U.S. patent application number 10/902967 was filed with the patent office on 2005-01-06 for total internal reflection illumination apparatus and microscope using this total internal reflection illumination apparatus.
This patent application is currently assigned to Olympus Optical Co., Ltd.. Invention is credited to Aono, Yasushi, Hochizuki, Tsuyoshi.
Application Number | 20050002092 10/902967 |
Document ID | / |
Family ID | 26624373 |
Filed Date | 2005-01-06 |
United States Patent
Application |
20050002092 |
Kind Code |
A1 |
Aono, Yasushi ; et
al. |
January 6, 2005 |
Total internal reflection illumination apparatus and microscope
using this total internal reflection illumination apparatus
Abstract
A total internal reflection illumination apparatus applied to a
microscope which illuminates a sample through an objective having a
numerical aperture enabling total internal reflection illumination,
comprises a first total internal reflection mirror which is
arranged in the vicinity of an outermost peripheral part of an
observation optical path of the microscope to reflect an incident
illumination light in a direction of the objective, a second total
internal reflection mirror which is arranged at a symmetrical
position with the first total internal reflection mirror to
sandwich an observation optical axis and reflects return light
reflected on a surface of the sample in a direction different from
the illumination optical path, and a return light dimming part
configured to dim the return light reflected by the second total
internal reflection mirror.
Inventors: |
Aono, Yasushi;
(Yokohama-shi, JP) ; Hochizuki, Tsuyoshi;
(Hachioji-shi, JP) |
Correspondence
Address: |
FRISHAUF, HOLTZ, GOODMAN & CHICK, PC
767 THIRD AVENUE
25TH FLOOR
NEW YORK
NY
10017-2023
US
|
Assignee: |
Olympus Optical Co., Ltd.
Tokyo
JP
|
Family ID: |
26624373 |
Appl. No.: |
10/902967 |
Filed: |
July 29, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10902967 |
Jul 29, 2004 |
|
|
|
10286639 |
Nov 1, 2002 |
|
|
|
6819484 |
|
|
|
|
Current U.S.
Class: |
359/368 |
Current CPC
Class: |
G02B 21/06 20130101;
G02B 21/16 20130101 |
Class at
Publication: |
359/368 |
International
Class: |
G02B 021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 6, 2001 |
JP |
2001-341007 |
Dec 7, 2001 |
JP |
2001-374426 |
Claims
What is claimed is:
1. A microscope comprising: a changing part configured to change an
incidence angle of illumination light which is emitted onto a
sample from a light source through an objective and enables
switching between total internal reflection illumination and
approximate total internal reflection illumination; and a
restricting part configured to restrict the incidence angle of the
illumination light to the sample through the objective to a range
where total internal reflection illumination and approximate total
internal reflection illumination can be obtained.
2. The microscope according to claim 1, further comprising a
dimming part configured to dim light reflected by the sample.
3. The microscope according to claim 1, wherein the light source
has an optical fiber having an outgoing end provided to be movable
in a direction vertical to an optical axis, and the restricting
part restricts a movement range of the outgoing end of the optical
fiber in the direction vertical to the optical axis to the range
where total internal reflection illumination and approximate total
internal reflection illumination can be obtained.
4. The microscope according to claim 3, further comprising an
optical element which is insertably and removably arranged the
light path between the light source and the objective and diffuses
the illumination light.
5. The microscope according to claim 1, wherein a light path
between the light source and the objective has a reflection member
provided so as to be capable of moving along the light path
direction, and the restricting part restricts a movement range of
the reflection member along the light path direction to the range
where total internal reflection illumination and approximate total
internal reflection illumination can be obtained.
6. The microscope according to claim 5 further comprising an
optical element which is insertably and removably arranged to the
light path between the light source and the objective and diffuses
the illumination light.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Divisional Application of U.S.
application Ser. No. 10/286,639, filed Nov. 1, 2002 which is based
upon and claims the benefit of priority from the prior Japanese
Patent Applications No. 2001-341007, filed Nov. 6, 2001; and No.
2001-374426, filed Dec. 7, 2001, the entire contents of both of
which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a total reflection
illumination apparatus applied to a microscope which illuminates a
sample through an objective having a numerical aperture enabling
total internal reflection illumination, and a microscope using this
total internal reflection illumination apparatus.
[0004] 2. Description of the Background Art
[0005] In recent years, functional analysis of a biological cell
has been vigorously carried out. In such function analysis of
cells, in order to observe a function of a cell membrane in
particular, attention is drawn to a total internal reflection
fluorescence microscopy (TIRFM) which acquires total internal
reflection fluorescence images from the cell membrane and a nearby
part.
[0006] In such a total internal reflection fluorescence microscopy
(which will be referred to as a "microscope" hereinafter), a total
internal reflection illumination which locally illuminates only a
sample (which may be also referred to as a "specimen" in some
cases) in the vicinity of the glass surface is used. In this
microscope, when the illumination light is totally reflected on the
interface between the cover glass and the specimen, a fluorescent
material is excited by using the light called evanescent light
which permeates in a small range of not more than several 100 nm on
the specimen side. Therefore, only the fluorescence in a small
range in the vicinity of the cover glass is observed. Accordingly,
the background (scattered light or the like) is very dark, and the
weak fluorescence can be observed (for example, observation of the
fluorescence of one molecule of the fluorescent dye).
[0007] Meanwhile, in the fluorescence observation by using such a
total internal reflection illumination, a permeation depth of the
evanescent light which permeates from the glass surface to the
sample side varies depending on a refractive index of the glass and
others. Further, this permeation depth means a depth along which
observation has been carried out, and it also varies depending on a
purpose of a speculum user.
[0008] Thus, there is also considered varying an incidence angle of
the illumination light from the glass to the sample in accordance
with conditions of the specimen or a depth along which observation
should be carried out.
[0009] Meanwhile, in case of performing fluorescence observation
using the total internal reflection illumination, there is known a
microscope which conducts the total internal reflection
illumination which illuminates the sample through an objective.
[0010] For example, in such a microscope, a mirror which reflects
the light from a light source to an objective side is moved, and an
incidence position of the illumination light to the objective is
continuously moved in a direction away from the optical axis of the
objective. As a result, the incidence angle from the glass to the
sample is continuously changed and the incident-light fluorescence
illumination and the total internal reflection illumination are
switched (see Jpn. Pat. Appln. KOKAI Publication No. 09-159922). It
is to be noted that a micrometer or the like is generally used for
movement of the mirror which reflects the illumination light,
namely, adjustment of the incidence angle from the glass to the
sample because fine adjustment is required.
[0011] Furthermore, in another microscope, a frame of the objective
has a dual structure consisting of an inner frame and an outer
frame, the light from the light source is reflected by an annular
mirror so that the illumination light can pass between the inner
frame and the outer frame in the dual structure. A sample is
illuminated with that light, and the return light from the sample
is observed through the objective (see Jpn. Pat. Appln. KOKAI
Publication No. 10-96861).
[0012] In the above-described structure, in the total internal
reflection illumination which performs illumination through the
objective, the illumination light with which the specimen is
irradiated returns to the objective in principle. Moreover, the
mirror used to lead the illumination light to the objective side is
provided in an observation optical path extending from the
objective to observing means.
[0013] Therefore, the observation optical path of the fluorescence
emitted from the specimen crosses the illumination light or the
totally reflected return light. Therefore, the self-fluorescence
generated on the illumination light or the totally reflected return
light beam enters the observation light beam, and there is a
possibility that a fluorescence observation image may be
deteriorated.
[0014] In addition, since the illumination light and the totally
reflected return light also cross each other, an interference
fringe may be generated due to crossing of the laser beams when the
laser beam is used as the illumination light, for example. An
excellent fluorescence observation image can not be obtained due to
the influence of the interference fringe.
[0015] Additionally, in the microscope disclosed in Jpn. Pat.
