U.S. patent application number 12/105137 was filed with the patent office on 2008-08-28 for microscope system and microscope focus maintaining device for the same.
This patent application is currently assigned to OLYMPUS CORPORATION. Invention is credited to Kenichi Koyama, Atsuhiro Tsuchiya, Takashi YONEYAMA.
Application Number | 20080204865 12/105137 |
Document ID | / |
Family ID | 34381788 |
Filed Date | 2008-08-28 |
United States Patent
Application |
20080204865 |
Kind Code |
A1 |
YONEYAMA; Takashi ; et
al. |
August 28, 2008 |
MICROSCOPE SYSTEM AND MICROSCOPE FOCUS MAINTAINING DEVICE FOR THE
SAME
Abstract
A microscope system has a stage on which an observation sample,
including an observation object and a transparent member, is to be
placed. An objective lens is placed to face the observation sample
placed on the stage, and a focusing unit moves at least one of the
stage and the objective lens to perform a focusing operation. An
autofocus unit controls a focusing driving unit by a so-called
Through-the-Lens: TTL system. After autofocus is performed for the
transparent member by the autofocus unit, the focusing driving unit
makes at least one of the stage and the objective lens move by a
predetermined constant amount.
Inventors: |
YONEYAMA; Takashi;
(Sagamihara-shi, JP) ; Tsuchiya; Atsuhiro;
(Hachioji-shi, JP) ; Koyama; Kenichi;
(Sagamihara-shi, JP) |
Correspondence
Address: |
FRISHAUF, HOLTZ, GOODMAN & CHICK, PC
220 Fifth Avenue, 16TH Floor
NEW YORK
NY
10001-7708
US
|
Assignee: |
OLYMPUS CORPORATION
Tokyo
JP
|
Family ID: |
34381788 |
Appl. No.: |
12/105137 |
Filed: |
April 17, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12018061 |
Jan 22, 2008 |
|
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|
12105137 |
|
|
|
|
10951175 |
Sep 27, 2004 |
7345814 |
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12018061 |
|
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Current U.S.
Class: |
359/381 ;
359/385; 359/392 |
Current CPC
Class: |
G02B 21/16 20130101;
G02B 21/367 20130101; G02B 21/245 20130101 |
Class at
Publication: |
359/381 ;
359/392; 359/385 |
International
Class: |
G02B 21/02 20060101
G02B021/02; G02B 21/06 20060101 G02B021/06; G02B 21/26 20060101
G02B021/26 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 29, 2003 |
JP |
2003-338489 |
Sep 30, 2003 |
JP |
2003-342088 |
Jul 8, 2004 |
JP |
2004-202040 |
Claims
1-4. (canceled)
5. A microscope focus maintaining device for a microscope including
a stage on which an observation sample is to be placed, an
objective lens which is placed to face the observation sample
placed on the stage, an observation optical system which includes
an objective lens for observation of the observation sample, and a
focusing unit which moves at least one of the stage and the
objective lens to perform focusing operation, comprising: a focus
detection optical system which includes the objective lens for
in-focus detection; and a control unit which controls a focusing
driving unit on the basis of information obtained by the focus
detection optical system, wherein the focus detection optical
system has the same focus drift as that of the observation optical
system due to a temperature change.
6. A device according to claim 5, wherein the focus detection
optical system and the observation optical system comprise
identical members in terms of optical characteristics.
7. A device according to claim 6, wherein the focus detection
optical system includes an imaging lens, and the observation
optical system includes another imaging lens, the two imaging
lenses having the same optical characteristics.
8. A device according to claim 6, wherein both the focus detection
optical system and the observation optical system comprise imaging
optical systems for objective lens primary images.
9. A device according to claim 5, wherein magnifications of the
focus detection optical system and the observation optical system
can be changed to the same magnification.
10. A device according to claim 9, wherein the focus detection
optical system and the observation optical system include variable
power lenses which can be inserted/withdrawn on/from an optical
path, the variable power lenses having the same magnification.
11. A device according to claim 9, wherein the control unit changes
focus detection parameters in accordance with a change in
magnification of the focus detection optical system and the
observation optical system.
12. A microscope focus maintaining device for a microscope
including a stage on which an observation sample is to be placed,
an objective lens which is placed to face the observation sample
placed on the stage, an observation optical system which includes
an objective lens for observation of the observation sample, and a
focusing unit which moves at least one of the stage and the
objective lens to perform focusing operation, comprising: a focus
detection optical system which includes the objective lens for
in-focus detection; and a control unit which controls a focusing
driving unit on the basis of information obtained by the focus
detection optical system, the focus detection optical system
including a laser light source which emits a laser light beam, a
beam diameter changing unit which changes a diameter of a laser
light beam, and a power changing unit which changes power of the
laser light source.
13. A device according to claim 12, wherein the beam diameter
changing unit includes beam diameter restriction stops, and a stop
control unit which selectively places one of the beam diameter
restriction stops on an optical path of a laser light beam, the
objective lens is selected from objective lenses prepared in
advance, and each of the beam diameter restriction stops has an
opening with a diameter coinciding with a pupil of a corresponding
one of the objective lenses.
14. A device according to claim 12, wherein the beam changing unit
changes a laser light beam diameter so as to make the laser light
beam diameter equal to a pupil diameter of the objective lens.
15. A device according to claim 12, wherein the power changing unit
changes the power of the laser light source so as to keep power of
a laser light beam striking the objective lens constant regardless
of a change in laser light beam diameter.
16. A device according to claim 14, wherein the power changing unit
changes the power of the laser light source so as to keep power of
a laser light beam striking the objective lens constant regardless
of a change in laser light beam diameter.
17. A microscope focus maintaining device for a microscope
including a stage on which an observation sample is to be placed,
an objective lens which is placed to face the observation sample
placed on the stage, an observation optical system which includes
an objective lens for observation of the observation sample, and a
focusing unit which moves at least one of the stage and the
objective lens to perform focusing operation, the objective lens
being selected from objective lenses prepared in advance,
comprising: a focus detection optical system which includes the
objective lens for in-focus detection; and a control unit which
controls a focusing driving unit on the basis of information
obtained by the focus detection optical system, the focus detection
optical system including a laser light source which emits a laser
light beam, the laser light beam having a diameter equal to a
smallest pupil diameter of the objective lenses.
18. A microscope focus maintaining device for a microscope
including a stage on which an observation sample is to be placed,
an objective lens which is placed to face the observation sample
placed on the stage, an observation optical system which includes
an objective lens for observation of the observation sample, and a
focusing unit which moves at least one of the stage and the
objective lens to perform focusing operation, the observation
sample including an observation object and a transparent member
which holds the observation object, comprising: a focus detection
optical system which includes the objective lens for in-focus
detection; and a control unit which controls a focusing driving
unit on the basis of information obtained by the focus detection
optical system, wherein the focus detection optical system applies
a laser light beam to the observation sample to detect an in-focus
state on the basis of light reflected by a boundary surface between
the observation object and the transparent member, and the laser
light beam has a diameter corresponding to an NA smaller than a
refractive index of the observation object.
19. A device according to claim 18, wherein the focus detection
optical system includes a laser light source which emits a laser
light beam, and a beam diameter changing unit which changes a
diameter of a laser light beam, and when an NA of the objective
lens is larger than the refractive index of the observation object,
the beam diameter changing unit reduces a diameter of a laser light
beam to a diameter smaller than the refractive index of the
observation object.
20. A device according to claim 19, wherein the beam diameter
changing unit includes a beam diameter restriction stop which
reduces a diameter of a laser light beam, and a stop control unit
which properly places the beam diameter restriction stop on an
optical path of a laser light beam.
21. A microscope focus maintaining device for a microscope
including a stage on which an observation sample is to be placed,
an objective lens which is placed to face the observation sample
placed on the stage, an observation optical system which includes
an objective lens for observation of the observation sample, a
focusing unit which moves at least one of the stage and the
objective lens to perform focusing operation, and an
epi-fluorescence illumination system for fluorescence observation,
comprising: a focus detection optical system which includes the
objective lens for in-focus detection; and a control unit which
controls a focusing driving unit on the basis of information
obtained by the focus detection optical system, wherein light used
for focus detection is guided from a side closer to the objective
lens than the epi-fluorescence illumination system.
22. A device according to claim 21, wherein a wavelength of light
used for focus detection differs from a wavelength of light used
for fluorescence observation.
23. A device according to claim 22, wherein the epi-fluorescence
illumination system includes a fluorescence filter cassette, and
the focus detection optical system includes a light guide element
which guides light used for focus detection to the objective lens,
the light guide element being located between the objective lens
and the fluorescence filter cassette.
24. A device according to claim 23, wherein the light guide element
comprises a dichroic mirror which reflects one of light used for
focus detection and light used for observation and transmits the
other.
25. A microscope focus maintaining device for a microscope
including a stage on which an observation sample is to be placed,
an objective lens which is placed to face the observation sample
placed on the stage, an observation optical system which includes
an objective lens for observation of the observation sample, and a
focusing unit which moves at least one of the stage and the
objective lens to perform focusing operation, the objective lens
comprising an infinity objective lens, and the observation optical
system including an imaging lens which forms an image of the
infinity objective lens, comprising: a focus detection optical
system which includes the objective lens for in-focus detection;
and a control unit which controls a focusing driving unit on the
basis of information obtained by the focus detection optical
system, wherein light used for focus detection is guided from
between the infinity objective lens and the imaging lens.
26. A device according to claim 25, wherein a wavelength of light
used for focus detection differs from a wavelength of light used
fluorescence observation.
27. A device according to claim 26, wherein the focus detection
optical system includes a light guide element which guides light
used for focus detection to the infinity objective lens, the light
guide element being located between the infinity objective lens and
the imaging lens.
28. A device according to claim 27, wherein the light guide element
comprises a dichroic mirror which reflects one of light used for
focus detection and light used for observation and transmits the
other.
29. A microscope focus maintaining device for a microscope
including a stage on which an observation sample is to be placed,
an objective lens which is placed to face the observation sample
placed on the stage, an observation optical system which includes
an objective lens for observation of the observation sample, and a
focusing unit which moves at least one of the stage and the
objective lens to perform focusing operation, comprising: a focus
detection optical system which includes the objective lens for
in-focus detection; and a control unit which controls a focusing
driving unit on the basis of information obtained by the focus
detection optical system, the focus detection optical system
including a light guide element which guides light used for focus
detection to the objective lens, and the light guide element being
located on an optical path only at the time of focus detection.
