflecting mirror array
30 in which a plurality of the minute deflecting mirrors 31 are
arranged in the two-dimensional matrix form is arranged at a
position conjugated with the sample 2 and, in this minute
deflecting mirror array 30, the angle of each minute deflecting
mirror 31 within the diffraction diameter .phi.D of the fluorescent
light spot is controlled so as to reflect the fluorescence in the
arrangement direction of the first and second photodetectors 24 and
26, whilst the angle of each minute deflecting mirror 31 outside
the diffraction diameter .phi.D of the fluorescent light spot is
controlled to be an angle different from that of each minute
deflecting mirror 31 within the diffraction diameter .phi.D of the
light spot. Therefore, the mechanical transmission unit using as a
source of power a motor and the like for switching the dimension of
the confocal pinhole diameter is substituted by angle switching of
the minute deflecting mirror array 30 manufactured by the
semiconductor process, and mechanical abrasion of the driving
section does not occur. Additionally, it is possible to realize
increase in speed of diameter correction of means for effectively
restricting the diffraction diameter.
[0172] Incidentally, the dimension of each minute deflecting mirror
31 is 10 .mu.m.times.10 .mu.m in the fourth embodiment. However,
when reducing the light intensity loss due to a gap between the
respective minute deflecting mirrors 31 as much as possible, it is
good enough to set the focal length of the confocal lens 19 longer
to increase the light spot on the minute deflecting mirror array 30
and increase the dimension of each minute deflecting mirror 31. If
the dimension of the gap between the respective minute deflecting
mirrors 31 is fixed, increase in size of each minute deflecting
mirror 31 can improve the efficiency of use of the light
intensity.
[0173] A fifth embodiment according to the present invention will
now be described. It is to be noted that the structure of the
scanning optical microscope is the same as that in FIG. 7, thereby
omitting illustration and description.
[0174] As to the dimension of the light spot in this example as
described in FIG. 11, it is good enough that the fluorescence
reflected on a part indicated by "a" enters the detector when
measuring the fluorescence having the central wavelength 520 nm
emitted from the fluorescent dye FITC and the fluorescence
reflected on parts indicated by "a" and "b" enters the detector
when measuring the fluorescence having the central wavelength 670
nm emitted from the fluorescent dye Cy5.
[0175] As described above, according to the fifth embodiment, as
similar to the fourth embodiment, the mechanical transmission unit
using as a source of power a motor and the like for switching the
dimension of the confocal pinhole diameter is substituted by angle
switching of the minute deflecting mirror array 30, and the
mechanical abrasion of the driving section does not occur. Further,
it is possible to realize increase in speed of diameter correction
of means for restricting the effective range of the diffraction
diameter. Furthermore, as similar to the first embodiment, when
observing the sample 2 double-fluorescent-dyed with the fluorescent
dyes FITC and Cy5, an image of the FITC is acquired by the first
photodetector 24 and an image of the Cy5 is acquired by the second
photodetector 26 by switching scanning in the outward route and the
inward route by the X galvanometer mirror 14a. Therefore, cross
talk of the two types of fluorescences can be prevented from
occurring, and an optimum diffraction diameter can be set with each
fluorescent light wavelength.
[0176] It is to be noted that a scanning frequency of the X
galvanometer mirror 14a is fast, i.e., 500 Hz. Furthermore, it is
desirable to carry out the set range of the minute deflecting
mirror array 30 in each of the outward route and the inward route,
namely, the time for switching the areas Q1 and Q2 shown in FIG. 11
at 100 .mu.sec or lower which is sufficiently faster than the
one-way scanning time 1 msec for one line in the horizontal
direction.
[0177] Since each minute deflecting mirror 31 has very small mass
and rarely has the inertia, it can accurately deal with this
switching speed. It is to be noted that each minute deflecting
mirror 31 can sufficiently cope with the electrically switching
speed of the first and second photodetectors 24 and 26.
[0178] The fifth embodiment can be modified as follows.
[0179] Although detection is performed by the first and second
photodetectors 24 and 26 in accordance with each fluorescent light
wavelength, the two fluorescences may be acquired by any one of the
first and second photodetectors 24 and 26. According to this
method, a detection signal from any one of the photodetectors, for
example, the first photodetector 24 is processed in the time
sharing manner in the control section 27. That is, the detection
signal in the outward route in the horizontal scanning by the X
galvanometer mirror 14a is processed as an optical signal of the
fluorescent dye FITC, and the detection signal in the inward route
is processed as an optical signal of the fluorescent dye Cy5,
thereby realizing the modification of the above embodiment.