Appln. KOKAI Publication No. 09-159922, the incident-light
fluorescence illumination may be turned on at the time of adjusting
the incidence angle from the glass to the sample when performing
the fluorescence observation by the total internal fluorescence
illumination in some cases. In this case, the mirror must be
freshly moved to a position of the total internal reflection
illumination. However, since the sample on the glass surface is
irradiated with the incident-light illumination with the strong
intensity as the exciting light during this movement, the entire
sample may lose its color.
[0016] Further, although the micrometer or the like is used to move
the mirror in the range from the incident-light fluorescence
illumination to the total internal reflection illumination, since
the micrometer has a small quantity of movement per one rotation of
a rotation operation portion, the number of times of rotation
increases when switching from the incident-light illumination to
the total internal reflection illumination. Therefore, a lot of
trouble is taken until this switching, thereby greatly reducing the
operability of the fluorescence observation. Furthermore, this
means that the entire sample may possibly lose its color during
this switching when trying to switch from the incident-light
fluorescence illumination to the total internal reflection
illumination while irradiating the sample with the illumination
exciting light.
BRIEF SUMMARY OF THE INVENTION
[0017] A total internal reflection illumination apparatus according
to the first aspect of the present invention apparatus applied to a
microscope which illuminates a sample through an objective having a
numerical aperture enabling total internal reflection illumination,
is characterized by comprising: a first total internal reflection
mirror which is arranged in the vicinity of an outermost peripheral
part of an observation optical path of the microscope to reflect an
incident illumination light in a direction of the objective; a
second total internal reflection mirror which is arranged at a
symmetrical position with the first total internal reflection
mirror to sandwich an observation optical axis and reflects return
light reflected on a surface of the sample in a direction different
from the illumination optical path; and a return light dimming part
configured to dim the return light reflected by the second total
internal reflection mirror.
[0018] A microscope according to the second aspect of the present
invention is characterized by comprising: a light source which
emits predetermined light; a total internal reflection illumination
apparatus according to above-mentioned total internal reflection
illumination apparatus, which irradiates a sample with the light
from the light source through an objective; and an image pickup
device which images the light from the sample as an image.
[0019] A microscope according to the third aspect of the present
invention is characterized by comprising: a changing part
configured to change an incidence angle of illumination light which
is emitted onto a sample from a light source through an objective
and enables switching between total internal reflection
illumination and approximate total internal reflection
illumination; and a restricting part configured to restrict the
incidence angle of the illumination light to the sample through the
objective to a range where total internal reflection illumination
and approximate total internal reflection illumination can be
obtained.
[0020] Additional objects and advantages of the invention will be
set forth in the description which follows, and in part will be
obvious from the description, or may be learned by practice of the
invention. The objects and advantages of the invention may be
realized and obtained by means of the instrumentalities and
combinations particularly pointed out hereinafter.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING
[0021] The accompanying drawings, which are incorporated in and
constitute a part of the specification, illustrate presently
preferred embodiments of the invention, and together with the
general description given above and the detailed description of the
preferred embodiments given below, serve to explain the principles
of the invention.
[0022] FIG. 1 is a schematic structural view for illustrating a
scheme of a microscope according to the present invention;
[0023] FIG. 2 is a schematic structural view for illustrating the
regular fluorescence incident-light illumination of the microscope
according to the present invention;
[0024] FIG. 3 is a view showing a schematic structure of a first
embodiment according to the present invention;
[0025] FIG. 4 is a view showing a schematic structure of a slider
main body used in the first embodiment;
[0026] FIG. 5 is an overview diagram when a total internal
reflection illumination apparatus according to the first embodiment
is applied to an inverted microscope;
[0027] FIG. 6 is an overview diagram when the total internal
reflection illumination apparatus according to the first embodiment
is applied to an microscope with upright frame;
[0028] FIG. 7 is a view showing a schematic structure of a second
embodiment according to the present invention;
[0029] FIG. 8 is a view showing a schematic structure of a slider
main body used in a third embodiment according to the present
invention;
[0030] FIG. 9 is a view showing a schematic structure of a slider
main body used in the third embodiment;
[0031] FIG. 10 shows a modification of the third embodiment;
[0032] FIG. 11 is a modification of the first embodiment to the
third embodiment;
[0033] FIGS. 12A to 12C are views showing a schematic structure of
a fourth embodiment according to the present invention;
[0034] FIGS. 13A and 13B are views showing a schematic structure of
a modification of the fourth embodiment according to the present
invention;
[0035] FIG. 14 is a view showing a schematic structure of a fifth
embodiment according to the present invention; and
[0036] FIGS. 15A to 15C show the case where the present invention
is applied to the microscope with upright frame.
DETAILED DESCRIPTION OF THE INVENTION
[0037] Embodiments according to the present invention will now be
described hereinafter with reference to the accompanying
drawings.
[0038] FIG. 1 is a view for illustrating a scheme of total internal
reflection fluorescence observation using a microscope. FIG. 1
shows an example where an inverted microscope is used and
observation is carried out by an objective 7 arranged below a
sample 2.
[0039] As shown in FIG. 1, a cover glass 3 is arranged on the lower
side of the sample 2. The objective 7 is provided below the cover
glass 3 through an immersion oil 5.
[0040] A mirror unit turret 9 which holds two or more fluorescence
mirror units 10a and 10b and is capable of rotating (or linearly
moving (only the rotational operation will be described thereafter)
is arranged on an optical axis 8 of the objective 7. The
fluorescence mirror units 10a and 10b corresponding to the total
internal reflection illumination or the incident-light fluorescence
illumination are selectively switched on the optical axis 8 by the
rotational operation by a rotary shaft 14 of the mirror unit turret
9. In FIG. 1, the fluorescence mirror unit 10a corresponding to the
total internal reflection illumination is switched on the optical
axis 8. A high-reflection mirror 24 is arranged in the incidence
light path of the fluorescence mirror units 10a and 10b. The
high-reflection mirror 24 is fixed to a mirror holding portion 22
by, e.g., an adhesive. A dovetail portion 22a is provided to the
mirror holding portion 22. The dovetail portion 22a is held in a
dovetail groove portion 20 provided in an incident-light projection
tube 17 so as to be capable of moving in a direction vertical to a
page space in FIG. 1. The high-reflection mirror 24 can be moved in
the direction vertical to the page space by moving in or out an
operation knob 23 along the dovetail groove portion 20. In this
case, as shown in FIG. 1, when the high-reflection mirror 24 is on
the optical axis 19 of the incident-light projection tube 17, the
light from a laser beam source 41 is reflected to the fluorescence
mirror units 10a and 10b side.
[0041] The laser beam from the laser beam source 41 is led from an
optical fiber incidence portion 40, and then outgoes from an
optical fiber outgoing portion 38. The outgoing light 21a from the
optical fiber outgoing portion 38 is converted into parallel light
21b by a collimator lens 29 of a fiber projection tube 28. The
parallel light 21b is reflected by the high-reflection mirror 24,
then condensed by a condenser lens 18, and led to the fluorescence
mirror unit 10a. The fluorescence mirror unit 10a includes a
dichroic mirror 11a and an absorption filter 12a. The light
condensed by the condenser lens 18 is reflected by the dichroic
mirror 11a and focalized at a rear side focal position 6 of the
objective 7. The outgoing light from the end of the objective 7
enters a sample 2 from the cover glass 3. Here, evanescent light 4
which permeates in a range of several hundred nm from the interface
with the cover glass 3 can be generated on the sample (low
refractive index side) 2 by shifting the optical axis 31 of the
outgoing light 21a from the optical fiber outgoing portion 38 in a
direction vertical to the optical axis 30 of the fiber projection
tube 28 in such a manner that an incidence angle of the
incident-light which is emitted from the end of the objective 7 and
enters the sample (low refractive index side) 2 from the cover
glass (high refractive index side) 3 becomes larger than a critical
angle.
[0042] A fluorescent material in the sample 2 which exists in the
vicinity of the surface of the cover glass 3 where the evanescence
light 4 is generated is excited by the evanescence light 4 as the
exciting light and produces the fluorescence. The fluorescence
passes through the objective 7 and the dichroic mirror 11a, and the
disadvantageous light in a wavelength band other than the
fluorescence is eliminated therefrom by the absorption filter 12a.