30. A device according to claim 29, wherein whether to enable or
disable operation of placing the light guide element on the optical
path only at the time of focus detection can be selected.
31. A device according to claim 30, wherein whether to enable or
disable operation of placing the light guide element on the optical
path only at the time of focus detection is automatically
determined upon switching of objective lenses and microscopic
examination methods on the basis of types of objective lens and
microscopic examination method.
32. A microscope focus maintaining device for a microscope
including a stage on which an observation sample is to be placed,
an objective lens which is placed to face the observation sample
placed on the stage, an observation optical system which includes
an objective lens for observation of the observation sample, and a
focusing unit which moves at least one of the stage and the
objective lens to perform focusing operation, the observation
optical system including a retardation changing element for
differential interference observation, comprising: a focus
detection optical system which includes the objective lens for
in-focus detection; and a control unit which controls a focusing
driving unit on the basis of information obtained by the focus
detection optical system, the focus detection optical system
comprising a laser light source which emits linearly polarized
light for focus detection, a photodetector which detects reflected
light from the observation sample, a polarizing beam splitter which
splits reflected light from the observation sample from linearly
polarized light from the laser light source, a .lamda./4 plate
which is placed between the polarizing beam splitter and the
retardation changing element, and a retardation correction device
which generates a retardation which cancels out a retardation
generated by the retardation changing element.
33. A device according to claim 32, wherein the retardation
correction device includes a motor which rotates the .lamda./4
plate around an optical axis.
34. A device according to claim 33, wherein the retardation
correction device further includes a retardation sensor which
detects a retardation generated by the retardation changing
element, and a control unit which automatically corrects a
retardation on the basis of the retardation detected by the
retardation sensor.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Divisional application of U.S.
application Ser. No. 12/018,061 filed Jan. 22, 2008, which is a
Divisional application of U.S. Ser. No. 10/951,175 filed Sep. 27,
2004 (now U.S. Pat. No. 7,345,814), which is based upon and claims
the benefit of priority from prior Japanese Patent Applications No.
2003-338489, filed Sep. 29, 2003, No. 2003-342088, filed Sep. 30,
2003; and No. 2004-202040, filed Jul. 8, 2004, the entire contents
of all of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a microscope system used
for, for example, an apparatus which automatically examines a
specimen and automatically records an image of the specimen by
using a microscope.
[0004] 2. Description of the Related Art
[0005] Recently, as the automation of various functions of an
examining apparatus using a microscope has progressed, an autofocus
function of focusing on a specimen has become an indispensable
function to be automated.
[0006] Microscope autofocus is also used for an examining apparatus
for a specimen encapsulated in a slide glass. For example, Jpn.
Pat. Appln. KOKAI Publication No. 58-83906 discloses a method of
forming an infrared reflecting film on a slide glass or cover glass
in which a specimen is encapsulated, and focusing on the film. The
composition of an infrared reflecting film is, in particular,
disclosed in Jpn. Pat. Appln. KOKAI Publication No. 8-82747. In
addition, Jpn. Pat. Appln. KOKAI Publication No. 2001-91821
discloses a method of accurately focusing on a specimen by using a
passive AF scheme after focusing on a slide glass or cover glass by
using an active AF scheme.
BRIEF SUMMARY OF THE INVENTION
[0007] A microscope system according to the present invention
includes a stage on which an observation sample, including an
observation object and a transparent member is to be placed, an
objective lens which is placed to face the observation sample
placed on the stage, a focusing unit which moves at least one of
the stage and the objective lens to perform focusing operation, and
an autofocus unit which controls a focusing driving unit by a
so-called Through-the-Lens: TTL system, wherein after autofocus is
performed for the transparent member by the autofocus unit, the
focusing driving unit makes at least one of the stage and the
objective lens move by a predetermined constant amount.
[0008] 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.
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
[0009] 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.
[0010] FIG. 1 is a block diagram showing the arrangement of a
microscope system according to the first embodiment of the present
invention;
[0011] FIG. 2A is a view showing a time lapse model for long-time
observation of the movement of an observation object;
[0012] FIG. 2B is a view showing a setting window of a host PC in
the microscope system according to the first embodiment of the
present invention;
[0013] FIG. 3 is a view showing a model of observation images
photographed during a predetermined photographing period;
[0014] FIG. 4 is a flowchart showing the operation of the
microscope system according to the first embodiment of the present
invention;
[0015] FIG. 5 is a view showing a model having measurement points
in an observation object encapsulated between a slide glass and a
cover glass;
[0016] FIG. 6 is a view showing a host PC setting window in a
microscope system according to the second embodiment of the present
invention;
[0017] FIG. 7 is a flowchart showing the operation of the
microscope system according to the second embodiment of the present
invention;
[0018] FIG. 8 is a flowchart showing operation control in a
microscope system according to the third embodiment of the present
invention;
[0019] FIG. 9 is a flowchart showing operation control in a
microscope system according to the fourth embodiment of the present
invention;
[0020] FIG. 10 is a view showing a slide glass model in a
microscope system according to the fifth embodiment of the present
invention;
[0021] FIG. 11 is a flowchart showing operation control in the
microscope system according to the fifth embodiment of the present
invention;
[0022] FIG. 12 is a view showing the arrangement of a microscope
system according to the sixth embodiment of the present
invention;
[0023] FIG. 13 is a view showing the overall arrangement of a
microscope according to the seventh embodiment of the present
invention;
[0024] FIG. 14 is a view showing portions around an observation
sample in FIG. 13;
[0025] FIG. 15A is a plan view of a sensor head shown on the right
side of FIG. 13;
[0026] FIG. 15B is a front view of the sensor head shown in FIG.
15A;
[0027] FIG. 16A is a plan view of a sensor head in the eighth
embodiment of the present invention;
[0028] FIG. 16B is a front view of the sensor head shown in FIG.
16A;
[0029] FIG. 17A is a plan view of a sensor head in the ninth
embodiment of the present invention;
[0030] FIG. 17B is a front view of the sensor head shown in FIG.
17A;
[0031] FIG. 18 is a view showing the overall arrangement of a
microscope according to the 10th embodiment of the present
invention;
[0032] FIG. 19A is a plan view of a sensor head shown on the right
side of FIG. 18;
[0033] FIG. 19B is a front view of the sensor head shown in FIG.
19A;
[0034] FIG. 20 is a view showing the overall arrangement of a
microscope according to the 11th embodiment of the present
invention;
[0035] FIG. 21A is a plan view of a sensor head shown on the right
side of FIG. 20;
[0036] FIG. 21B is a front view of the sensor head shown in FIG.
21A;
[0037] FIG. 22 is a view showing the overall arrangement of a
microscope according to the 12th embodiment of the present
invention;
[0038] FIG. 23A is a plan view of a sensor head shown on the right
side of FIG. 22;
[0039] FIG. 23B is a front view of the sensor head shown in FIG.
23A;
[0040] FIG. 24 is a view showing the overall arrangement of a
microscope according to the 13th embodiment of the present
invention;
[0041] FIG. 25 is a view showing the overall arrangement of a
microscope according to the 14th embodiment of the present
invention;
[0042] FIG. 26A is a plan view of a sensor head shown on the right
side of FIG. 25;
[0043] FIG. 26B is a front view of the sensor head shown in FIG.
26A; and
[0044] FIG. 27 is a graph in which the ordinate represents the
outputs of the two-segments photodiode as the A and B phases; and
the abscissa, the movement of the focus in the optical axis
direction on the observation sample side.
DETAILED DESCRIPTION OF THE INVENTION
[0045] The embodiments of the present invention will be described
below with reference to the views of the accompanying drawing.
<Microscope System>
[0046] An embodiment of a microscope system will be described
first.
FIRST EMBODIMENT
[0047] This embodiment is directed to a microscope system including
an upright microscope. FIG. 1 shows the arrangement of the
microscope system according to the first embodiment. As shown in
FIG. 1, a microscope system 100 according to this embodiment
comprises a stage 101 on which an observation sample 102 is placed,
and an objective lens 109 which is placed to face the observation
sample 102 placed on the stage 101.
[0048] The objective lens 109 is mounted on a revolver 108. The
revolver 108 can be motor-driven and controlled. The objective lens
109 with a desired magnification can be placed on the optical path
by controlling the rotation of the revolver 108 using a revolver
driving control unit (not shown).
[0049] The position of the stage 101 in the X-Y-Z direction can be
motor-driven and controlled. The microscope system 100 comprises a
stage X-Y control unit 115 which controls the position of the stage
101 in the X-Y direction, and a stage Z control unit 116 which
controls the position of the stage 101 in the Z direction. The
stage Z control unit 116 forms a focusing unit which moves at least
one of the stage and the objective lens to perform focusing
operation.
[0050] For example, as shown in FIG. 2A, the observation sample 102
comprises a slide glass 102A, a cover glass 102B, and an
observation object encapsulated between them. The observation
object comprises, for example, a specimen (e.g., a cell) and a
culture solution. The observation object is positioned between the
slide glass 102A and the cover glass 102B, and the objective lens
109 is positioned above the cover glass 102B.
[0051] Referring back to FIG. 1, the microscope system 100
comprises a known active type autofocus unit 118 for a microscope.
The autofocus unit 118 controls the focusing driving unit, i.e.,
the stage Z control unit 116, by the so-called Through The Lens:
TTL system. That is, the autofocus unit 118 optically detects,
through the objective lens 109, the focus position of an
observation optical system including the objective lens 109, and
sends the resultant information to the stage Z control unit 116.
The stage Z control unit 116 moves the observation sample 102 to
the focus position in accordance with information from the
autofocus unit 118.
[0052] The microscope system 100 further comprises a light source
103 for transmitted illumination of the observation sample 102 or a
light source 103' for fluorescence illumination of the observation
sample 102. In transmitted observation, illumination light from the
light source 103 is applied to the observation sample 102 through
an ND filter 104 for light attenuation, a field stop (FS) 105, and
a condenser 106 incorporating an aperture stop (AS). In
fluorescence observation, illumination light from the light source
103' is returned back by a fluorescence cube 107 and applied as
excitation light to the observation sample 102. Each optical
element is motor-driven by a corresponding driving control unit
(not shown) to perform optical element conversion. With this
arrangement, the illumination optical system for fluorescence
observation including the light source 103' and fluorescence cube
107 can selectively apply excitation light beams having different
wavelengths. In other words, the illumination optical system for
fluorescence observation can select excitation light.