[0180] As described above, when the two fluorescences are acquired
in the time division manner in the control section 27 and the
detection signals from the photodetectors are subjected to signal
processing by dividing the detection time for each fluorescence of
each of the fluorescent dyes FITC and Cy5, one photodetector
substitutes for a plurality of the photodetectors, and the
photodetectors can be cut back. In this case, the barrier filter to
be used is provided with a characteristic for cutting the two
excitation wavelengths 488 nm and 633 nm and transmitting the
wavelengths through the both fluorescent light wavelength regions
of the fluorescent dyes FITC and Cy5.
[0181] Alternatively, in the fifth embodiment, an image of the
fluorescent dye FITC is acquired in the outward route of the X
galvanometer mirror 14a performing scanning in the horizontal
direction, and an image of ant the fluorescent dye Cy5 is acquired
in the inward route of the same. However, a difference in time of
approximately 1 msec is produced between the two types of acquired
fluorescent images. If this difference in time is a problem,
switching between the fluorescent dyes FITC and Cy5 may be carried
out during scanning of one pixel (one point) instead of the
horizontal line. That is, this is setting required for observation
of each of the fluorescent dyes FITC and Cy5. It is good enough to
carry out switching by the acousto-optic device 9 for selecting the
excitation wavelength, angle control of each minute deflecting
mirror 31 within the diffraction diameter .phi.D according to the
fluorescent light wavelength in the minute deflecting mirror array
30, and switching of the first or second photodetector 24 or 26 for
preventing the detector which does not effect detection from
electrically measuring during scanning of one pixel according to
one light spot in the process of scanning on the sample 2.
[0182] According to the fifth embodiment in which the
above-described modifications are combined, since the two types of
fluorescent wavelengths can be obtained in the time division manner
during acquisition of one image and the optimum diffraction
diameter (.phi.D) according to each fluorescent light wavelength
can be set, there occurs no fluorescent light cross talk and the
same confocal effect can be obtained in accordance with each
wavelength.
[0183] A sixth embodiment according to the present invention will
now be described. Incidentally, like reference numerals denote the
same parts as those in FIG. 7, thereby omitting the detailed
explanation.
[0184] FIG. 12 is a block diagram showing a scanning optical
microscope according to a sixth embodiment of the present
invention. This scanning optical microscope acquires three
fluorescences of the fluorescent dyes FITC, PI and Cy5 by one
photodetector 24 in the time division manner. The sixth embodiment
substitutes the minute deflecting mirror array 30 for the confocal
pinhole 20 in the second embodiment. In the sixth embodiment,
description will be given as to the case of acquisition of an image
of the sample 2 triple-dyed with the fluorescent dyes FITC, PI and
Cy5.
[0185] The control section 27 has a function of performing in
accordance with each pixel: the angle control of each minute
deflecting mirror 31 within the diffraction diameter .phi.D
(.apprxeq.100 .mu.m) of the fluorescent spot light by the
fluorescent dye FITC in the minute deflecting mirror array 30 when
the Ar laser device 3 is first selected from the laser unit 1; the
angle control of each minute deflecting mirror 31 within the
diffraction diameter .phi.D (.apprxeq.114 .mu.m) of the fluorescent
spot light by the fluorescent pixel PI in the minute deflecting
mirror array 30 when the HeNe-G laser device 4 is then selected;
and the angle control of each minute deflecting mirror 31 within
the diffraction diameter .phi.D (.apprxeq.130 .mu.m) of the
fluorescent spot light by the fluorescent dye Cy5 in the minute
deflecting mirror array 30 when subsequently selecting the HeNe-R
laser device 5.
[0186] Furthermore, the control section 27 fetches and accumulates
a signal from the photodetector 24 as a signal by the fluorescent
dye FITC; then fetches and accumulates a signal from the
photodetector 24 as a signal by the fluorescent dye PI; and
thereafter fetches and accumulates a signal from the photodetector
24 as a signal by the fluorescent dye Cy5 in synchronization with
selection of the Ar laser device 3, the HeNe-G laser device 4 or
the HeNe-R laser device 5. Moreover, the control section 27
performs these operations with respect to all the pixels while
scanning using the X/Y galvanometer mirrors 14a and 14b. Then, the
control section 27 distinguishes the respective signals by the
fluorescent dyes FITC, PI and Cy5 accumulated in accordance with
each pixel by using different colors, and displays them as one
multi-dye fluorescent image on, e.g., a monitor.