Then, the fluorescence is led to an observation image formation
system 15, divided into two light paths by a half mirror 60. One
divided fluorescence is image-formed on a high-sensitivity camera
(CCD or the like) 16. The other one is transmitted through the half
mirror 60, reflected by the mirror 61, and observed by an eyepiece
63. As a result, the fluorescent material in the sample 2 can be
imaged or visually observed.
[0043] In case of performing the regular incident-light
fluorescence illumination, as shown in FIG. 2, the high-reflection
mirror 24 is removed from the optical axis 19 of the incident-light
projection tube 17, and the fluorescence mirror unit 10a is
switched to the fluorescence mirror unit 10b for the incident-light
fluorescence illumination. The fluorescence mirror unit 10b
includes a dichroic mirror 11b, an absorption filter 12b and an
exciting filter 13b. The exciting filter 13b transmits therethrough
only the exciting light in the light beams from a mercury burner 26
of a mercury lamp house 25. The exciting light transmitted through
the exciting filter 13b is reflected by the dichroic mirror 11b and
enters the objective 7. The outgoing light from the end of the
objective 7 enters the sample 2 from the cover glass 3. The
fluorescence from the fluorescent material in the sample 2 is
transmitted through the dichroic mirror 11b, and the
disadvantageous light in the wavelength band other than the
fluorescence is removed therefrom by the absorption filter 12b.
Then, the fluorescence is led to the observation image formation
system 15, and divided into two light paths by the half mirror 60.
One divided fluorescence is image-formed on the high-sensitivity
camera (CCD or the like) 16. The other one is transmitted through
the half mirror 60, reflected by the mirror 61 and observed by the
eyepiece 63 through a relay optical system 62. As a result, the
fluorescent material can be imaged or visually observed.
[0044] Description will now be given as to embodiments of the total
internal reflection illumination apparatus according to the present
invention applied to the microscope having the above-described
structure.
[0045] (First Embodiment)
[0046] FIG. 3 is a view showing a schematic structure of a
microscope to which the total internal reflection illumination
apparatus according to a first embodiment of the present invention
is applied. FIG. 3 is a view showing only the total internal
reflection illumination apparatus and the vicinity of the objective
to which the total internal reflection illumination apparatus is
attached for the convenience's sake. It is to be noted that any
other structure is basically the same as the above-described
structure, thereby omitting illustration and explanation. FIG. 4 is
a view showing a schematic structure of a slider main body used in
the first embodiment.
[0047] In FIG. 3, an objective revolver holding base 71 is attached
to a non-illustrated microscope main body.
[0048] A fixing portion 72a of the objective revolver 72 is held to
the objective revolver holder base 71. A rotation portion 72b is
rotatably provided to the fixing portion 72a of the objective
revolver 72. A plurality of (only one in the drawing) objectives 7
are screwed and fixed to the rotation portion 72b. By rotating the
rotation portion 72b, a desired objective 7 can be moved onto an
observation optical axis O. In this case, circular hole portions
71a and 72al having at the center thereof the observation optical
axis O running through the center of the objective 7 are provided
to the objective revolver holding base 71 and the fixing portion
72a of the objective revolver 72.
[0049] The objective 7 has the numerical aperture enabling the
total internal reflection illumination. An objective of the
spotlight 7a is provided at the end of the objective 7. The
objective of the spotlight 7a is arranged through the immersion oil
5 at a position opposed to the cover glass 3 having the specimen 2
mounted thereon.
[0050] A concave portion 71b is formed on the top face of the
objective revolver holding base 71. A box-like slider main body 80
as a support member is provided to the concave portion 71b so as to
be capable of being inserted or removed along the surface
orthogonal to the observation optical axis O. Hole portions 6a and
76b through which the observation optical path a runs are formed to
the slider main body 80 on the upper and lower surfaces thereof
positioned on the observation optical axis O while being attached
to the concave portion 71b of the objective revolver holding base
71.
[0051] An optical fiber outgoing portion 38 is fixed to the side
surface of the slider main body 80.
[0052] A convex lens 81 is arranged in the slider main body 80 at a
position opposed to the optical fiber outgoing portion 38. A first
total internal reflection mirror 82 is arranged on the light path
of the light which is transmitted through a convex lens 81 and
converged.
[0053] The convex lens 81 converts the scattered light beam emitted
from the optical fiber 39 into the convergent light beam. The
convex lens 81 has such a focal distance as that the light
reflected in the direction of the objective 7 by the first total
internal reflection mirror 82 is focalized at the rear side focal
position 7b of the objective 7.
[0054] Furthermore, the first total internal reflection mirror 82
is arranged in the vicinity of the outermost peripheral part of the
observation optical path a and reflects the convergent light beam
transmitted through the convex lens 81 in the direction of the
objective 7. The reflected light moves along the observation
optical axis O in the vicinity of the outermost peripheral part of
the inner space of the objective 7.
[0055] A second total internal reflection mirror 83 is arranged at
a position symmetrical with respect to the first total internal
reflection mirror 82 so as to sandwich the observation optical axis
O. The second total internal reflection mirror 83 reflects in the
direction different from the observation optical path a the return
light reflected on the surface of the specimen 2 and returned along
the observation optical axis O in the vicinity of the outermost
peripheral part of the inner space of the objective 7.
[0056] A light trap 85 as return light dimming part is arranged in
the reflection direction of the second total internal reflection
mirror 83. The light trap 85 dims the return light reflected by the
second total internal reflection mirror 83.
[0057] As shown in FIG. 4, a first guide shaft 821 is provided to
the first total internal reflection mirror 82. The first guide
shaft 821 has a linear portion 821a and a bent portion 821b
obtained by the end portion of the linear portion 821a
substantially at right angles. The first total internal reflection
mirror 82 is attached to the bent portion 821b. Moreover, the
linear portion 821a of the first guide shaft 821 is supposed by a
pair of guides 822a and 822b. By linearly moving the linear portion
821a along the guides 822a and 822b, the first total internal
reflection mirror 82 can be moved toward or away from the
observation optical axis O.
[0058] A second guide shaft 831 arranged in parallel with the first
guide shaft 821 is provided to the second total internal reflection
mirror 83. The second guide shaft 831 has a linear portion 831a and
a bent portion 831b obtained by bending the end portion of the
linear portion 831a substantially at right angles. The second total
internal reflection mirror 83 is attached to the bent portion 831b.
In addition, the linear portion 831a of the second guide shaft 831
is supported by a pair of guides 832a and 832b. By linearly moving
the linear portion 831a along the guides 832a and 832b, the second
total internal reflection mirror 83 can be moved toward or away
from the observation optical axis O.
[0059] A link 84 is provided between the first guide shaft 821 and
the second guide shaft 831. The intermediate portion of the link 84
is rotatably supposed on the bottom surface of the slider main body
80. The both ends of the link 84 are rotatably attached to the
linear portion 821a of the first guide shaft 821 and the linear
portion 831a of the second guide shaft 831, respectively. As a
result, the second guide shaft 831 moves in the direction opposed
to the movement direction of the first-guide shaft 821 with respect
to movement of the first guide shaft 821.
[0060] Additionally, a spring 84a is provided to the link 84 at a
position between the link 84 and the bottom surface of the slider
main body 80. This spring 84a constantly gives the link 84 a
running torque in the counterclockwise direction.
[0061] An operation shaft 823 pierces a screw portion 823a and is
provided to the side surface of the slider main body 80. The
operation shaft 823 is in contact with the end portion of the first
guide shaft 821. A quantity of screwing of the screw portion 823a
is adjusted by operation of a knob 823b, and the first guide shaft
821 is linearly moved. As a result, the first total internal
reflection mirror 82 and the second total internal reflection
mirror 83 can be moved close to or away from the observation
optical axis O while maintaining the positional relationship of
symmetry so as to sandwich the observation optical axis O.
[0062] The operation of the first embodiment having the is
above-described structure will now be described.