[0053] A transmitted illumination image or fluorescence image from
the observation sample 102 passes through the objective lens 109
and is partly guided to an eyepiece lens 111 through a lens barrel
110. The remaining light beams enter a TV camera 112.
[0054] The microscope is controlled by a host PC 113 through a
microscope controller 114. The microscope controller 114 performs
actual driving control on the portions to be motor-driven and
controlled through the corresponding control units.
[0055] The light source 103, ND, AS, and FS can be controlled by
the corresponding control units (not shown) from the microscope
controller 114; the light source voltage, stop diameter, and the
like are controlled.
[0056] An image of the observation sample 102 sensed by the TV
camera 112 is acquired by the host PC 113 through a video capture
board 117. The host PC 113 can store acquired images in an image
memory (not shown).
[0057] FIG. 2A shows a so-called time lapse model for long-time
observation of the movement of a specimen. FIG. 2A indicates that
the specimen (or a specific region such as a nucleus in the
specimen) in the observation object encapsulated between the slide
glass 102A and the cover glass 102B moves from a position A to a
position B in a long period of time. Observation is performed by
performing photography at intermittent intervals at the positions
indicated by the chain double-dashed lines from the upper limit of
the photographing area to its lower limit while moving the stage
from the photographing area center position indicated by the chain
line in FIG. 2A by a predetermined amount in the Z direction.
[0058] FIG. 3 shows an observation image model obtained by
performing photography during a period from photographing time t0
to photographing time t4 under the above conditions. Referring to
FIG. 3, each image information in the vertical axis direction
indicates the distance the specimen indicated by the crosshatched
circle has moved, i.e., indicates that the specimen has moved a
distance Z4 from t0 to t4. Likewise, the X-Y position in each image
indicates that the specimen has moved in the X-Y direction. The
model shown in FIG. 3 indicates that the specimen located in the
center of the image at t0 has moved to the right until t4.
[0059] FIG. 2B shows the setting window of the host PC 113 in the
microscope system 100, which is used to perform the above
photographing operation. The setting window comprises a portion G1
which displays a specimen image from the TV camera 112, an
operation display portion G2 associated with control on the
microscope, and a display portion G3 for setting photographing
conditions for a specimen image.
[0060] The photographing condition setting method will be described
with reference to FIGS. 2A and 2B.
[0061] In setting photographing conditions, first of all, a button
G4 is pressed to perform autofocus (AF) for the slide glass 102A or
cover glass 102B. FIG. 2A shows a case wherein AF is performed for
the cover glass 102B. When AF is complete, the stage 101 is located
at the reference position in FIG. 2A. An observer operates a button
G5 to vertically drive the stage 101 so as to focus on a specimen
as a photographing target. A portion G6 is displaying the address
of the current stage position, from which the distance from the
position of the cover glass (the position where AF is complete) to
the position of the specimen can be read. After the specimen is
brought into focus, a button G10 is pressed to register a
photographing area center position like photographing count 3 in
FIG. 2A in the Z direction of the stage at which photography is to
be performed. The distance between this cover glass position and
the center position becomes an offset. After the photographing area
center position is registered, buttons G7 and G8 are operated to
set a pitch and frame count in the Z direction of the stage for
Z-direction slice photography for the specimen like those indicated
by a photographing Z interval and photographing count in FIG. 2A. A
frame count and pitch can also be set by setting a range and frame
count in the Z direction in which photography is performed. In this
case, a button G8 and the button G9 are operated to make settings.
If a ranged in the Z direction in which photography is performed is
set beyond the slide glass 102A or cover glass 102B, warning
display is performed against the set conditions. After a Z
photographing count and two-direction pitch are set, a time
interval for photography is set by using a button G11, and a
photographing period is set by using a button G12. If a
photographing interval is set to be shorter than the photographing
time in the Z direction set with the buttons G7 and G9, warning
display is performed against the set photographing interval.
[0062] In this embodiment, after autofocus is performed for the
cover glass 102B, the position of the photographing target is set.
However, the same effect can be obtained by performing autofocus
for the cover glass 102B after the position of the photographing
target is set.
[0063] The operation of the microscope system 100 set in the above
manner will be described with reference to FIG. 4.
[0064] When measurement is started (S1), it is checked whether
photography is completed by a predetermined frame count and time
lapse measurement corresponding to the photographing time (total)
set by the host PC is completed (S2). If the measurement is
completed, the processing is terminated (S10). If the measurement
is not completed, the system is set in the standby state until the
time lapse measurement interval set by the time lapse interval set
by the host PC is reached (S3).
[0065] When the measurement interval is reached, AF control is
performed for the cover glass 102B to control the position of the
cover glass 102B to the reference position shown in FIG. 2A (S4).
After the cover glass 102B is positioned at the reference position,
the stage 101 is driven to the photographing area center position
set in advance by the host PC, i.e., by the offset amount (S5).
[0066] That is, after the autofocus unit 118 performs autofocus for
the cover glass 102B, the stage Z control unit 116 moves the stage
101 by a predetermined constant amount, i.e., an offset amount.
[0067] In order to acquire a Z-direction image, the stage 101 is
driven to the upper limit of the photographing area (S6), and
photography is performed (S7). Of the operations in steps S5 and
S6, the operation in step S5 can be omitted (internally processed)
by calculating the upper limit of the photographing area in advance
from the set value of the photographing area center position. In
these driving operations, in order to minimize a position shift due
to so-called backlash, position control is preferably performed in
one direction.
[0068] After the photographing is performed, photography is
performed by the frame count set by the host PC while the stage is
driven by the photographing Z interval set by the host PC (S8, S9).
This operation is repeated until measurement is finished (S2).
[0069] The microscope system 100 of this embodiment which has the
above arrangement and is controlled in the above manner can
reliably measure the movement of a specimen because a specimen
photographing position is set with an offset by performing
autofocus for the cover glass 102B while a reference position is
fixed even if the objective lens focus position changes with a
change in ambient temperature.
[0070] The microscope system 100 of this embodiment is configured
to vertically move the stage 101. However, the system may be
configured to vertically move the objective lens 109. In this case
as well, similar effects can be obtained. In addition, in the
microscope system 100 of this embodiment, the microscope is an
upright microscope. However, a so-called inverted microscope may be
used, which has an objective lens placed below the stage 101. In
this case as well, similar effects can be obtained. In this
embodiment, an active type autofocus system is used. However, a
known passive type autofocus system may be used. In this case as
well, similar effects can be obtained.
[0071] Various modifications can be made to this embodiment without
departing from the object of the embodiment, i.e., temporarily
performing autofocus for the cover glass 102B or slide glass 102A,
then driving the stage by a predetermined amount, and performing
photography.
[0072] In this embodiment, the stage 101 need not be capable of
moving in the X-Y direction. That is, the stage 101 may be capable
of moving in only the Z direction, and the stage X-Y control unit
115 may be omitted. In addition, the objective lens 109 need not be
interchangeable. That is, the microscope need not have the function
of a revolver capable of rotatably holding objective lenses and
selectively placing one of them on an optical path.
SECOND EMBODIMENT
[0073] This embodiment is directed to time lapse measurement for
measurement points differing in X-Y position. Since the arrangement
of a microscope system of this embodiment is the same as that of
the first embodiment, a description thereof will be omitted. In
this embodiment, offset driving operation is performed at each X-Y
position of a specimen.
[0074] FIG. 5 shows a model having measurement points XY1, XY2, and
XY3 in an observation object encapsulated between a slide glass
102A and a cover glass 102B. In this model, the measurement points
XY1, XY2, and XY3 have different Z-direction positions C1, C2, and
C3 with respect to the cover glass 102B. The following is a
sequence for time lapse measurement on the movement of each
measurement point.
[0075] FIG. 6 shows a host PC setting window for setting offset
driving for each X-Y position in this embodiment. As compared with
the first embodiment, several functions are added to this setting
window. The added functions will be described.
[0076] Reference symbol G20 denotes an overall image of the slide
glass 102A. An observer operates a button G23 to move a stage 101
in the X-Y direction to determine measurement points, e.g., nuclei,
for time lapse observation. The X-Y position information of the
stage 101 is displayed on a portion G24. Measurement points are
determined by operation a button G21 while the X-Y position of the
stage is fixed. When position setting is done, the registered
measurement points are indicated by crosses in the overall
observation object image G20. The offset amounts of the respective
measurement points and photographing conditions are set in the same
sequence as that in the first embodiment. Assume that the same
photographing conditions as those which have already been
registered for other measurement points are to be set. In this
case, registering the photographing conditions allows the observer
to call them by operating a button G22 and can unify photographing
conditions for all measurement points or specific measurement
points.
[0077] The operation of the microscope system for which settings
have been made in the above manner will be described with reference
to FIG. 7.
[0078] When measurement is started (S20), it is checked whether
photography is completed by a predetermined frame count, and time
lapse measurement corresponding to the photographing time (total)
set by the host PC is finished (S21). If the measurement is
finished, the processing is terminated (S31). If the measurement is
not finished, the system is set in the standby state until the time
lapse measurement interval set by the time lapse interval set by
the host PC is reached (S22).
[0079] When the measurement interval is reached, the stage is
driven in the X-Y direction to set measurement points (S23). When
the stage is driven to the stage X-Y position of each measurement
point, AF control is performed for the cover glass 102B at the
stage X-Y position to control the position of the cover glass 102B
to the reference position (S24). After the cover glass 102B is
positioned at the reference position, the stage 101 is driven to
each photographing center area position set in advance by the host
PC, i.e., by an offset amount (S25).
[0080] That is, after an autofocus unit 118 performs autofocus for
the cover glass 102B, a stage Z control unit 116 moves the stage
101 by a predetermined constant amount, i.e., an offset amount.
[0081] The stage is further driven to the upper limit of the
photographing area to acquire a Z-direction image (S26), and
photography is performed (S27). Of the operations in steps S25 and
S26, the operation in step S25 can be omitted (internally
processed) by calculating the upper limit of the photographing area
in advance from the set value of the photographing area center
position as in the first embodiment.