[0187] It is to be noted that the barrier filter 25 arranged in
front of the photodetector 24 is the same as one which has been
described in connection with the third embodiment.
[0188] According to the sixth embodiment, as similar to the fourth
embodiment, the mechanical transmission unit using as a source of
power a motor and the like for switching the dimension of the
confocal pinhole diameter is substituted for angle switching of the
minute deflecting mirror array 30 manufactured by the semiconductor
process, and the mechanical abrasion of the driving section does
not occur. Moreover, it is possible to realize increase in speed
for diameter correction of means for restricting the effective
range of the diffraction diameter. In addition, when observing the
sample 2 triple-fluorescent-dyed with the fluorescent dyes FITC, PI
and Cy5, the optimum diffraction diameter can be set in accordance
with each fluorescent light wavelength relative to each of these
fluorescent dyes FITC, PI and Cy5 and its multi-dye fluorescent
image can be acquired.
[0189] A seventh embodiment according to the present invention will
now be described. It is to be noted that like reference numerals
denote the same parts as those in FIG. 7, thereby omitting the
detailed explanation.
[0190] FIG. 13 is a block diagram showing a scanning optical
microscope according to the seventh embodiment of the present
invention. This scanning optical microscope is provided with a
first function of angle control of each minute deflecting mirror 31
within the diffraction diameter .phi.D of the fluorescent spot
light in the minute deflecting mirror array 30 when switched to the
objective lens 18 or 40, and a second function for correcting the
displacement of the light spot on the minute deflecting mirror
array 30 caused due to each mounting angle error when the
excitation dichroic mirror 13a is switched to the excitation
dichroic mirror 13b or 13c having another wavelength
characteristic.
[0191] The objective lenses 18 and 40 have different magnifications
B and numerical aperture NA. The present invention is not
restricted to these objective lenses, and there are prepared
multiple objective lenses having different magnifications B and
numerical aperture NA.
[0192] An objective lens switching mechanism 41 has a function for
switching the objective lens 18 or 40, arranging it in the optical
axis, and informing the control section 27 of a type of the
switched objective lens 18 or 40.
[0193] The excitation dichroic mirror switching sensor 42 has a
function for detecting the excitation dichroic mirror 13a, 13b or
13c arranged in the fluorescent optical path by the switching
operation of the respective excitation dichroic mirror 13a, 13b or
13c and transmitting its detection signal to the control section
27.
[0194] When the control section 27 is informed of a type of the
switched objective lens 18 or 40 from the objective lens switching
mechanism 41, the control section 27 functions to calculate the
diffraction diameter .phi.D of the light spot in the minute
deflecting mirror array 30 based on the fluorescent light
wavelength to be observed, the magnification B and numerical
aperture NA of the switched objective lens 18 or 40.
[0195] Assuming that the fluorescent light wavelength to be
observed is .lambda., the focal length of the confocal lens 19 is
Fc, the focal length of the pupil projection lens 15 is Fp, the
magnification of each of the objective lens 18 or 40 is B, and
numerical aperture is NA, the diffraction diameter .phi.D of the
light spot can be obtained by calculating the following expression
(and so forth in this embodiment):
.phi.D=1.22.times.(B.multidot.Fc.multidot..lambda.)/(NA.multidot.Fp)
[0196] Therefore, the control section 27 has a function for
controlling an angle of each minute deflecting mirror 31 within the
diffraction diameter .phi.D of the light spot calculated by the
above expression in the minute deflecting mirror array 30 so that
the fluorescence from the sample 2 is reflected on the optical path
33 which is an arrangement direction of the first and second
photodetectors 24 and 26, and controlling an angle of each minute
deflecting mirror 31 so that an angle of each minute deflecting
mirror 31 outside the diffraction diameter .phi.D of the light spot
is reflected on the optical path 32 different from the arrangement
direction of the first and second photodetectors 24 and 26, when
switched to the objective lens 18 or 40.