[0063] When the light is emitted from the optical fiber outgoing
portion 38, it becomes the scattered light beam and enters the
convex lens 81. The scattered light beam which has entered the
convex lens 81 is converted into the convergent light beam, then
reflected in the direction of the objective 7 by the first total
internal reflection mirror 82, and moves along the observation
optical axis O in the vicinity of the outermost part of the inner
space of the objective 7. Then, the light beam is focalized at the
rear side focal position 7b of the objective 7, again becomes the
scattered light beam, and is transmitted through the objective of
the spotlight 7a. Thereafter, the light beam becomes the parallel
light beam inclined with respect to the observation optical axis O
and outgoes from the objective 7. Then, it is transmitted through
the immersion oil 5 and the cover glass 3, and the specimen 2 is
irradiated with this light.
[0064] Here, when the operation shaft 823 is rotated by the
operation of the knob 823b, the first guide shaft 821 and the
second guide shaft 831 linearly move in the opposite directions in
accordance with a quantity of screwing of the screw portion 823a.
The first total internal reflection mirror 82 ad the second total
internal reflection mirror 83 are subjected to positional
adjustment in the direction to move closer to or away from the
observation optical axis O while maintaining the positional
relationship of symmetry with respect to the observation optical
axis O. As a result, the distance of the light beam which moves in
the inner space of the objective 7 from the observation optical
axis O varies, and the inclination angle of the parallel light beam
outgoing from the objective 7 changes. That is, the total internal
reflection illumination can be obtained by changing the inclination
angle of the parallel light beam outgoing from the objective 7 by
rotating the knob 823b of the operation shaft 823 and setting this
inclination angle equal to or above a critical angle.
[0065] On the other hand, the light beam totally reflected on the
surface of the specimen 2 again enters the objective 7 as the
return light, is focalized at the rear side focal position 7b of
the objective 7, all reflected by the second total internal
reflection mirror 83, led to the light trap 85 and terminated.
[0066] In this state, when the fluorescence is emitted from the
specimen 2, this florescence moves in the observation optical path
a along the observation optical axis O as an observation light beam
O'. The observation light beam O' at this moment is led to the
observation side without crossing the illumination light beam
reflected by the first total internal reflection mirror 82 or the
return light beam which enters the second total internal reflection
mirror 83. Additionally, the illumination light beam with which the
specimen 2 is irradiated and the return light beam after total
internal reflection do not cross each other on the observation
optical path a and they are eliminated from the observation optical
path a.
[0067] Therefore, according to the above-described structure, the
observation light beam O' of the fluorescence emitted from the
specimen 2 can be set so as not to cross the illumination light
beam reflected by the first total internal reflection mirror 82 or
the return light beam entering the second total internal reflection
mirror 83 on the observation optical path a. Accordingly, the
self-fluorescence generated on the illumination light beam or the
return light beam can be prevented from entering the observation
light beam O', thereby greatly suppressing deterioration of the
fluorescence observation image. Further, since the illumination
light beam with which the specimen 2 is irradiated and the return
light beam after total internal reflection do not cross each other,
the interference fringe generated due to crossing of the laser
beams can be prevented, thereby constantly assuring the excellent
fluorescence observation image.
[0068] It is to be noted that the interlocking mechanism
constituted by the first guide shaft 821, the second guide shaft
831 and the link 84 are used as a movement part of the first total
internal reflection mirror 82 and the second total internal
reflection mirror 83 is used in the first embodiment but the
present invention can be embodied by using any other moving parts,
e.g., using an electric motor in place of the interlocking
mechanism. Further, the convex lens 81 is of the fixed type, but it
may be capable of moving along the illumination light beam, thereby
enabling the precise optical adjustment.
[0069] FIGS. 5 and 6 show the schematic structures obtained when
the total internal reflection illumination apparatus according to
the first embodiment having the above-described structure is
applied to inverted microscope and microscope with upright frame.
FIG. 5 is a view when the total internal reflection illumination
apparatus is applied to the inverted microscope, and FIG. 6 is a
view when the total internal reflection illumination apparatus is
applied to the microscope with upright frame. Incidentally, in
FIGS. 5 and 6, like reference numerals denote parts equal to those
in FIGS. 1 to 4, thereby omitting the detailed description.
[0070] As shown in FIG. 5, in the inverted microscope, a light
source for incident-light illumination is arranged above a stand
100, and regular observation of a sample is carried out with the
eyepiece 63 by illuminating the sample 2 on the cover glass 3
through an incident-light optical system 96. In this case, the
sample 2 is not illuminated with the laser beam from the laser beam
source 41 using the slider main body 80. Further, when performing
observation by the total internal reflection illumination using the
total internal reflection illumination apparatus, the fluorescence
from the sample 2 is observed by performing the total internal
reflection illumination to the sample 2 with the laser beam from
the laser beam source 41 (or image pickup is carried out by a
non-illustrated image pickup device).
[0071] In case of FIG. 6, the illumination from a non-illustrated
light source is emitted toward the upper direction from the lower
part of the stand 100' along the optical axis of the objective 7.
Any other structure is substantially the same with as of FIG. 5,
thereby omitting the description.
[0072] (Second Embodiment)
[0073] FIG. 7 is a view showing a schematic structure of a
microscope to which a total internal reflection illumination
apparatus according to a second embodiment of the present invention
is applied. In FIG. 7, like reference numerals denote parts equal
to those in FIG. 3, thereby omitting the detailed explanation.
[0074] In the second embodiment, the convex lens 851 is arranged on
the reflected light path of the second total internal reflection
mirror 83. The convex lens 851 is used to again converge the return
light subjected to total internal reflection by the second total
internal reflection mirror 83. An incidence end 852a of the optical
fiber 852 is positioned at a focal position of the return light
obtained by the convex lens 851. In this case, the incidence end
852a of the optical fiber 852 has a sufficiently large core
diameter in such a manner that the light can be assuredly led into
the fiber even if the focal position of the return light slightly
deviates. Furthermore, the light trap 853 is connected to an
outgoing end 22b of the optical fiber 852. This light trap 853 is
set outside the microscope.
[0075] Description will be given as to the operation of the second
embodiment having the above-described structure.
[0076] The light beam totally reflected by the surface of the
specimen 2 again enters the objective 7 as the return light, and is
focalized at the rear side focal position 7b of the objective 7.
Thereafter, the return light is all reflected by the second total
internal reflection mirror 83 and enters the convex lens 851. Then,
the return light is transmitted through the convex lens 851, again
becomes the convergent light beam, enters the incidence end 852a of
the optical fiber 852, and is terminated at the light trap 853
provided outside the microscope.
[0077] By doing so, the advantages like those in the first
embodiment can be obtained. Moreover, since the return light beam
reflected by the second total internal reflection mirror 83 is
positively taken out to the outside of the microscope by using the
optical fiber, the possibility of permeation of the scattered light
of the return light beam into the observation optical path a can be
greatly reduced, thereby assuring the further excellent
fluorescence observation image.
[0078] (Third Embodiment)
[0079] FIGS. 8 and 9 are views showing a schematic structure of a
slider main body according to a third embodiment of the present
invention. In FIGS. 8 and 9, like reference numerals denote parts
equal to those in FIG. 4, thereby omitting the detailed
explanation.
[0080] In FIGS. 8 and 9, a pair of click grooves 825 and 826
provided with a predetermined gap therebetween along the side
surface which is in contact with the concave portion 71b to the
slider main body 8b disposed so as to be capable of being inserted
to or removed from the concave portion 71b of the objective
revolver holding base 71. Moreover, a ball plunger 827 is provided
on the side surface of the objective revolver holding base 71 on
the concave portion 71b side, with which the slider main body 80 is
in contact.
[0081] The ball plunger 827 constantly presses the side surface of
the slider main body 80, and positions the slider main body 80 when
fitted in the click grooves 825 and 826. When the slider main body
80 is inserted to reach the observation optical axis O as shown in
FIG. 8, the ball plunger 827 is fitted in the click groove 825.