[0082] After the photographing is performed, photography is
performed by the frame count set by the host PC while the stage is
driven by the photographing Z interval set by the host PC (S28,
S29). This operation is repeated until photography is complete at
all the measurement points and the time lapse measurement finish
conditions are satisfied (S30, S31).
[0083] The microscope system of this embodiment which has the above
arrangement and is controlled in the above manner can reliably
measure the movement of a specimen even in measurement operations
because a specimen photographing position is set with an offset by
performing autofocus for the cover glass 102B for each measurement
point while a reference position is fixed even if the objective
lens focus position changes with a change in ambient
temperature.
[0084] In addition, at measurement points in the X-Y direction, if
autofocus conditions acquired in previous time lapse measurement,
e.g., a region for determining a specimen search range, are updated
during AF control indicated by S24 in FIG. 7, AF control can be
performed at a higher speed and with high reliability.
[0085] In this embodiment, an objective lens 109 need not be
interchangeable. That is, the microscope need not have the function
of a revolver capable of rotatably holding objective lenses and
selectively placing one of them on an optical path.
THIRD EMBODIMENT
[0086] This embodiment is directed to time lapse measurement
including the exchange of objective lenses. Since the arrangement
of a microscope system of this embodiment is the same as that of
the first embodiment, a description thereof will be omitted.
[0087] If the conditions for time lapse measurement include the
exchange of objective lenses, since different imaging lenses are to
be used, the offset amount may need to be changed. In this
embodiment, time lapse measurement is performed upon setting an
offset amount for each objective lens.
[0088] An offset amount for each objective lens is set by operating
an offset amount setting button for each objective lens, which is
not shown in a host PC window, as in the first or second
embodiment.
[0089] FIG. 8 shows operation control on the microscope system in
which an offset amount is set for each objective lens.
[0090] When measurement is started (S40), it is checked whether
photography is completed by a predetermined frame count, and time
lapse measurement corresponding to the photographing time (total)
set by the host PC is finished (S41). If the measurement is
finished, the processing is terminated (S51). If the measurement is
not finished, the system is set in the standby state until the time
lapse measurement interval set by the time lapse interval set by
the host PC is reached (S42).
[0091] When the measurement interval is reached, the set objective
lens is inserted on an optical path (S43). When the objective lens
is inserted on the optical path, AF control is performed for the
cover glass 102B to control the position of the cover glass 102B to
the reference position (S44). After the cover glass 102B is
positioned at the reference position, the stage 101 is driven by
the offset amount set for each objective lens by the host PC
(S45).
[0092] That is, after an autofocus unit 118 performs autofocus for
the cover glass 102B, a stage Z control unit 116 moves the stage
101 by a predetermined constant amount, i.e., an offset amount.
[0093] The stage is further driven to the upper limit of the
photographing area to acquire a Z-direction image (S46), and
photography is performed (S47).
[0094] After the photographing is performed, photography is
performed by the photographing count set by the host PC while the
stage is driven by the photographing Z interval set by the host PC
(S48, S49). This operation is repeated until photography is
complete with all set objective lenses and the time lapse
measurement finish conditions are satisfied (S50, S51).
[0095] The microscope system of this embodiment which has the above
arrangement and is controlled in the above manner can reliably
measure the movement of a specimen even in measurement operations
by performing autofocus using an objective lens on each optical
path when time lapse measurement conditions includes observation
with objective lenses, even if a focus shift including the
autofocus side exists between the objective lenses, because a
specimen photographing position is set with an offset while a
reference position is fixed.
[0096] In this embodiment, the stage 101 need not be capable of
moving in the X-Y direction. That is, the stage 101 may be capable
of moving in only the Z direction, and a stage X-Y control unit 115
may be omitted.
FOURTH EMBODIMENT
[0097] This embodiment is directed to time lapse measurement
including excitation light conversion. Since the arrangement of a
microscope system of this embodiment is the same as that of the
first embodiment, a description thereof will be omitted.
[0098] If the conditions for time lapse measurement include
fluorescence cube conversion, i.e., excitation light conversion,
since a specimen has different luminescent portions, the offset
amount may need to be changed. In this embodiment, time lapse
measurement is performed after an offset amount is set for each
excitation light conversion.
[0099] FIG. 9 shows operation control on the microscope system in
which an offset amount is set for each excitation light
conversion.
[0100] When measurement is started (S60), it is checked whether
photography is completed by a predetermined frame count, and time
lapse measurement corresponding to the photographing time (total)
set by the host PC is finished (S61). If the measurement is
finished, the processing is terminated (S71). If the measurement is
not finished, the system is set in the standby state until the time
lapse measurement interval set by the time lapse interval set by
the host PC is reached (S62).
[0101] When the measurement interval is reached, the fluorescence
cube is inserted on an optical path (S63). When the fluorescence
cube is inserted on the optical path and the excitation light is
switched to another kind of excitation light, AF control is
performed for the cover glass 102B to control the position of the
cover glass 102B to the reference position (S64). After the cover
glass 102B is positioned at the reference position, the stage 101
is driven by the offset amount set for each excitation light by the
host PC (S65).
[0102] That is, after an autofocus unit 118 performs autofocus for
a cover glass 102B, a stage Z control unit 116 moves a stage 101 by
a predetermined constant amount, i.e., an offset amount.
[0103] The stage is further driven to the upper limit of the
photographing area to acquire a Z-direction image (S66), and
photography is performed (S67).
[0104] After the photographing is performed, photography is
performed by the frame count set by the host PC while the stage is
driven by the photographing Z interval set by the host PC (S68,
S69). This operation is repeated until photography is complete with
all set kinds of excitation light and the time lapse measurement
finish conditions are satisfied (S70, S71).
[0105] The microscope system of this embodiment which has the above
arrangement and is controlled in the above manner can, when the
movement of an object with different luminescent points of a
specimen is to be observed under time lapse measurement conditions
for fluorescence observation, reliably measure the movement of the
specimen for each excitation light by performing autofocus for a
slide glass 102A or the cover glass 102B because a specimen
photographing position is set with an offset while a reference
position is fixed.
[0106] In this embodiment, autofocus is performed for the slide
glass 102A or cover glass 102B for each observation. However, the
photographing time may be shortened by performing autofocus at
intermittent time intervals.
[0107] Although offset setting for each X-Y position of the stage
in the second embodiment, offset setting for each objective lens in
the third embodiment, and offset setting for each excitation light
in the fourth embodiment are so described as to be independently
performed, any two of these setting operations or all the three
setting operations may be combined. In this case, measurement can
be done with higher precision.
FIFTH EMBODIMENT
[0108] This embodiment is directed to AF control on a slide glass
102A or cover glass 102B. Since the arrangement of a microscope
system of this embodiment is the same as that of the first
embodiment, a description thereof will be omitted.
[0109] FIG. 10 shows a model of the slide glass 102A used in this
embodiment. As shown in FIG. 10, the slide glass 102A has an AF
marking 102C. The AF marking 102C is positioned in a range where it
has no influence on an observation object 102D, and has been
subjected to a process for improving the precision of autofocus
control. This embodiment will exemplify an active AF. The AF
marking 102C has been coated with a film which reflects laser light
used for active AF at high reflectance.
[0110] FIG. 11 explains operation control in the above microscope
system.
[0111] When measurement is started (S80), it is checked whether
photography is completed by a predetermined frame count, and time
lapse measurement corresponding to the photographing time (total)
set by the host PC is finished (S81). If the measurement is
finished, the processing is terminated (S91). If the measurement is
not finished, the system is set in the standby state until the time
lapse measurement interval set by the time lapse interval set by
the host PC is reached (S82).
[0112] When the measurement interval is reached, the stage is
driven to the AF marking position in the X-Y direction (S83). When
the stage is moved to the AF marking position, autofocus control is
performed to control the position of the cover glass 102B to the
reference position (S84). After the cover glass 102B is positioned
at the reference position, the stage is moved to an X-Y position
where actual measurement is to be performed (S85). Then, the stage
101 is driven by the offset amount set in advance by the host PC
(S86).
[0113] That is, an autofocus unit 118 performs autofocus for the AF
marking 102C of the slide glass 102A, a stage Z control unit 116
moves the stage 101 by a predetermined constant amount, i.e., an
offset amount.
[0114] In order to acquire a Z-direction image, the stage 101 is
driven to the upper limit of the photographing area (S87), and
photography is performed (S88).
[0115] After the photographing is performed, photography is
performed by the frame count set by the host PC while the stage is
driven by the photographing Z interval set by the host PC (S89,
S90). This operation is repeated until photography is complete with
all set kinds of excitation light and the time lapse measurement
finish conditions are satisfied (S89, S91).
[0116] In the microscope system of this embodiment, since
high-precision AF can be performed for the slide glass 102A, the
measurement precision can be improved. In addition, since an
autofocus target is known, optimal autofocus conditions can be set.
This makes it possible to shorten the focusing time and improve the
throughput of measurement.
[0117] This embodiment has exemplified the case wherein autofocus
is performed by the active AF scheme, and a reflecting film is used
as the AF marking 102C accordingly. However, autofocus may be
performed by a passive AF scheme. In this case, a high-contrast
marking is used as the AF marking 102C, and autofocus control such
as a known hill-climbing scheme may be performed.
SIXTH EMBODIMENT
[0118] This embodiment is directed to a microscope system including
an inverted microscope. FIG. 12 shows the arrangement of the
microscope system according to the sixth embodiment of the present
invention.
[0119] As shown in FIG. 12, a microscope system 600 according to
this embodiment comprises a stage 621 on which an observation
sample 610 is to be placed, and an observation optical system
including an objective lens 631 which is placed to face the
observation sample 610 placed on the stage 621.
[0120] The observation sample 610 comprises an observation object
611 and a vessel 612 (so-called dish) which houses the observation
object 611. The observation object 611 comprises, for example, a
specimen (e.g., a cell) and a culture solution. The vessel 612
comprises a dish-like transparent vessel body 613 having an opening
in its bottom portion and a cover glass 614 sealing the opening of
the vessel body 613. Therefore, the observation object 611 is
positioned above the cover glass 614, and the objective lens 631 is
positioned below the cover glass 614.
[0121] The stage 621 is capable of moving in the X-Y direction. In
order to motor-drive and control the position of the stage 621 in
the X-Y direction, the microscope system 600 comprises a stage
driving motor 622 for moving the stage 621 in the X-Y direction and
a stage X-Y control unit 623 which controls the position of the
stage 621 in the X-Y direction.