[0197] It is to be noted that in the memory 43 is stored data of
the fluorescent light wavelength .lambda., the focal length Fc of
the confocal lens 19, the focal length Fp of the pupil projection
lens 15, the magnification B and the numerical aperture NA of each
of the objective lenses 18 and 40.
[0198] Further, the control section 27 has a function for
correcting the displacement of the light spot on the minute
deflecting mirror array 30 caused due to each mounting angle error
in accordance with the excitation dichroic mirror 13a, 13b or 13c
upon receiving the detection signal indicative of switching of each
excitation dichroic mirror 13a, 13b or 13c from the excitation
dichroic mirror switching sensor 42. The displacement of the light
spot on the minute deflecting mirror array 30 with respect to each
excitation dichroic mirror 13a, 13b or 13c is stored in the memory
43 in advance.
[0199] The effect of the scanning optical microscope having the
above-described structure will now be explained.
[0200] Description will be first given as to the angle control of
each minute deflecting mirror 31 within the diffraction diameter
.phi.D of the fluorescent spot light in the minute deflecting
mirror array 30 when switched to the objective lens 18 or 20.
[0201] For example, when the control section 27 issues a selection
command of the Ar laser device 3 to the acousto-optic device 9 of
the laser unit 1, this acousto-optic device 9 selects a laser beam
having an excitation wavelength of 488 nm outputted from the Ar
laser device 3 and leads it to the single-mode fiber 10.
[0202] The laser beam having the excitation wavelength 488 nm is
transmitted through the single-mode fiber 10 and led to the
scanning unit 11. This laser beam is then formed into a parallel
beam by the collimator lens 12, reflected by the excitation
dichroic mirror 13, and scanned by the X/Y galvanometer mirrors 14a
and 14b. Furthermore, it is transmitted through the pupil
projection lens 15, reflected downwards by the mirror 16, and
imaged as a light spot on the sample 2 through the image formation
lens 17 and the objective lens 18.
[0203] At this moment, the light spot is scanned in the horizontal
direction by the X galvanometer mirror 14a of the X/Y galvanometer
mirrors 14a and 14b, then scanned for one pixel in the vertical
direction by the Y galvanometer mirror 14b, and again scanned in
the horizontal direction by the X galvanometer mirror 14a. This
process is repeated.
[0204] The fluorescence having the central wavelength 520 nm by the
fluorescent dye FITC generated when scanned on the sample 2 in the
above-described manner proceeds in a direction opposite to the
illumination optical path, namely, proceeds from the objective lens
18 to the image formation lens 17, the mirror 16, the pupil
projection lens 15, the X/Y galvanometer mirrors 14a and 14b, and
enters the confocal lens 19 through the excitation dichroic mirror
13a. The fluorescence is then condensed by the confocal lens 19 and
imaged as a light spot on the minute deflecting mirror array
30.
[0205] At this moment, if the objective lens 18 is set on the
optical axis, the objective lens switching mechanism 41 informs the
control section 27 of a type of the objective lens 18.
[0206] When this control section 27 is informed of a type of the
objective lens 18 from the objective lens switching mechanism 41,
the control section 27 calculates the diffraction diameter .phi.D
of the light spot in the minute deflecting mirror array 30 based on
the selected excitation wavelength (fluorescent light wavelength
.lambda. generated by 488 nm), the confocal length Fc of the
confocal lens 19, the focal length Fp of the pupil projection lens
15, and the magnification B and a number of openings NA of the
objective lens 18.
[0207] Thus, when the objective lens 18 is set on the optical axis,
the control section 27 controls an angle of each minute deflecting
mirror 31 within the diffraction diameter .phi.D of the light spot
in the minute deflecting mirror array 30 so that the fluorescence
from the sample 2 is reflected on the optical path 33 which is an
arrangement direction of the first and second photodetectors 24 and
26, and controls an angle of each minute deflecting mirror 31 so
that an angle of each minute deflecting mirror 31 outside the
diffraction diameter .phi.D of the light spot is reflected on the
optical path 32 different from the arrangement direction of the
first and second photodetectors 24 and 26.
[0208] With the angle setting of each minute deflecting mirror 31
on the minute deflecting mirror array 30 mentioned above, the
fluorescent light spot from the focusing surface of the sample 2 is
reflected on each minute deflecting mirror within the diffraction
diameter .phi.D of the light spot and proceeds on the optical path
33. Moreover, the fluorescent light spot is reflected by the
reflecting mirror 35 and enters the spectral dichroic mirror 22. As
a result, the minute deflecting mirror array 30 serves as the
reflecting type confocal pinhole.