When the slider main body 80 is moved away from the observation
optical axis O as shown in FIG. 9, the ball plunger 827 is fitted
in the click groove 826.
[0082] A micro switch 86 is arranged to the concave portion 71b of
the objective revolver holding base 71 at a position where it is
pressed by the end of the slider main body 80. The micro switch 86
is pressed and becomes conductive when the slider main body 80 is
being inserted in the observation optical axis O as shown in FIG.
8.
[0083] An interlocking terminal 41a of a laser oscillator 41 is
connected to an output terminal of the micro switch 86 through a
cable 87. The laser oscillator 41 oscillates when interlocking is
released by conduction of the micro switch 86, and generates the
laser beam. The laser beam is led to the slider main body 80
through the optical fiber 39.
[0084] The operation of the third-embodiment having such a
structure will now be described.
[0085] As shown in FIG. 8, when the slider main body 80 is inserted
onto the observation optical axis O, the ball plunger 827 is fitted
in the click groove 825, and the slider main body 80 is held so as
not to easily move. When the micro switch 86 is pressed by the end
of the slider main body 80 and becomes conductive in this state,
the interlocking of the laser oscillator 41 is released, and the
laser beam is generated. The laser beam is led to the slider main
body 80 through the optical fiber 39, and the operation like that
described in connection with the first embodiment can be
obtained.
[0086] On the other hand, as shown in FIG. 9, when the slider main
body 80 is moved away from the observation optical axis O, the ball
plunger 827 is fitted in the click groove 826, and the slider main
body 80 is held so as not to easily move. In this state, since the
micro switch 86 is opened, the interlocking of the laser oscillator
41 connected to the cable 87 is actuated, thereby generating no
laser beam.
[0087] Therefore, the advantages like those described in connection
with the first embodiment can be obtained by doing so. Furthermore,
when the slider main body 80 is removed from the observation axis
O, the laser beam from the laser oscillator 41 can be automatically
stopped surely. Thus, the scattered light by the reflection of the
laser beam never permeates observation light axis O' and excellence
can be maintained.
[0088] It is to be noted that the cable 87 is directly connected to
the interlocking terminal 41a of the laser oscillator 41 in the
third embodiment, the similar advantages can be obtained by
inserting an electric shutter between the laser oscillator 41 and
the optical fiber 39, connecting the cable 87 to the electric
shutter and using a part of, e.g., combining the opening/closing
operation of the electric shutter with the on/off operation of the
micro-switch 86.
[0089] Further, in the third embodiment, the micro switch 86 is
configured to be pressed and conductive with the slider main body
80 being inserted into the observation optical axis O, but the
micro switch 86 may be provided on the side surface of the inner
wall as shown in FIG. 10. It is to be noted that the micro switch
86 may be of a contact type or it may be a proximity switch or the
like in this case.
[0090] In the first to third embodiments mentioned above, in the
first guide shaft 821 and the second guide shaft 831 which is
arranged in parallel with the first guide shaft 821, there is no
structure which restricts a quantity of movement of these shafts.
Accordingly, as shown in FIG. 11, there may be provided a stopper
which restricts a quantity of movement of the first guide shaft 821
and the second guide shaft 831. This stopper can prevent the
reflection mirror from moving in the optical axis direction in
particular.
[0091] Although the micro switch 86 is arranged at a position where
it is pressed by the end of the slider main body 80 in the third
embodiment, the micro switch 86 may be provided at such a position
as that it is pressed by the side surface of the slider main body
80 as shown in FIG. 10. The micro switch 86 may be provided at any
position as long as it can detect insertion and removal of the
slider main body 80 in this way. Moreover, any kind of the micro
switch 86, e.g., a mechanical type, a proximity sensor or the like
can be used.
[0092] In addition, in the first to third embodiments, the first
guide shaft 821 and the second guide shaft 831 can be moved by the
operation shaft 823 and the link 84, but a quantity of movement of
the first guide shaft 821 and the second guide shaft 83 is
restricted by providing a stopper 88 so as to come into contact
with one of the second guide shaft 831 as shown in FIG. 11, and the
first total internal reflection mirror 82 and the second total
internal reflection mirror 83 do not move close to the optical axis
more than necessary, thereby preventing damages to these
members.
[0093] (Fourth Embodiment)
[0094] FIGS. 12A to 12C are views showing a schematic structure of
a micro scope to which a fourth embodiment according to the present
invention is applied. In FIGS. 12A to 12C, like reference numerals
denote parts equal to those in FIG. 1, thereby omitting the
detailed description.
[0095] The incident-light projection tube 17 is fixed to the
inverted microscope main body (not shown). This incident-light
projection tube 17 has a connection portion 17 relative to a fiber
projection tube 28, and a connection portion 17b relative to a
mercury lamp house 25 which holds the mercury burner 26. These
members are respectively connected in such a manner that an optical
axis 30 of the fiber projection tube 28 becomes orthogonal to an
optical axis 19 of the incident-light projection tube 17 and an
optical axis 27 of the mercury lamp house 25 coincides with the
optical axis 19 of the incident-light projection tube.
[0096] In the incident-light projection tube 17, the
high-reflection mirror 24 is fixed to the mirror holding portion 22
by an adhesive or the like so as to reflect the parallel beam 21b
of the fiber projection tube 28 on the optical axis 19 of the
incident-light projection tube 17. A dovetail portion 22a is
provided to the mirror holding portion 22. The dovetail portion 22a
is held in the dovetail groove portion 20 provided to the
incident-light projection tube 17 so as to be capable of moving in
the direction vertical to the page space, and the high-reflection
mirror 24 can be moved in the direction vertical to the page space
by moving in or out the operation knob 23 in the direction vertical
to the page space from the outside of the incident-light projection
tube 17. Moreover, the mirror holding portion 22 has a light
shielding portion 22b provided on the side surface thereof on the
mercury lamp house 25 side.
[0097] The fiber projection tube 28 is constituted by a collimator
lens 29 and a fiber lead-in portion 32. The fiber lead-in portion
32 is connected to the end of the fiber projection tube 28 on the
side opposite to the incident-light projection tube 17.
[0098] On the other hand, the outgoing light from the laser beam
source 41 enters the optical fiber 39 from the optical fiber
incidence portion 40, and the outgoing light 21a outgoes from the
optical fiber outgoing portion 38. The optical fiber outgoing
portion 38 is fixed to the movement portion 37 by a screw or the
like (not shown). An outer side surface portion 37a of the movement
portion 37 is fitted with an inner side surface portion 32a1 of the
fiber lead-in portion 32 and can move in the horizontal direction
of the page space 54.
[0099] Here, a screw hole 32a having a central line 36 in parallel
with the horizontal direction of the page space 54 of the movement
portion 37 and a fitting hole 32b are formed to the fiber lead-in
portion 32, and a lid cylinder 33 having a screw portion is engaged
with the screw hole 32a, and an adapter 35 is fitted in the fitting
hole 32b.
[0100] In addition, a compression coil spring 34, which is
compressed to be shorter than a natural length, as an elastic body
is sandwiched between the lid cylinder 33 and the adapter 35, and
the adapter 35 comes into contact with the outer side surface
portion 37a of the movement portion 37. On the other hand, a slant
surface contact portion 37b is provided on the opposite side to the
outer side surface portion 37a of the movement portion 37.
[0101] A micrometer holding portion 42 is fixed to the fiber
lead-in portion 32 by a screw or the like (not shown). The
micrometer main body 44 is held to the micrometer holding portion
42 by a screw or the like (not shown).
[0102] The micrometer main body 44 has a knob 46 engage with the
rotation portion 44a by a screw or the like (not shown).
Additionally, a screw hole 32a and a cylindrical hole 32c which has
a central line orthogonal to the central line of the fitting hole
32b are provided to the fiber lead-in portion 32, and a capsule
adapter 43 is arranged being fitted in the cylindrical hole 32c and
sandwiched between the slant surface contact portion 37b and the
end portion 44b of the rotation portion 44a of the micrometer main
body 44.