[0122] The objective lens 631 is mounted on a moterized revolver.
The moterized revolver comprises a revolver body 632 which can hold
objective lenses 631, a revolver motor 633 for rotating the
revolver body 632, and a revolver motor driving unit 634 for
controlling the revolver motor 633.
[0123] The moterized revolver can selectively place one of the
objective lenses 631 held on the revolver body 632 on an optical
path by controlling the rotation of the revolver body 632 using the
revolver motor driving unit 634.
[0124] The revolver body 632 has objective lens mount holes in
which the objective lenses 631 can be mounted. The moterized
revolver further includes a mount hole position detection unit 635
for detecting the objective lens mount hole of the revolver body
632 in which the objective lens 631 placed on the optical path is
mounted.
[0125] The revolver body 632 can move along the observation optical
axis. The microscope system 600 further comprises a focusing
driving motor 636 which moves the revolver body 632 along the
observation optical axis for focusing operation and a focusing
motor driving unit 637 which controls the focusing driving motor
636. The focusing driving motor 636 and focusing motor driving unit
637 constitute a focusing unit which moves at least the stage or
the objective lens for focusing operation.
[0126] The microscope system 600 comprises an active AF device
based on an active pupil division method. The AF device comprises a
reference light source 671, laser driving unit 672, collimator lens
673, light-emitting side stopper 674, polarizing beam splitter
(PBS) 675, condenser lens group 676, chromatic aberration
correction lens group 677, .lamda./4 plate 678, dichroic mirror
679, light-receiving sensor 681, signal processing unit 682,
chromatic aberration lens group driving motor 683, and chromatic
aberration lens driving unit 684.
[0127] The microscope system 600 comprises a microscope controller
691 which controls the stage X-Y control unit 623, revolver motor
driving unit 634, focusing motor driving unit 637, and chromatic
aberration lens driving unit 684. The microscope controller 691 is
a known CPU circuit. An operation unit 692 including various kinds
of operation SWs is connected to the microscope controller 691. An
observer can perform operations such as starting/stopping AF or
switching objective lenses, or can input necessary information
associated with AF, e.g., a glass thickness, through the operation
unit 692. A jog dial 693 is connected to the microscope controller
691 through a pulse counter 695 and jog encoder 694. The observer
can move the revolver body 632 vertically through the jog dial
693.
[0128] The microscope system 600 comprises a transmitted
illumination optical system which provides transmitted illumination
for the observation sample 610. The transmitted illumination
optical system includes an illumination light source 641, lens 642,
mirror 643, and lens 644.
[0129] The microscope system 600 also comprises an illumination
optical system for fluorescence observation. The fluorescence
illumination optical system includes a light source 651 which emits
excitation light and a fluorescence cube 652. The fluorescence cube
652 can be exchanged with another one by motor-driven control. The
illumination optical system for fluorescence observation can
selectively apply excitation light beams having different
wavelengths. In other words, the illumination optical system for
fluorescence observation can select excitation light.
[0130] The microscope system 600 need not comprise both a
transmitted illumination optical system and an illumination optical
system for fluorescence observation, and may comprise one of the
illumination optical systems in accordance with an observation
purpose.
[0131] The microscope system 600 further comprises a TV camera 661
for sensing an observation image, a video capture board 662, and a
host PC 663.
[0132] In transmitted illumination observation, illumination light
from the illumination light source 641 passes through the lens 642
and is reflected by the mirror 643. The reflected light is
condensed by the lens 644 and illuminates the observation sample
610 from above. The light transmitted through the observation
sample 610 passes through the objective lens 631 and is transmitted
through the dichroic mirror 679 to strike the TV camera 661.
[0133] In fluorescence observation, the excitation light emitted
from the light source 651 is reflected by the fluorescence cube
652, passes through the objective lens 631, and strikes the
observation sample 610. The fluorescence emitted from the
observation sample 610 passes through the objective lens 631 and is
wavelength-selected by the fluorescence cube 652. The resultant
light passes through the dichroic mirror 679 and strikes the TV
camera 661.
[0134] The images sensed by the TV camera 661 are acquired by the
host PC 663 through the video capture board 662 as in the first
embodiment. The host PC 663 can store the acquired images in an
image memory (not shown).
[0135] In the microscope system 600, the AF device based on the
active pupil division method applies a laser beam to the
observation sample, and detects the reflected light, thereby
adjusting the focus.
[0136] The reference light source 671 comprises a light source
which emits light in an invisible wavelength range, i.e., an
infrared laser. The reference light source 671 is controlled by the
laser driving unit 672. The laser driving unit 672 controls the
intensity of the reference light source 671 by performing pulse
lighting of the reference light source 671.
[0137] The laser beam emitted from the reference light source 671
passes through the collimator lens 673 to become a parallel light
beam. Half of the light beam diameter is cut by the light-emitting
side stopper 674, and only a P-polarized light component is
reflected by the polarizing beam splitter (PBS) 675.
[0138] The light beam from the polarizing beam splitter (PBS) 675
is converged once by the condenser lens group 676 and then passes
through the chromatic aberration correction lens group 677. The
light passing through the chromatic aberration correction lens
group 677 is polarized by 45.degree. when it passes through the
.lamda./4 plate 678 and strikes the dichroic mirror 679. The
dichroic mirror 679 reflects only light in the infrared region, and
hence the laser light beam is reflected. The reflected light beam
passes through the objective lens 631 and strikes the observation
sample 610 to form a light spot.
[0139] The light beam reflected by the observation sample 610
strikes the objective lens 631 and is reflected by the dichroic
mirror 679. The reflected light beam is polarized by 45.degree. to
be converted into an S-polarized light component when it passes
through the .lamda./4 plate 678 again. The light beam further
passes through the chromatic aberration correction lens group 677
and condenser lens group 676 and strikes the PBS 675. The light
beam is transmitted through the PBS 675 because the light beam has
become an S-polarized light component. The transmitted light beam
passes through a condenser lens group 680 and is then formed into
an image on the light-receiving sensor 681.
[0140] The light-receiving sensor 681 is a two-segments photodiode
having two adjacent light-receiving portions, and is placed such
that an optical axis is located on a boundary line between the two
light-receiving portions. The signal processing unit 682 acquires a
so-called S-shaped curve which allows determination of a focus
position and focusing direction. The microscope controller 691
performs autofocus control on the observation optical system by
controlling the focusing motor driving unit 637 on the basis of the
information (S-shaped curve) obtained by the signal processing unit
682.
[0141] This AF device can perform chromatic aberration correction
for the infrared laser by moving the chromatic aberration
correction lens group 677 along the optical axis by using the
chromatic aberration lens group driving motor 683, and can perform
so-called optical offset driving of shifting the focus position.
This can compensate for the focus drift difference between the
observation optical system and the AF optical system which is
caused by, for example, a temperature change. In other words, this
makes it possible to equalize the focus drifts of the two
systems.
[0142] In the microscope system 600 of this embodiment, as in the
first embodiment, after autofocus is performed for the cover glass
614 by active AF, the revolver body 632 is moved by the focusing
motor driving unit 637 to move the objective lens 631 by a
predetermined constant amount, i.e., an offset amount. This makes
it possible to obtain the same advantages as those of the first
embodiment.
[0143] As in the same manner that the second to fifth embodiments
are applied to the first embodiment, the second to fifth
embodiments may be applied to the microscope system 600 of the
sixth embodiment. In such cases, the same advantages as those of
the second to fifth embodiments can be obtained.
<Microscope Focus Maintaining Device>
[0144] An embodiment of a microscope focus maintaining device which
can be applied to the above microscope system will be described
next. Although a microscope focus maintaining device applied to an
inverted microscope will be representatively described below, the
microscope focus maintaining device to be described below may be
applied to an upright microscope.
SEVENTH EMBODIMENT
[0145] The seventh embodiment of the present invention will be
described with reference to FIGS. 13, 14, 15A, and 15B. This
embodiment is directed to a microscope comprising a microscope
focus maintaining device. FIG. 13 shows the overall arrangement of
the microscope according to the seventh embodiment of the present
invention. FIG. 13 shows a side surface of the microscope on the
left side, together with portions around the objective lens of the
microscope on the right side. FIG. 14 shows portions around an
observation sample in FIG. 13. FIG. 15A is a plan view of a sensor
head shown on the right side in FIG. 13. FIG. 15B is a front view
of the sensor head shown in FIG. 15A.
[0146] The arrangement and function of the microscope will be
described first.
[0147] The microscope includes an X-Y stage 1129 on which an
observation sample is to be placed. The observation sample includes
a cover glass 1104 as a transparent member and an observation
object 1103 which is held on the cover glass. The observation
object 1103 includes a cell as a specimen and a culture solution
which cultures the cell. The microscope further includes an
objective lens 1105 placed below the X-Y stage 1129, a CCD 1136 for
the observation of the observation object 1103 through the
objective lens 1105, and an optical path switching prism 1121 which
optically couples the CCD 1136 to the objective lens 1105. The
microscope further includes an eyepiece lens 1110 for the
observation of the observation object through the eyes, a mirror
1122 which optically couples the eyepiece lens 1110 to the
objective lens 1105, and a relay lens 1109 placed between the
mirror 1122 and the eyepiece lens 1110. In addition, the microscope
includes a transmitted illumination optical system including a
light source 1111 and condenser lens 1113, and an epifluorescence
illumination system including a mercury lamp 1114, an
epi-fluorescence emitting tube 1115, and a fluorescence filter
cassette 1119.
[0148] Illumination light from the light source 1111 illuminates
the observation object 1103 and cover glass 1104 through
transmitted illumination optical elements provided in a transmitted
illumination pillar 1112 and the condenser lens 1113.
[0149] Of illumination light from the mercury lamp 1114 for
epi-fluorescence illumination, only light having a wavelength which
can efficiently excite a fluorescent dye staining the cell in the
observation object 1103 is transmitted through an excitation filter
1116 provided in the fluorescence filter cassette 1119 through the
epi-fluorescence emitting tube 1115 to become excitation light.
This light is coaxially guided along an observation optical axis
1108 through a dichroic mirror 1117 and is reflected toward the
objective lens 1105 to excite the fluorescent dye staining the
observation object 1103 through the objective lens 1105. In this
case, four types of each of fluorescence filters, including the
excitation filter 1116, the dichroic mirror 1117, and an absorption
filter 1118, are mounted in the fluorescence filter cassette 1119.