[0209] The fluorescence of the fluorescent dye FITC which has
entered the spectral dichroic mirror 22 is reflected here, and its
unnecessary laser reflection light is cut by the barrier filter 23.
Thus, only the fluorescence of the FITC enters the first
photodetector 24.
[0210] The control section 27 fetches a signal from the first
photodetector 24 and finally acquires a fluorescent image of the
sample 2.
[0211] Subsequently, when the objective lens 18 is switched to the
objective lens 40, the objective lens switching mechanism 41
informs the control section 27 of a type of the objective lens
40.
[0212] Upon being informed of a type of the objective lens 40 from
the objective lens switching mechanism 41, the control section 27
calculates the diffraction diameter .phi.D of the light spot in the
minute deflecting mirror array 30 based on the selected excitation
wavelength (fluorescent wavelength .lambda. generated by 488 nm),
the focal length Fc of the confocal lens 19, the focal length Fp of
the pupil projection lens 15, the magnification B and numerical
aperture NA of the objective lens 40.
[0213] Thus, when the objective lens 40 is switched, the control
section 27 controls an angle of each minute deflecting mirror 31
within the diffraction diameter .phi.D of the light spot in the
minute deflecting mirror array 30 so that the fluorescence from the
sample 2 is reflected on the optical path 33 which is an
arrangement direction of the first and second photodetectors 24 and
26, and controls an angle of each minute deflecting mirror 31 so
that an angle of each minute deflecting mirror 31 outside the
diffraction diameter .phi.D of the light spot is reflected on the
optical path 32 different from the arrangement direction of the
first and second photodetectors 24 and 26.
[0214] With the angle setting of each minute deflecting mirror 31
in the minute deflecting mirror array 30, the fluorescent light
spot from the focusing surface from the sample 2 is reflected by
each minute deflecting mirror within the diffraction diameter
.phi.D of the light spot. In addition, the fluorescent light spot
is reflected by the reflecting mirror 35 and enters the spectral
dichroic mirror 22. As a result, the minute deflecting mirror array
30 serves as the reflecting type confocal pinhole.
[0215] The fluorescence of the fluorescent dye FITC which has
entered the spectral dichroic mirror 22 is reflected here, and its
unnecessary laser reflected light is cut by the barrier filter 23.
Thus, only the fluorescence of the FITC enters the first
photodetector 24.
[0216] The control section 27 fetches a signal from the first
photodetector 24 and finally acquires a fluorescent image of the
sample 2.
[0217] As described above, since a type of the currently used
objective lens is recognized by the control section 27, it is
possible to rapidly and assuredly control the dimension of the
reflecting type pinhole which can be matched with the diffraction
diameter determined by the magnification and NA of the objective
lens and the fluorescent light wavelength even when the objective
lens is switched.
[0218] Description will now be given as to correction of the
displacement of the light spot on the minute deflecting mirror
array 30 which is caused due to each mounting angle error when the
excitation dichroic mirror 13a is switched to the excitation
dichroic mirror 13b or 13c having another wavelength
characteristic.
[0219] The acousto-optic device 9 is first used to select the laser
beam having the excitation wavelength of 488 nm outputted from the
Ar laser device 3 for example, and the excitation dichroic mirror
13a is included in the optical path. This laser beam is transmitted
through the single-mode fiber 10 and led to the scanning unit 11.
This laser beam is then imaged as a light spot on the sample 2
which is dyed to, for example, the fluorescent dye FITC, through
the collimator lens 12, the excitation dichroic mirror 13a,the X/Y
galvanometer mirrors 14a and 14b, the pupil projection lens 15, the
mirror 16, the image formation lens 17, and the objective lens
18.
[0220] The fluorescence having the central wavelength 520 nm by the
fluorescent dye FITC generated in the sample 2 proceeds in a
direction opposite to the illumination optical path, namely,
proceeds from the objective lens 18 to the image formation lens 17,
the mirror 16, the pupil projection lens 15 and the X/Y
galvanometer mirrors 14a and 14b, passes through the excitation
dichroic mirror 13a, and enters the confocal lens 19. The
fluorescence is then condensed by the confocal lens 19 and imaged
as a light spot on the minute deflecting mirror array 30. FIG. 14
shows the center of the light spot which is imaged on the minute
deflecting mirror array 30 when the excitation dichroic mirror 13a
is set in the optical path. The spot center is determined as
P1.