[0103] As a result, the knob 46 of the micrometer main body 44 is
rotated, the slant surface contact portion 37b of the movement
portion 37 is pressed by the capsule adapter 43 of the end portion
44b of the rotation portion 44a, and the movement portion 37 is
moved against the pressing force of the compression coil spring 34.
Consequently, the optical axis 31 of the outgoing light 21a from
the optical fiber outgoing portion 38 can be shifted from the
optical axis 30 of the fiber projection tube 28 (to the left side
in the drawing) so as to be capable of adjusting an incidence angle
of the incident light which outgoes from the end of the objective 7
and enters the sample (low refractive index side) 2 from the cover
glass (high refractive index side) 3.
[0104] An opening portion 45a of a notch stopper 45 which has the
opening portion 45a and an U-shaped notch portion 45c is fitted to
the fixing portion 44c of the micrometer main body 44 as
restricting part as shown in FIG. 12C. Additionally, a screw 47
which adjusts a gap of the U-shaped notch portion 45c is
provided.
[0105] The operation of the fourth embodiment having such a
structure will now be described.
[0106] The adapter 35 is in the state that the outer side surface
portion 37a of the movement portion 37 is pressed by the
compression coil spring 34. On the other hand, the end portion 44b
can be moved in the vertical direction of the page space 55 by
rotating the knob 46 of the micrometer main body 44, and the
movement portion 37 can be moved in the horizontal direction of the
page space 54 through the capsule adapter 43. As a result, the
optical axis 31 of the outgoing light 21a from the optical fiber
outgoing portion 38 can be adjusted in the vertical direction
relative to the optical axis 30 of the fiber projection tube 28
while maintaining the horizontal state with respect to the optical
axis 30 of the fiber projection tube 28.
[0107] At the same time, an angle of the incident light which is
emitted from the end of the objective 7 and enters the sample 2
from the cover glass 3 can be also adjusted. Here, a position of
the movement portion 37 is adjusted by rotating the knob 46 in such
a manner that the incidence angle from the cover glass 3 to the
sample 2 becomes slightly larger than the critical angle, and the
side surface portion 45d of the notch stopper 45 is pressed against
a contact portion 44aa of the rotation portion 44a of the
micrometer main body 44 at this position. Further, a gap of the
notch portion 45c of the notch stopper 45 is constricted by a screw
47, and the fixing portion 44c of the micrometer main body 44 is
shut in by the opening portion 45a, thereby positioning and fixing
the notch stopper 45 with respect to the micrometer main body
44.
[0108] Therefore, the movement portion 37 can not thereafter move
the optical fiber outgoing portion 38 to the optical axis 30 side
of the fiber projection tube 28 by restriction of the notch stopper
45, and movement is limited to that only in a range of the total
internal reflection illumination that the incidence angle from the
cover glass 3 to the sample 2 is larger than the critical
angle.
[0109] On the other hand, although the light beam (not shown) from
the mercury lamp house 25 is prevented by the light shielding
portion 22b of the mirror holding portion 22, light beam (not
shown) from the mercury burner 26 can be led to the incident-light
projection tube 17 by drawing the operation knob 23 toward the
front side in the vertical direction of the page space and removing
the mirror holding portion 22 from the light path. At this moment,
by rotating the mirror unit turret 9 around the rotary shaft 14,
the regular incident-light fluorescence illumination observation
can be enabled by arranging the fluorescence mirror unit 10b
including the exciting filter 13b, the dichroic mirror 11b and the
absorption filter 12 on the optical axis.
[0110] Therefore, according to the fourth embodiment, when
adjusting the incidence angle from the cover glass 3 to the sample
2 by the notch stopper 45 fixed to the micrometer main body, this
incidence angle is restricted in a range where it becomes larger
than the critical angle, and hence the fluorescence observation can
be performed by only the total internal reflection illumination.
Therefore, it is possible to prevent color degradation of the
entire sample due to the strong light of the incident-light
fluorescence illumination, thereby obtaining the stable
fluorescence observation by the total internal reflection
illumination. Furthermore, since the incidence angle from the cover
glass 3 to the sample 2 can be constantly adjusted in the range of
the total internal reflection illumination, the operability can be
improved. Moreover, the total internal reflection illumination and
the incident-light fluorescence illumination can be rapidly
switched by insertion and removal of the high-reflection mirror 24,
and hence a factor of color degradation of the sample can be
avoided when switching from the incident-light fluorescence
illumination to the total internal reflection illumination (or vice
versa) while keeping illumination of the sample with the
illumination light.
[0111] It is to be noted that the inverted microscope has been
described in the fourth embodiment but the similar advantages can
be obtained when the present invention is applied to the microscope
with upright frame.
[0112] (Modification of Fourth Embodiment)
[0113] A modification of the fourth embodiment will now be
described. FIGS. 13A and 13B illustrate a modification of the
fourth embodiment, and like reference numerals denote parts equal
to those in FIGS. 12A to 12C, thereby omitting the detailed
description.
[0114] In FIGS. 13A and 13B, a slide opening portion 28a is
provided to the fiber projection tube 28 on the rear side
(incident-light projection tube 17 side) of the collimator lens 29.
A slider 48 having an opening 48a and an opening 48b is provided to
the slide opening portion 28a so as to be capable of moving in the
horizontal direction of the page space 54. The slider 48 has a
diffused plate 49 fixed to the opening 48a by a ring screw (not
shown).
[0115] On the other hand, the mirror holding portion 22 of the
high-reflection mirror 24 is directly fixed to a fixing portion 50
of the incident-light projection tube 17 by a screw or the like
(not shown).
[0116] With such a structure, when the opening 48b of the slider 48
is arranged on the optical axis 30 of the fiber projection tube 28
with the total internal reflection illumination being set as
described in connection with the fourth embodiment, this state is
maintained, but the parallel light 21b is diffused and the
illumination is switched to the incident-light fluorescence
illumination when the slider 48 is moved and the opening 48a having
the diffused plate 49 is arranged on the optical axis 30.
[0117] By doing so, the advantages like those in the fourth
embodiment can be obtained, and switching between the total
internal reflection illumination and the incident-light
fluorescence illumination can be realized without using the mercury
lamp house 25.
[0118] Moreover, in the fourth embodiment, the light stopper 85
which introduces return light of the total reflected laser beam to
an outside of the observation optical path is provided between the
objective 7 and the fluorescence mirror unit 10a like the first to
third embodiments. As a result, the unnecessary light can be
prevented from entering the observation optical path, thereby
obtaining a further excellent image.
[0119] It is to be noted that the high-reflection mirror 24 can be
omitted by providing the fiber projection tube 28 coaxially with
the incident-light projection tube 17 in the modification of the
fourth embodiment, thereby obtaining the further inexpensive
structure.
[0120] (Fifth Embodiment)
[0121] The fifth embodiment according to the present invention will
now be described. FIG. 14 illustrates the fifth embodiment, and
like reference numerals denote parts equal to those in FIG.
12A.
[0122] In FIG. 14, a dovetail portion 51 is provided to the mirror
holding portion 22 of the high-reflection mirror 24, and this
dovetail portion 51 is held so as to be capable of moving in the
horizontal direction of the page space 54 in a dovetail groove
portion 52 provided to the incident-light projection tube 17.
[0123] In the mirror holding portion 22, a contact portion 51a of
the dovetail portion 51 is constantly being pressed by the
compression coil spring 34 through the adapter 35. On the other
hand, a micrometer holding portion 53 is provided on the side
surface of the incident-light projection tube 17 on the side
opposite to a position where the adapter 35 is arranged, and the
micrometer main body 44 is provided to the micrometer holding
portion 53. In this case, the micrometer main body 44 brings the
end portion 44b of the rotation portion 44a rotated by rotation of
the knob 46 into contact with the contact portion 51b of the
dovetail portion 51.
[0124] By rotating the knob 46 of the micrometer main body 44 in
this state, the mirror holding portion 22 can be moved along the
reflected light path of the high-reflection mirror 24 by the
dovetail portion 51, and a reflection position of the parallel
light 21b on the high-reflection mirror 24 can be adjusted.