These filters can be inserted/withdrawn on/from the optical path
and switched in accordance with the fluorescent dye to be used by a
known switching mechanism such as a turret system.
[0150] The observation object 1103 placed on the cover glass 1104
is placed on the X-Y stage 1129 and can be moved to a desired
observation position of the observation object 1103 by operating an
X-Y handle (not shown). The objective lens 1105 which projects an
image of the observation object 1103 at infinity is screwed into
the revolver 1106 below the observation object 1103. Although only
one objective lens 1105 is mounted in the revolver in FIG. 13, five
objective lenses can be mounted in the revolver. When a button or
the like provided on an input unit 1172 is pressed, the outer
surface of the revolver is rotated by a motor 1174 under the
control of a control unit 1138, thereby switching to a desired
objective lens. Although not shown, the rotational position of a
revolver 1106 is detected by a sensor to allow the control unit
1138 to recognize an objective lens on the optical path. The
revolver 1106 is held on a vertical guide 1125 through a revolver
raising member 1126 and is vertically moved by a motor 1123 so as
to focus the observation object 1103. That is, the revolver 1106,
revolver raising member 1126, vertical guide 1125, and motor 1123
constitute a focusing driving unit. Although the motor 1123 is
controlled by the control unit 1138, the revolver can be vertically
moved by rotating a focusing handle 1124.
[0151] Parallel light which exists from the objective lens 1105 and
is projected at infinity is formed into an image on objective lens
primary image planes 1130a and 1130b by an imaging lens 1120. In
the case of fluorescence observation, the fluorescence emitted from
the observation object 1103 exits from the objective lens 1105 and
is transmitted through the dichroic mirror 1117. Of this light,
light having a wavelength necessary for observation is selectively
transmitted through the absorption filter 1118, guided to the
imaging lens 1120, and formed into an image on the objective lens
primary image planes 1130a and 1130b.
[0152] The optical path switching prism 1121 is detachably held on
the observation optical axis 1108. In observation with the CCD
1136, the optical path switching prism 1121 is inserted on the
observation optical axis 1108, an objective lens primary image
1130b is reflected by the optical path switching prism 1121 and can
be observed through the CCD 1136. In observation with the eyes, the
optical path switching prism 1121 is removed from the observation
optical axis 1108, and an objective lens primary image plane 1133a
is reflected by the optical path switching prism 1121 toward the
eyepiece lens 1110. The objective lens primary image plane is
relayed by the relay lens 1109 to be observed through the eyepiece
lens 1110.
[0153] Transmitted illumination observation is the same as
fluorescence observation except that observation is performed upon
withdrawal of a fluorescence filter from the optical path.
[0154] The arrangement and function of the microscope focus
maintaining device will be described next.
[0155] The microscope focus maintaining device roughly comprises a
sensor head 1137 which performs focus detection with a laser light
beam, and a microscope-side optical path switching unit which
optically couples the sensor head 1137 to the objective lens 1105.
The microscope-side optical path switching unit will be described
first.
[0156] The optical path switching unit includes a dichroic mirror
1134 and a movable guide 1132 which movably holds the dichroic
mirror 1134 on a fixed guide 1131.
[0157] The dichroic mirror 1134 has the property of reflecting only
800-nm laser light from the sensor head 1137 and transmitting all
visible light necessary for observation. The dichroic mirror 1134
coaxially guides a laser light beam from a sensor head optical axis
1170 of the sensor head 1137 to the observation optical axis 1108
and reflects it toward the objective lens 1105. The dichroic mirror
1134 is fixed to the movable guide 1132 with adhesive, screws, or
the like. An IR cut filter is bonded and fixed to the movable guide
1132 at a position below the dichroic mirror 1134. In order to
prevent laser light from entering the eyes, this IR cut filter cuts
800-nm laser beam and transmits only visible light necessary for
observation.
[0158] The movable guide 1132 is designed to be capable of moving
in the direction indicated by the arrow shown on the left side of
FIG. 13 (in the back-and-forth direction relative to the observer)
with respect to a guide portion 1131a of the fixed guide 1131
through a guide portion 1132a. By operating an operation lever 1133
back and forth, the dichroic mirror 1134 and an IR cut filter 1135
can be inserted/withdrawn on/from the observation optical axis
1108. A light-shielding plate 1171 is fixed to the movable guide
1132. When the dichroic mirror 1134 is withdrawn from the optical
path as indicated by the left portion of FIG. 13, the
light-shielding plate 1171 is inserted onto the sensor head optical
axis 1170 to prevent a laser light beam from exiting to the right
side of a microscope body 1169. There is therefore no possibility
of causing any harmful laser light to strike the observer.
[0159] Note that the fixed guide 1131 is held on the microscope
body 1169 while being fixed to a lower front side stage raising
member 1127a and lower back side stage raising member 1127b with
screws, and holds and fixes the sensor head 1137 through a fitting
portion 1150 of the sensor head 1137.
[0160] The switching mechanism for the dichroic mirror 1134 and the
mount of the sensor head 1137 are arranged between the revolver
1106 and the fluorescence filter cassette 1119 in this manner. In
order to ensure a space for this arrangement, the revolver 1106 is
raised by the revolver raising member 1126 as described above, and
the X-Y stage 1129 and transmitted illumination pillar are also
raised. The X-Y stage 1129 is raised by the lower side stage
raising members 1127a and 1127b fixed to the microscope body 1169
with screws and by upper side stage raising members 1128a and 1128b
fixed to the lower side stage raising members 1127a and 1127b with
screws. The X-Y stage 1129 is fixed to the upper side stage raising
members 1128a and 1128b with screws.
[0161] In this case, the raising members constitute two units for
the following reason. If the above raising operation is to be
performed by using the screws with which the X-Y stage 1129 is
fixed to the microscope body 1169, the raising members must be
fastened together with the X-Y stage 1129. As a consequence, just
unfastening the X-Y stage 1129 will unfasten the raising members
and also unfasten the fixed guide 1131 and sensor head 1137 fixed
to the raising members. This makes it impossible to stably maintain
the focus because the optical center of the sensor head 1137
deviates from that of the microscope body 1169.
[0162] The sensor head 1137 will be described next with reference
to FIGS. 15A and 15B.
[0163] The sensor head 1137 includes a laser diode (LD) 1145, a
beam diameter restriction stop 1144 which restricts the diameter of
a laser light beam from the LD 1145, a pupil division stop 1142
which changes a circular beam from the beam diameter restriction
stop 1144 into a semicircular beam, a mirror 1140 which deflects
the laser light beam, and an imaging lens 1139 which collimates the
laser light beam. The sensor head 1137 further includes a beam
splitter 1141 which splits exit light from return light, a mirror
1148 which deflects the return light beam from the beam splitter
1141, and a photodiode (PD) 1149 which detects the return light.
The sensor head 1137 further includes a high-NA objective lens
dedicated beam restriction stop 1143 and a motor 1175 for properly
placing the stop on an optical path.
[0164] An 800-nm laser light beam 1146a emitted from the laser
diode 1145 is restricted to a necessary beam angle by a beam
diameter restriction stop 1144 having a circular opening in its
center, and the circular beam is restricted to a semicircular beam
by a pupil division stop 1142. This laser light beam is transmitted
through the beam splitter 1141, reflected by the mirror 1140, and
collimated by the imaging lens 1139. Referring to FIG. 15A,
reference numeral 1146b denotes a light beam on the laser
projection side. In this case, the beam diameter restriction stop
1144 prevents a focus detection error due to flare or the like by
restricting unnecessary light beams of the laser light beams
exiting from the LD 1145.
[0165] The collimated laser light beam exiting from the sensor head
1137 is reflected by the dichroic mirror 1134 and condensed by the
objective lens 1105. This laser light beam is then reflected by the
boundary surface between the cover glass 1104 and the observation
object 1103, and returns to the sensor head 1137 through the
objective lens 1105 and dichroic mirror 1134. Referring to FIG.
15A, reference numeral 1146c denotes a laser light beam which is
reflected by the observation sample and has returned therefrom, and
indicates an optical path on the detection side.
[0166] The laser light which has returned to the sensor head 1137
is reflected by the mirror 1140, beam splitter 1141, and mirror
1148, and is condensed and strikes the photodiode 1149 serving as a
light-receiving element having a photoelectric conversion function.
In this case, a pupil division stop 1147 is placed on the
detection-side optical path to cut any laser light beam harmful to
focus detection, e.g., flare produced midway along the optical
path.
[0167] As is obvious from the above description, in the microscope
focus maintaining device of this embodiment, laser light used for
focus detection is guided from a side closer to the objective lens
1105 than the epi-fluorescence illumination system. More
specifically, the laser light used for focus detection is guided to
the objective lens 1105 by the dichroic mirror 1134 placed between
the objective lens 1105 and the fluorescence filter cassette
1119.
[0168] When the conventional focus maintaining device based on the
TTL system is applied to a fluorescence microscope, light used for
focus detection is guided from a side closer to the image than the
fluorescence illumination device. For this reason, the focus
detection optical path passes through the fluorescence filter
cassette. In general, invisible light which is not generally used
for observation is used as light used for focus detection so as not
to become stray light to observation light. The fluorescence filter
cassette generally has the property of not transmitting light with
wavelengths other than the wavelength required for fluorescence
observation. For this reason, the fluorescence filter cassette may
not transmit light used for focus detection. In such a case, focus
detection cannot be done. The fluorescence filter cassette may be
withdrawn from the optical path at the time of focus detection.
This, however, causes a loss of time, resulting in a decrease in
throughput in a case wherein information about many places in an
observation object is to be obtained or time lapse images are to be
obtained at short time intervals, in particular.
[0169] In this embodiment, laser light used for focus detection is
guided from a side closer to the objective lens 1105 than the
epi-fluorescence illumination system, and hence is not transmitted
through the fluorescence filter cassette. Therefore, there is only
a small loss of focus detection light which strikes the
light-receiving element for focus detection. This makes it possible
to stably maintain the focus.
[0170] In addition, the wavelength of laser light used for focus
detection is different from that of light used for fluorescence
observation. For this reason, there is no possibility that light
for focus detection enters as stray light fluorescence observation
light. This allows fluorescence observation with high contrast.
Furthermore, since laser light is guided by the dichroic mirror
1134, the losses of light used for focus detection and light used
for fluorescence observation can be minimized. This makes it
possible to obtain a bright observation image.