[0221] When switching to the excitation dichroic mirror 13b in the
state where the excitation dichroic mirror 13a is set in the
optical path, the excitation dichroic mirror switching sensor 42
detects the excitation dichroic mirror 13b switched and arranged in
the fluorescent optical path and transmits its detection signal to
the control section 27.
[0222] The control section 27 receives the detection signal
indicative of switching of the excitation dichroic mirror 13b from
the excitation dichroic mirror switching sensor 42, and corrects
the displacement of the light spot on the minute deflecting mirror
array 30 caused due to each mounting angle error in accordance with
this excitation dichroic mirror 13b.
[0223] That is, when the excitation dichroic mirror 13a is switched
to 13b in the state where the excitation dichroic mirror 13a is set
in the optical path, an angle of the fluorescence incident upon the
confocal lens 19 is shifted, e.g., approximately 80" in the lateral
direction due to an angle error of the excitation dichroic mirror
13b relative to the excitation dichroic mirror 13a.
[0224] Therefore, the central position of the fluorescent light
spot on the minute deflecting mirror array 30 is moved from the
spot center P1 when the excitation dichroic mirror 13a is set in
the optical path to the spot center P2 by switching to the
excitation dichroic mirror 13b.
[0225] Assuming that the focal length of the confocal lens 19 is,
for example, 200 nm, the distance S between the excitation dichroic
mirrors 13a and 13b is as follows: 5 S = 200 tan ( 80 " ) = 80
m
[0226] It is to be noted that a position of the spot center P2 is
previously stored in the memory 43 as a position corresponding to
the excitation dichroic mirror 13b.
[0227] At this moment, since the sample 2 is excited by the laser
beam having the excitation wavelength of 488 nm and the
fluorescence having the central wavelength of 520 nm of the
fluorescent dye FITC is generated, the diffraction diameter .phi.D
on the minute deflecting mirror array 30 is 100 .mu.m.
[0228] Therefore, the control section 27 controls an angle of each
minute deflecting mirror 31 within the diffraction diameter .phi.D
(within the area Q4) with the spot center P2 at the center thereof
in the minute deflecting mirror array 30, and causes the
fluorescence reflected by each minute deflecting mirror 31 to
proceed on the optical path 33 which is in the arrangement
direction of the first photodetector 24.
[0229] As a result, the fluorescence from the focusing surface of
the sample 2 is reflected by each minute deflecting mirror 31 in
the area Q4 and proceeds on the optical path 33. This fluorescence
is further reflected by the reflecting mirror 35 and enters the
spectral dichroic mirror 22 on which the fluorescence is reflected.
The unnecessary laser beam is cut by the barrier filter 23, and
only the fluorescence of the FITC enters the first photodetector
24.
[0230] The control section 27 fetches a signal from the first
photodetector 24 and finally acquires a fluorescent image of the
sample 2.
[0231] Subsequently, it is assumed that the acousto-optic device 9
selects the laser beam having the excitation wavelength 543 nm
outputted from the HeNe-G laser device 4 by a selection command
from the control section 27 and the excitation dichroic mirror is
switched to the counterpart 13c.
[0232] The excitation dichroic mirror switching sensor 42 detects
the excitation dichroic mirror 13c which has been switched and
arranged in the fluorescent optical path and transmits its
detection signal to the control section 27.
[0233] This control section 27 receives the detection signal
indicative of switching to the excitation dichroic mirror 13c from
the excitation dichroic mirror switching sensor 42, and corrects
the displacement of the light spot on the minute deflecting mirror
array 30 caused due to each mounting angle error in accordance with
this excitation dichroic mirror 13c.
[0234] That is, when the excitation dichroic mirror 13a is switched
to the counterpart 13c in the state where the excitation dichroic
mirror 13a is set in the optical path, an angle of the fluorescence
incident upon the confocal lens 19 is shifted, e.g., approximately
-60"in the lateral direction due to an angle error of the
excitation dichroic mirror 13c relative to the excitation dichroic
mirror 13a.