[0125] As a result, the reflected light 21c obtained from the
parallel light 21b on the high-reflection mirror 24 can be shifted
from the optical axis 19, and an angle of the incident light which
outgoes from the objective 7 and enters the sample 2 from the cover
glass 3 can be adjusted.
[0126] Here, a position of the mirror holding portion 22 is also
adjusted by rotating the knob 46 in such a manner that the
incidence angle from the cover glass 3 to the sample 2 becomes
slightly larger than the critical angle, the notch stopper 45 is
brought into contact with the rotation portion 44a of the
micrometer main body 44 at this position, and the notch stopper 45
is fastened, thereby performing positioning and fixing with respect
to the micrometer main body 44.
[0127] As a result, the mirror holding portion 22 can not
thereafter move the reflected light 21c from the high-reflection
mirror 24 to the optical axis 19 side by the restriction of the
notch stopper 45, and movement is restricted to that only in the
range of the total internal reflection illumination that the
incidence angle from the cover glass 3 to the sample 2 is larger
than the critical angle.
[0128] In this case, like the modification of the fourth
embodiment, the parallel light 21b is diffused by providing the
slider 48 and arranging the opening 48a having the diffused plate
49 on the optical axis 30, thereby obtaining the incident-light
fluorescence illumination utilizing the laser beam source 41.
[0129] In the fifth embodiment, although the mirror holding portion
22 is moved along the reflected light path of the high-reflection
mirror 24, the similar advantages can be obtained by moving it
along the incident-light path of the high-reflection mirror 24.
[0130] It is to be noted that the micrometer main body 44 and the
notch stopper 45 which restricts movement of the rotation portion
44a of the micrometer main body 44 are used in the fifth
embodiment. However, instead of not using these members, a light
shielding plate having a slit hole shifted to the right or left
side from the center of the optical axis 30 of the fiber projection
tube 28 may be arranged in front of the optical fiber outgoing
portion 38, and the optical axis of the outgoing light emitted from
the optical fiber outgoing portion 38 may be moved to the optical
axis 30 side of the fiber projection tube 28. Even in this case, by
preventing the light on the optical axis center side of the
outgoing light by using the light shielding plate, switching to the
incident-light illumination can be avoided, and movement can be
restricted to that only in the range of the total internal
reflection illumination. In this case, switching between the total
internal reflection illumination and the incident-light
fluorescence illumination utilizing the laser beam source 41 can be
performed by moving the high-reflection mirror 24 so as to be
inserted into or removed from the light path as with the fourth
embodiment.
[0131] Description will now be given as to a method for realizing
the total internal reflection illumination. In the confirmation
method when the incidence angle of the illumination light from the
cover glass 3 to the sample 2 exceeds the critical angle, the
eyepiece (not shown) attached to the body tube (not shown) is
replaced with a CT (centering telescope) (not shown), and a lens
group (not shown) in the vicinity of the rear side focal position 6
of the objective 7 is watched by using this CT. The
self-fluorescence of the lens group is generated by the light
obtained by transmission of the illumination light through the lens
group (not shown) of the objective 7, and a bright spot can be
observed by the lens group in the vicinity of the rear side focal
position 6 at which the light is condensed.
[0132] When the incidence angle from the cover glass 3 to the
sample 2 is smaller than the critical angle, only one bright spot
on the incidence angle side of the illumination light is observed
at the rear side focal position 6 of the objective 7, but two
bright spots which are symmetric so as to sandwich the optical axis
can be observed in the vicinity of the inner side of the outer
periphery of the rear side focal position 6 of the objective 7 when
the incidence angle is larger than the critical angle. The second
bright spot is obtained because the illumination light totally
reflected on the interface between the cover glass 3 and the sample
2 returns to the objective 7 from the end of the objective 7.
[0133] By gradually changing from the state that the incidence
angle of the illumination light from the cover glass 3 to the
sample 2 is smaller than the critical angle to the state that it is
larger than the critical angle, one bright spot first gradually
moves in the outer peripheral direction from the central side of
the optical axis of the objective. Then, when the incidence angle
exceeds the critical angle, the second bright point appears
symmetrically so as to sandwich the optical axis of the objective,
and the notch stopper 45 is fixed to the micrometer main body 44
when the second bright spot appears.
[0134] Description will now be given as to the definition of the
approximate total internal reflection illumination and effects and
advantages obtained when the present invention is applied to the
fourth embodiment based on the fourth embodiment. Since the
structure is similar to that of the fourth embodiment, the
explanation thereof is omitted. Further, in regard to the effects
and the advantages, parts equal to those in the fourth embodiments
are omitted, and description will be given as to only different
parts.
[0135] The approximate total internal reflection illumination is
first defined as follows. In the total internal reflection
illumination, the evanescent light 4 is generated in the range of
several hundred nm on the sample 2 side which is the low refractive
index medium side on the interface between the cover glass 3 and
the sample 2. By setting the incidence angle from the cover glass 3
to the sample 2 slightly smaller than the critical angle, the
refracted light from the cover glass 3 to the sample 2 is emitted
from the cover glass 3 along the vicinity on the interface of the
sample 2. In this illumination method, a range of several nm in the
sample 2 in the vicinity of the cover glass 3 can be illuminated.
This is one type of dark field illumination, and this illumination
method is referred to as "approximate total internal reflection
illumination" in this specification.
[0136] The effects of the approximate total internal reflection
illumination will now be described. Here, description will be given
as to only the approximate total internal reflection illumination
using the laser beam source 41, and the explanation about the
incident-light fluorescence illumination using the mercury burner
26 as a light source will be omitted. Like the fourth embodiment,
the knob 46 of the micrometer 44 is first rotated, and illumination
is switched to the total internal reflection illumination that the
incidence angle from the cover glass 3 to the sample 2 is larger
than the critical angle. Then, the knob 46 of the micrometer main
body 44 is gradually rotated in the direction that the incidence
angle from the cover glass 3 to the sample 2 becomes smaller, and
the illumination light is adjusted to be the above-described
approximate total internal reflection illumination.
[0137] In this state, like the fourth embodiment, movement of the
optical fiber outgoing portion 38 is restricted to only the range
of the approximate total internal reflection illumination and the
total internal reflection illumination by positioning and fixing
the notch stopper 45 to the micrometer main body 44.
[0138] The advantages will now be described. The illumination range
is only the sample 2 in the range of several .mu.m in the vicinity
of the top face of the cover glass 3 even in case of the
approximate total internal reflection illumination, and color
degradation of the entire sample 2 can be avoided like the fourth
embodiment. Also, the illumination light can be moved in the
direction away from the optical axis in order to change to the
total internal reflection illumination.
[0139] Besides the above advantages, an area which can not be
observed with the evanescent light 4 generated by the total
internal reflection illumination or the sample 2 which can not be
observed because the generated fluorescence is too weak can be
observed. Furthermore, the fluorescence is generated from the
entire sample 2 in the incident-light fluorescence illumination
using the mercury burner 26 as a light source, whereas the
unnecessary fluorescence can be eliminated and observation with
less background noise is enabled since the illumination range can
be restricted to the range of several .mu.m in the vicinity of the
cover glass 3 in the approximate total internal reflection
illumination.
[0140] Although description has been give as to the inverted type
microscope in the fourth and fifth embodiments mentioned above, the
present invention can be of course applied to the erect type
microscope. FIGS. 15A to 15C show the case where the present
invention is applied to the microscope with upright frame. In FIGS.
15A to 15C, since respective structures are the same as above, like
reference numerals are given, thereby omitting the explanation.
[0141] The following inventions can be extracted from each of the
foregoing embodiments. It is to be noted that each of the following
inventions may be appropriately combined and applied or it may be
applied independently.