[0171] In addition, in this embodiment, light used for focus
detection is guided from between the infinite objective lens 1105
and the imaging lens 1120. More specifically, laser light used for
focus detection is guided to the objective lens 1105 by the
dichroic mirror 1134 placed between the objective lens 1105 and the
imaging lens 1120.
[0172] When a focus detection optical path is to be guided from a
side closer to the image than the imaging lens in the inverted
microscope, accurate focus detection cannot be performed in the
optical path contrast detection scheme unless a telecentric optical
system in which a chief ray is perpendicular to an image plane is
formed. On the other hand, a primary image of the objective lens
which is formed by the imaging lens is not a telecentric optical
system. For this reason, the objective lens primary image must
relayed to near the light-receiving element for focus detection so
as to form a telecentric optical system. Although a new space is
required to form a relay optical system, various devices such as an
imaging device, a manipulator for operating a cell, and a laser
stimulating device are arranged around the microscope body in the
inverted microscope, and hence it is difficult to form a focus
maintaining device in the limited space.
[0173] In a laser projection type focus detection scheme, a
light-receiving element for focus detection may be placed on an
objective lens primary image plane unlike in the optical path
difference contrast detection scheme. However, the distance from
the imaging lens to the image plane is limited to about 180 to 200
mm. Since much of this distance is in the inverted microscope body,
it is difficult in consideration of the space to form a focus
maintaining device outside the inverted microscope body. Although
this problem can be solved by relaying an objective lens primary
image, it is difficult to form a focus maintaining device within a
limited space free from interference with various devices around
the inverted microscope, as in the case of the optical path
difference contrast detection scheme described above.
[0174] In this embodiment, light used for focus detection is guided
from between the infinite objective lens and the imaging lens. In
the optical path difference contrast detection scheme, therefore,
there is no need to relay an objective lens primary image. In
addition, in the case of the laser projection type focus detection
scheme, only placing the imaging lens on the focus detection
optical path in accordance with the position of the light-receiving
element for focus detection can eliminate the necessity to relay an
objective lens primary image. In addition, since the imaging lens
can be arbitrarily positioned, the light-receiving element can be
arbitrarily positioned, thus eliminating the possibility of
interference with various devices around the inverted microscope
body.
[0175] Furthermore, the wavelength of laser light used for focus
detection is different from the wavelength of light used for
observation. This eliminates the possibility that light for focus
detection enters as stray light observation light, thus allowing
observation with high contrast. Moreover, since laser light is
guided by the dichroic mirror 1134, losses of light used for focus
detection and light used for observation can be minimized. This
makes it possible to obtain a bright observation image.
[0176] An outline of the focus detection method in this embodiment
will be briefly described next.
[0177] The PD 1149 is a two-segments photodiode whose output is
divided into two regions corresponding to the A and B phases with a
pupil division plane, as a boundary, which is perpendicular to the
direction of pupil division by the pupil division stops 1142 and
1127. The laser light beam which strikes the PD 1149 is
photoelectrically converted by the PD 1149, and the output of the
PD 1149 changes as shown in FIG. 27 together with variations in
focus position. FIG. 27 is a graph in which the ordinate represents
the outputs of the two-segments photodiode as the A and B phases;
and the abscissa, the movement of the focus in the optical axis
direction on the observation sample side. Referring to FIG. 27,
"A+B" represents the value obtained by adding the output in the A
phase and the output in the B phase.
[0178] The control unit 1138 performs various predetermined
computations on outputs from the PD 1149. At the same time, the
control unit 1138 finally performs focus detection by determining a
focus position as an in-focus position at which the A-phase output
coincides with the B-phase output in FIG. 27 by controlling the
motor 1123 for focus driving.
[0179] More specifically, first of all, a proper threshold value is
set in advance for each objective lens type or each observation
object reflectance with respect to the value of "A+B". The control
unit 1138 then monitors, while moving the focus, whether or not
"A+B" exceeds this threshold. Only after "A+B" exceeds the
threshold, computation for focus detection is performed. For
example, computation is performed to check whether (A-B)/(A+B)
becomes 0.
[0180] In this case, a focus range in which "A+B" exceeds the
threshold will be referred to as a focus capture range. If the
threshold becomes too low, the focus capture range widens, and
hence the range in which computation for focus detection is
performed widens. As a consequence, the computation time increases,
and hence it takes much time for focus detection. In addition,
since a focus signal (an output from the light-receiving element)
with a low level in a greatly defocused state must be computed,
this operation becomes relatively susceptible to noise components.
As a consequence, operation errors tend to occur.
[0181] If the threshold is too high, the focus capture range
narrows. If the focus capture range becomes too narrow, smaller
feed steps must be set in the focus direction so as not to miss the
focus capture range when it is monitored, while the focus is moved,
whether or not "A+B" exceeds the threshold (this operation will be
referred to as focus search herein after). This increases the total
focus detection time. As described above, problems arise when the
focus capture range is too narrow or wide, and hence a proper focus
capture range must be set.
[0182] There are various parameters for focus detection, e.g., the
magnification, NA, and WD of the objective lens 1105. Such
parameters include, for example, "threshold value" corresponding to
the total value of "A+B" of A-phase and B-phase outputs from the PD
1149 described with reference to FIG. 27, "focus capture range"
determined from the threshold value, "focus feed step" for
monitoring (focus search) whether or not "A+B" exceeds the
threshold, "focus search range" for focus search, "in-focus
allowable range" for final determination of an in-focus state, and
"integral time" of the PD 1149 which changes in accordance with the
power of laser light striking the PD 1149.
[0183] In this embodiment, the diameter of a laser light beam on
the pupil plane of the objective lens 1105 (between the imaging
lens 1139 and the objective lens 1105) is set to about 11 mm. With
a 10.times. objective lens, the pupil diameter is about 14 mm. With
a 100.times. objective lens, the pupil diameter is about 5 mm. With
the 10.times. objective lens, since the diameter of a laser light
beam is smaller than the pupil diameter, all the light beam strikes
the objective lens without being vignetted by the frame of the
objective lens. With the 100.times. objective lens, however, the
diameter of a laser light beam is larger than the pupil diameter,
the light beam is vignetted by the frame of the objective lens.
That is, the power of laser light finally striking the PD 1149
varies depending on whether the 10.times. objective lens or
100.times. objective lens is used. Consequently, the value of "A+B"
as an output from the PD 1149 varies. Obviously, therefore, the
above "threshold value", "focus capture range", and "integral time"
must be changed for each objective lens.
[0184] In addition, the depth of focus on the observation sample
side varies depending on the NA and magnification of an objective
lens. Obviously, therefore, "in-focus allowable range" and "focus
feed step" must be changed for each objective lens. "Focus search
range" must also be properly set for each objective lens because,
for example, as the WDs of objective lenses differ from each other,
the risk of collision between an observation sample and the distal
end of the objective lens varies. It is therefore necessary to
properly set "focus search range" for each objective lens.
[0185] The control unit 1138 automatically sets parameters for
focus detection to optimal values for a given objective lens on the
basis of the type of objective lens on the optical path which is
detected by the revolver 1106. That is, focus detection parameters
can be changed in accordance with changes in the magnifications of
the focus detection optical path and observation optical path. In
this case, the observation optical path is an optical path from the
objective lens 1105 to the CCD 1136 through the optical path
switching prism 1121.
[0186] Note that these parameters for focus detection vary in
proper value depending on the reflectance of an observation sample
other than the objective lens. In this embodiment, however, since
laser light reflected by the boundary surface between the cover
glass 1104 and the observation object 1103 is detected, the
reflectance does not greatly vary depending on the observation
sample, and the focus detection parameters are not changed. When,
however, observation samples which greatly differ in reflectance
are to be observed, the parameters must be changed and set
independently to proper values.
[0187] The high-NA objective lens dedicated beam restriction stop
1143 will be described next.
[0188] As described above, focus detection is performed by using
the reflection of laser light by the boundary surface between the
cover glass 1104 and the observation object 1103. However, a laser
light beam with NA larger than the refractive index of an
observation object is totally reflected by the boundary surface
between the cover glass 1104 and the observation object 1103. The
reflectance of the observation object is several hundred times
higher than that of laser light with NA smaller than the refractive
index of the observation object. For example, the NA of an oil
objective lens with a magnification of about 60.times. or
100.times. is about 1.45. Since an observation object (e.g., a cell
or culture solution) has a refractive index of about 1.33 to 1.38,
laser light with an NA of 1.38 to 1.45 is totally reflected by the
boundary surface between the cover glass 1104 and the observation
object 1103. Laser light with an NA within this range, in
particular, tends to become stray light due to scattering by the
frame of the objective lens or the like because the light is
transmitted through a region around the pupil of the objective
lens, i.e., a portion inside the objective lens which is near its
frame. The generated stray light adversely affects the focus
precision.
[0189] If, therefore, the objective lens inserted on the optical
path by the revolver 1106 is detected and has an NA larger than the
refractive index of the observation object, the control unit 1138
inserts the high-NA objective lens dedicated beam restriction stop
1143 in the optical path through the motor 1175. The high-NA
objective lens dedicated beam restriction stop 1143 restricts only
light beams with NAs larger than the refractive index of the
observation object, and hence no flare or the like is generated,
thus eliminating adverse influences on the focus precision.
[0190] In other words, the diameter of a laser light beam used for
focus detection is equivalent to an NA smaller than the refractive
index of the observation object. If the NA of the objective lens is
larger than the refractive index of the observation object, the
high-NA objective lens dedicated beam restriction stop 1143 is
inserted in the optical path by the control unit 1138 and motor
1175 to make the diameter of a laser light beam smaller than the
refractive index of the observation object. The high-NA objective
lens dedicated beam restriction stop 1143 is a beam diameter
restriction stop for reducing the diameter of a laser light beam.
The motor 1175 and control unit 1138 constitute a stop control unit
which properly places the high-NA objective lens dedicated beam
restriction stop 1143 on the optical path of a laser light beam. In
addition, the high-NA objective lens dedicated beam restriction
stop 1143, motor 1175, and control unit 1138 constitute a beam
diameter changing unit which changes the diameter of a laser light
beam.