[0235] Therefore, the central position of the fluorescent light
spot on the minute deflecting mirror array 30 is the spot center P1
when the excitation dichroic mirror 13a is set in the optical path
as shown in FIG. 14, but it moves to the spot center P3 when the
excitation dichroic mirror is switched to the counterpart 13b.
[0236] Assuming that the focal length of the confocal lens 19 is,
e.g., 200 mm, the distance S between the excitation dichroic
mirrors 13a and 13c is as follows: 6 S = 200 tan ( - 60 " ) = - 60
m ( lateral direction )
[0237] It is to be noted that a position of the spot center P3 is
previously stored in the memory 43 as a position corresponding to
the excitation dichroic mirror 13c.
[0238] At this moment, since the sample 2 is excited by the
excitation laser beam having the excitation wavelength 543 nm and
the fluorescence having the central wavelength of approximately 590
nm by the fluorescent dye PI is generated, the diffraction diameter
.phi.D on the minute deflecting mirror array 30 is 114 .mu.m.
[0239] Therefore, the control section 27 controls an angle of each
minute deflecting mirror 31 within the diffraction diameter .phi.D
(area Q5) with the spot center at its center and causes the
fluorescence reflected on each minute deflecting mirror 31 to
proceed on the optical path 33 which is in the arrangement
direction of the second photodetector 26.
[0240] Consequently, the fluorescence from the focusing surface of
the sample 2 is reflected on each minute deflecting mirror 31 in
the area Q5 and proceeds on the optical path 33. The fluorescence
is further reflected by the reflecting mirror 35 and passes through
the spectral dichroic mirror 22. The barrier filter 25 is used to
cut the unnecessary laser beam, and only the fluorescence of the PI
enters the second photodetector 26.
[0241] The control section 27 fetches a signal from the second
photodetector 26 and finally acquires a fluorescent image of the
sample 2.
[0242] As described above, in the seventh embodiment, there are
provided a first function for controlling an angle of each minute
deflecting mirror 31 within the diffraction diameter .phi.D of the
fluorescent spot light in the minute deflecting mirror array 30
when the fluorescent light wavelength or the objective lens is
switched, and a second function for correcting the displacement of
the light spot on the minute deflecting mirror array 30 caused due
to each mounting angle error when the excitation dichroic mirror is
switched to 13a,13b or 13c. Therefore, it is possible to control
and obtain the diffraction diameter .phi.D which is optimum for the
fluorescent spot light in the minute deflecting mirror array 30
even when the objective lens is switched, and this embodiment can
thereby serve as the reflecting type confocal pinhole.
[0243] Furthermore, when the excitation dichroic mirror is switched
to 13a, 13b or 13c, it is possible to correct the displacement of
the light spot on the minute deflecting mirror array 30 caused due
to each mounting angle error. Thus, one minute deflecting mirror
array which does not require a mechanical transmission unit using a
motor and the like as a source of power can realize opening/closing
of the pinhole which needs the high accuracy, position correction
of two axes within a plane and a total of three drive mechanisms,
thereby obtaining the high reliability with the reduced abrasion of
the driving section. Moreover, it is possible to eliminate a number
of design steps and reduce the size of the apparatus.
[0244] Incidentally, the present invention is not restricted to the
first to seventh embodiments, and various modifications are
possible without departing from its scope on the embodying
stage.
[0245] In addition, the above-described embodiments include the
invention on the various stages, and a variety of the inventions
can be extracted by appropriately combining these stages in a
plurality of disclosed structural requirements. For example, even
if some structural requirements are deleted from all the structural
requirements shown in the embodiments, the problems described in
the section "problems to be solved by the invention" can be solved.
When the effects described in the section "effects of the
invention" an be obtained, the structure in which these structural
requirements are deleted can be extracted as the present
invention.
[0246] For example, the seventh embodiment can be modified as
follows. That is, the confocal lens and the minute deflecting
mirror array may be provided in accordance with each detection
channel at the rear of a plurality of the switching type spectral
dichroic mirrors for dividing the lights to the first and second
photodetectors 24 and 26 for each wavelength.
[0247] FIG. 15 is a partial block diagram showing a scanning
optical microscope when the confocal lens and the minute deflecting
mirror array are provided at the rear of such a switching type
spectral dichroic mirror. The switching type spectral dichroic
mirror 50 is arranged on the optical path of the fluorescence which
has passed through, e.g., the excitation dichroic mirror 13a among
the excitation dichroic mirrors 13a, 13b and 13c.