[0142] A total internal reflection illumination apparatus according
to the first aspect of the present invention apparatus applied to a
microscope which illuminates a sample through an objective having a
numerical aperture enabling total internal reflection illumination,
is characterized by comprising: a first total internal reflection
mirror which is arranged in the vicinity of an outermost peripheral
part of an observation optical path of the microscope to reflect an
incident illumination light in a direction of the objective; a
second total internal reflection mirror which is arranged at a
symmetrical position with the first total internal reflection
mirror to sandwich an observation optical axis and reflects return
light reflected on a surface of the sample in a direction different
from the illumination optical path; and a return light dimming part
configured to dim the return light reflected by the second total
internal reflection mirror.
[0143] In the first aspect the flowing modes are preferable. The
following modes may be applied independently or in combining
them.
[0144] (1) The return light dimming part has an external set
portion which is set outside the microscope.
[0145] (2) The return light dimming part is connected to the
external set portion through an optical fiber.
[0146] (3) At least a part of the total internal reflection
illumination apparatus is set to a support member which insertable
or removable to the observation optical path of the microscope.
[0147] (4) The first total internal reflection mirror and the
second total internal reflection mirror are arranged at positions
where they are symmetrical with each other to sandwich an optical
axis of the objective, and the first total internal reflection
mirror and the second total internal reflection mirror move in such
a manner a distance between the optical axis of the objective and
the first total internal reflection mirror and a distance between
the optical axis of the objective and the second total internal
reflection mirror become equal to each other.
[0148] (5) One of the microscope and the support member has a
switching part configured to turn on or turn off the illumination
light in cooperation with insertion or removal of the support
member.
[0149] (6) A switching part configured to switch total internal
reflection illumination and approximate total internal reflection
illumination by changing an incidence angle of the illumination
light with which the sample is irradiated; and a restricting part
configured to restrict the incidence angle of the illumination
light to the sample to a range where total internal reflection
illumination and approximate total internal reflection illumination
can be obtained are further provided.
[0150] A microscope according to the second aspect of the present
invention is characterized by comprising: a light source which
emits predetermined light; a total internal reflection illumination
apparatus according to above-mentioned total internal reflection
illumination apparatus, which irradiates a sample with the light
from the light source through an objective; and an image pickup
device which images the light from the sample as an image.
[0151] In the second aspect the flowing modes are preferable. The
following modes may be applied independently or in combining
them.
[0152] (1) The light source has an optical fiber having an outgoing
end provided to be movable in a direction vertical to the optical
axis, and the restricting part restricts a movement range of the
outgoing end of the optical fiber in the direction vertical to the
optical axis to the range where total internal reflection
illumination and approximate total internal reflection illumination
can be obtained.
[0153] (2) An optical element which is insertably and removably
arranged to the light path between the light source and the
objective and diffuses the illumination light is further
provided.
[0154] (3) A light path between the light source and the objective
has a reflection member arranged movably along a direction of the
light path, and the restricting part restricts a movement range of
the reflection member along the direction of the light path to the
range where total internal reflection illumination and approximate
total internal reflection illumination can be obtained.
[0155] (4) An optical element which is insertably and removably
arranged to the light path between the light source and the
objective and diffuses the illumination light is further
provided.
[0156] A microscope according to the third aspect of the present
invention is characterized by comprising: a changing part
configured to change an incidence angle of illumination light which
is emitted onto a sample from a light source through an objective
and enables switching between total internal reflection
illumination and approximate total internal reflection
illumination; and a restricting part configured to restrict the
incidence angle of the illumination light to the sample through the
objective to a range where total internal reflection illumination
and approximate total internal reflection illumination can be
obtained.
[0157] In the third aspect the flowing modes are preferable. The
following modes may be applied independently or in combining
them.
[0158] (1) A dimming part configured to dim light reflected by the
sample is further provided.
[0159] (2) The light source has an optical fiber having an outgoing
end provided to be movable in a direction vertical to an optical
axis, and the restricting part restricts a movement range of the
outgoing end of the optical fiber in the direction vertical to the
optical axis to the range where total internal reflection
illumination and approximate total internal reflection illumination
can be obtained.
[0160] (3) An optical element which is insertably and removably
arranged the light path between the light source and the objective
and diffuses the illumination light is further provided.
[0161] (4) A light path between the light source and the objective
has a reflection member provided so as to be capable of moving
along the light path direction, and the restricting part restricts
a movement range of the reflection member along the light path
direction to the range where total internal reflection illumination
and approximate total internal reflection illumination can be
obtained.
[0162] (5) An optical element which is insertably and removably
arranged to the light path between the light source and the
objective and diffuses the illumination light is further
provided.
[0163] Moreover, the respective foregoing embodiments include the
following inventions, for example.
[0164] (1) A total internal reflection illumination apparatus
applied to a microscope which illuminates a sample through an
objective having a numerical aperture enabling the total internal
reflection illumination, wherein the illumination light incident
from the outside of the microscope is reflected in a direction of
an objective by a first total internal reflection mirror arranged
in the vicinity of an outermost part of an observation optical
path, the return light totally reflected on the surface of the
specimen is reflected by a second total internal reflection mirror
arranged symmetrical with the first total internal reflection
mirror so as to sandwich the observation optical axis and it is
ended by light trapping part.
[0165] (2) In The total internal reflection illumination apparatus
according to (1), wherein the light trapping part is arranged
outside the microscope through an optical fiber.
[0166] (3) In The total internal reflection illumination apparatus
according to (1), wherein the total internal reflection
illumination apparatus of the microscope can be integrally moved
away from the observation optical path, and the illumination light
is turned on/off in cooperation with insertion to or removal from
the observation optical path.
[0167] The present invention is not restricted to each of the
foregoing embodiments, and it can be modified on the embodying
stage in many ways without changing its gist. In addition, the
respective foregoing embodiments can be appropriately combined and
applied.
[0168] Additionally, the foregoing embodiments include various
kinds of inventions, and a variety of inventions can be extracted
from appropriate combinations of a plurality of disclosed
structural requirements. For example, even if some of the
structural requirements are deleted from all the structural
requirements disclosed in the embodiments, the problems described
in the section "problems to be solved by the invention" can be
solved, and the structures obtained by deleting the structural
requirements can be extracted as inventions when the advantages
described in the section "advantages of the invention" can be
obtained.
[0169] According to each of the embodiments of the present
invention, it is possible to provide the total internal reflection
illumination apparatus of the microscope which can constantly
assure a good fluorescence observation image.
[0170] As a result, the observation light beam of the fluorescence
generated from the specimen can be set so as not to cross the
illumination light beam reflected by the first total internal
reflection mirror on the observation optical path or the return
light beam entering the second total internal reflection mirror,
and the illumination light beam with which the specimen is
irradiated and the return light beam after total internal
reflection can be set so as not to cross each other.
[0171] Further, according to the embodiments of the present
invention, since the return light beam reflected by the second
total internal reflection mirror is positively removed to the
outside of the microscope, the possibility that the scattered light
of the return light beam may permeate the observation optical path
can be further reduced.
[0172] Furthermore, according to the present invention, since the
illumination light can be turned on/off in cooperation with
insertion and removal of the support member, the illumination light
can be automatically stopped when the support member is
removed.
[0173] As a result, according to the present invention, the
incidence angle of the illumination light to the sample through the
objective can be constantly restricted to the range where the total
internal reflection illumination and the approximate total internal
reflection illumination can be obtained. Therefore, color
degradation of the entire sample due to the strong light of the
incident-light fluorescence illumination can be prevented, and the
stable fluorescence observation based on the total internal
reflection illumination and the approximate total internal
reflection illumination can be obtained. Furthermore, since the
incidence angle of the illumination light to the sample from the
objective can be constantly adjusted in the range of the total
internal reflection illumination and the approximate total internal
reflection illumination, the operability can be improved.
[0174] As described above, according to the present invention, it
is possible to provide the microscope which can realize the stable
fluorescence observation based on the total internal reflection
illumination and the approximate total internal reflection
illumination and improve the operability.
[0175] Additional advantages and modifications will readily occur
to those skilled in the art. Therefore, the invention in its
broader aspects is not limited to the specific details and
representative embodiments shown and described herein. Accordingly,
various modifications may be made without departing from the spirit
or scope of the general invention concept as defined by the
appended claims and their equivalents.
* * * * *