[0248] The minute deflecting mirror array 30a is arranged on the
transmission optical path of the switching type spectral dichroic
mirror 50 through the confocal lens 19a as the 1CH side.
Additionally, the first photodetector 24 is arranged on the
reflection optical path 33a of the minute deflecting mirror array
30a through the barrier filter 23.
[0249] On the other hand, the minute deflecting mirror array 30b is
arranged on the reflection optical path of the switching type
spectral dichroic mirror 50 through the confocal lens 19b as the
2CH side. Further, the second photodetector 26 is arranged on the
reflection optical path 33b of the minute deflecting mirror array
30b through the barrier filter 25.
[0250] In this case, when the spectral dichroic mirror 50 is
switched, its angle error also affects the displacement of the
light spot on the minute deflecting mirror array 30a or 30b.
Accordingly, when either or both of the respective excitation
dichroic mirrors 13a, 13b and 13c and the spectral dichroic mirror
50 are switched, the position correction can be executed with
respect to the displacement of the light spot in the minute
deflecting mirror array 30a or 30b.
[0251] Moreover, for example, although the minute deflecting mirror
array 30 is used as the minute device group, the present invention
is not restricted thereto. For example, liquid crystal may be used
as the minute device group. In this case, the minute device for
transmitting the light therethrough or the minute device for
blocking the light is selected and controlled by controlling the
electrode of each element (defined as the minute device) aligned in
the matrix form.
[0252] Therefore, when the control is effected in such a manner
that the electrode of each minute device in the minute device group
positioned in the area of the light spot is in the transmission
mode and the electrode of any other minute device is in the
blocking mode, the light transmitted thorough the liquid crystal
can be detected by the photodetector 24 or 15, and the light from
any other element (minute device) is blocked and is not detected by
the photodetector 24 or 15. Thus, the minute device group can be
used as the transmission type confocal pinhole means. Furthermore,
in the liquid crystal, the speed for controlling each element can
be increased.
[0253] When the minute device group using the liquid crystal is
applied to the scanning optical microscope shown in FIG. 7, it is
needless to say that the optical path 33 reflected by the minute
deflecting mirror array 30 becomes the path of the light which has
passed through the liquid crystal used instead of the minute
deflecting mirror array 30 in case of using the liquid crystal.
[0254] Moreover, as the minute device group, a two-dimensional CCD
in which a plurality of photo acceptance pixels (defined as minute
devices) are arranged in the form of a matrix may be used. In this
case, a sum total of accepted light intensity of pixels in a pixel
group (minute device group) positioned in the area of the light
spot is used as a detection signal, and any other pixel can not be
electrically detected, or its detection signal is not added even if
such a pixel is detected. Thus, selection and control of any other
minute devices which are controlled so as to be detected by the
photodetector can be carried out by choosing the detection signals
of the respective pixels. Accordingly, the optical path 33 to the
photodetector and the following structures after the minute
deflecting mirror array 30 shown in FIG. 7 are no longer
necessary.
[0255] In addition, the microscope can be realized by a simple
structure which can share the minute device group and the
photodetector by one device. Incidentally, when this modification
is applied to FIG. 7, it is needless to say that the minute
deflecting mirror array 30 substitutes for the CCD and the
following optical path 33 is no longer necessary.
[0256] Additionally, although the minute deflecting mirror array 30
has a plurality of minute deflecting mirrors 31 being arranged in
the two-dimensional matrix as shown in FIG. 8, the minute
deflecting mirrors 31 may be arranged at random positions and form
a group.
[0257] Further, although the first to seventh embodiments have
described the scanning optical microscope for exciting the sample 2
dyed with the fluorescent dyes and observing its fluorescences
through the confocal pinhole, these embodiments can be of course
applied to the scanning optical microscope for observing the
transmission lights and the reflection lights from the sample 2
through the confocal pinhole.
[0258] Furthermore, although the sample is used in a biosystem dyed
with the fluorescent dyes in the foregoing embodiments, the present
invention is not restricted thereto, and the sample may be used in,
for example, an industrial system.
[0259] Additional advantages and modifications will readily occur
to those skilled in the art. Therefore, the present invention in
its broader aspects is not limited to the specific details,
representative devices, and illustrated examples 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.
* * * * *