[0191] Even objective lenses with the same NA equivalent to the
refractive index of an observation object differ in pupil diameter
if they have different magnifications. In this case, therefore,
high-NA objective lens dedicated beam restriction stops 1143 with
different stop diameters must be prepared. Referring to FIG. 15A,
although only one type of high-NA objective lens dedicated beam
restriction stop 1143 is prepared, high-NA objective lens dedicated
beam restriction stops with different stop diameters may be
prepared to be inserted/withdrawn on/from the optical path.
[0192] In this embodiment, since the diameter of a laser light beam
used for focus detection is equivalent to an NA smaller than the
refractive index of an observation object, the laser light beam is
not totally reflected by the boundary surface between a transparent
member (e.g., the cover glass or slide glass) and the observation
object. This suppresses the generation of stray light. That is,
stray light originating from a laser light beam is minimized. This
makes it possible to reduce noise on a focus signal, thus
maintaining the focus more stably.
[0193] In addition, only when the NA of an objective lens is larger
than the refractive index of an observation object, the diameter of
a laser light beam is changed to become smaller than the refractive
index of the observation object. When, therefore, an objective lens
having an NA larger than the refractive index of the observation
object and a large pupil diameter is used, a laser light beam with
a large diameter can be ensured in accordance with the pupil
diameter can be ensured, and a laser light beam with sufficient
power for focus detection can be applied to an observation sample.
This makes it possible to stably maintain the focus.
[0194] The influence of a temperature change at the time of focus
maintenance will be described next.
[0195] As the imaging lens 1139 which collimates a laser light beam
exiting from the LD 1145 and condenses the laser light beam onto
the PD 1149, a lens which has the same curvature and thickness and
is made of the same material as those of the imaging lens 1120 in
the microscope body 1169 is used. Therefore, the emission point of
the LD 1145 is optically conjugate to the objective lens primary
image planes 1130a and 1130b. That is, even if an objective lens
primary image plane moves (defocuses) in the optical axis direction
due to a change in the temperature of the lens, since the objective
lens primary image plane or the focus detection optical path and
observation optical path move by the same amount, no relative focus
shift occurs between the focus detection optical path and the
observation optical path as long as the microscope focus
maintaining device operates normally. This makes it possible to
maintain the focus even with a change in room temperature or a
change in temperature due to heat generated by the light source or
power supply for the microscope. In this case, the observation
optical path is applied to an optical path on the CCD 1136 side on
which observation is performed with an objective lens primary image
plane.
[0196] In this embodiment, the focus drift of the focus detection
optical system due to a temperature change is the same as that of
the observation optical system. For this reason, even if the room
temperature changes, no focus drift difference occurs between the
focus detection optical system and the observation optical system
due to a temperature change. This makes it possible to stably
maintain the focus in long-time lapse observation with a change in
room temperature. In addition, since both the focus detection
optical system and the observation optical system are formed
through the objective lens, even if a focus drift occurs on the
objective lens itself, the drift of the focus detection optical
system becomes the same as that of the observation optical system.
This makes it possible to stably maintain the focus even if the
objective lens is exchanged for a different objective lens.
[0197] In addition, since the focus detection optical system and
observation optical system are formed from identical members, the
focus drift of the focus detection optical system due to a
temperature change can be reliably and easily made equal to that of
the observation optical system.
[0198] Furthermore, since both the focus detection optical system
and the observation optical system are imaging optical systems for
objective lens primary images, the focus drift of the focus
detection optical system due to a temperature change can be made
equal to that of the observation optical system at low cost and
with space saving.
[0199] In this embodiment, since the focus drift of the focus
detection optical path due to a temperature change is equal to that
of the observation optical path, there is no need to use the
chromatic aberration correction mechanism (the chromatic aberration
correction lens group 677, chromatic aberration lens group driving
motor 683, and chromatic aberration lens driving unit 684) in the
sixth embodiment. This also applies to the following
embodiments.
[0200] A sequence in which an examiner performs time lapse
observation of a living cell by using the device of this embodiment
will be described next.
[0201] The mercury lamp 1114 serving as a light source for
fluorescence illumination is turned on in advance in the case of
fluorescence observation, or the light source 1111 is turned on in
advance in the case of transmitted illumination observation. In
addition, a desired objective lens 1105, a fluorescence filter
corresponding to a fluorescent dye staining a cell in the
observation object 1103 in the case of fluorescence observation,
and the dichroic mirror 1134 which guides a laser light beam for
focus detection are placed on the optical path in advance.
Obviously, the control unit 1138 automatically recognizes
parameters for focus detection in accordance with the type of
objective lens detected by the revolver 1106, and the high-NA
objective lens dedicated beam restriction stop 1143 is
automatically inserted/withdrawn as needed. The optical path
switching prism 1121 is placed outside the observation optical axis
1108 at first to allow the examiner to observe with the eyes.
[0202] The examiner sets the observation object 1103, placed on the
cover glass 1104, on the X-Y stage 1129. The examiner then adjusts
the focus by rotating the focusing handle 1124, and at the same
time searches for a place to be observed by moving the X-Y stage
1129. In general, the observer can observe a wider range with the
eyes than with the CCD 1136, and hence searches for a place to be
observed with the eyes. However, no problem arises if the examiner
searches for a place with the CCD 1136. The optical path switching
prism 1121 is then inserted in the observation optical axis 1108 to
switch to the optical path of the CCD 1136. By pressing a focus
maintenance button provided on the input unit 1172 connected to the
control unit 1138, the focus maintaining function is activated to
accurately focus on the boundary surface between the observation
object 1103 and the cover glass 1104, thereby displaying a
microscopic image of the observation object 1103 on the monitor of
the CCD 1136 (not shown). The observer then moves a place to be
precisely observed to the center of the monitor by using the X-Y
stage 1129.
[0203] Subsequently, the examiner operates the input unit 1172 to
input time intervals at which images of the observation object 1103
are to be acquired and a total time for a time lapse experiment.
When the examiner presses a time lapse experiment start button
provided on the input unit 1172, the focus maintaining function is
activated at the set time intervals. Thereafter, exposure on the
CCD 1136 is performed, and the resultant image is automatically
stored in a memory 1173. This cooperative operation is controlled
by the control unit 1138.
[0204] In this embodiment, the X-Y stage 1129 is manually operated.
If, however, this stage is motorized and the positions of cells in
different places are stored, the above time lapse observation can
be performed on many cells at many regions.
[0205] According to this embodiment, since microscopic images can
be acquired and stored at predetermined time intervals even if the
examiner does not always stay at the microscope, the load on the
examiner can be greatly reduced. In addition, since laser light
used for focus detection has a wavelength of 800 nm, which is
different from the wavelength of light used for fluorescence
observation and transmitted illumination observation, there is no
possibility that observation light becomes stray light.
Furthermore, since the dichroic mirror which reflects the
wavelength of laser light and transmits visible light as
observation light is used when a laser light beam used for focus
detection is to be guided to the observation optical axis, no loss
of both laser light and observation light occurs. This allows
observation with high brightness and can maintain the focus more
stably. Moreover, since a laser light beam is guided from a side
closer to the objective lens than the fluorescence filter, no laser
light is transmitted through the filter, and hence no loss of light
occurs. This makes it possible to maintain the focus more
stably.
[0206] In this embodiment, the lenses having the same optical
characteristics are used for the focus detection optical path and
observation optical path. However, identical lenses may not be used
as long as lenses exhibiting the same optical focus drift. In this
embodiment, the LD 1145 and PD 1149 are arranged on an objective
lens primary image plane, and an objective lens primary image is
observed with the CCD 1136. However, an objective lens primary
image need not always be used. If identical lenses or lenses
exhibiting the same optical focus drift are used for the focus
detection optical path and observation optical path, the same
effects as described above can be obtained.
[0207] In addition, if an observation optical path with the eyes is
formed in the same manner, the focus can be maintained in the same
manner not only by observation with the CCD 1136 but also by
observation with the eyes. Although not shown, the same arrangement
as that for observation with the eyes is formed, the same effects
as described above can be obtained regardless of whether the
observation optical path is a confocal optical system based on a
disk scan scheme or a detection optical system for a laser
microscope.
[0208] Note that a detection optical system for a laser microscope
is often mounted in place of, for example, an epi-fluorescence
illumination device or the CCD 1136. In this case, however, if the
objective lens is raised, a galvano mirror which scans a laser
light beam becomes not conjugate to the pupil position of the
objective lens, resulting in insufficiency of the amount of
peripheral light or the like. This problem can be solved by moving
a pupil projection lens which projects the galvano mirror on the
pupil of the objective lens in the optical axis direction or
designing the galvano mirror to be movable in the optical axis
direction.
[0209] In addition to optical focus drifts, there exist focus
drifts due to the thermal expansion of the holding members of
optical elements and the like. If the same material used for the
holding members of optical elements on the focus detection optical
path and observation optical path to eliminate the influences of
such focus drifts, the focus maintenance precision can be further
improved.
[0210] This embodiment has exemplified the microscope focus
maintaining device based on the pupil division laser projection
scheme. If, however, a focus detection optical path and observation
optical path are independent of each other as in the case of a
confocal scheme in which a pinhole is formed in an image plane to
detect a focus position where the amount of light transmitted
through the pinhole is maximized, the same function and effect can
be obtained as described above.
EIGHTH EMBODIMENT
[0211] The eighth embodiment of the present invention will be
described next with reference to FIGS. 16A and 16B. This embodiment
is directed to a sensor head which can replace the sensor head in
the seventh embodiment. FIG. 16A is a plan view of the sensor head
according to the eighth embodiment of the present invention. FIG.
16B is a front view of the sensor head shown in FIG. 16A.
[0212] A microscope and an optical path switching unit on the
microscope side of a microscope focus maintaining device are the
same as those in the seventh embodiment shown in FIG. 13, and a
sensor head 1137 differs only partly from that in the seventh
embodiment. Therefore, only different portions will be described
below. With regard to other arrangements, the same reference
numerals as in the eighth embodiment denote the same parts in the
seventh embodiment, and a detailed description thereof will be
omitted.
[0213] For the sensor head 1137 in this embodiment, beam diameter
restriction stops 1144a and 1144b with different stop diameters are
arranged immediately after an LD 1145 in place of the high-NA
objective lens dedicated beam restriction stop 1143 in the seventh
embodiment. Although FIG. 13 representatively shows the two beam
diameter restriction stops 1144a and 1144b, the number of beam
diameter restriction stops is not limited to two. The stop diameter
of each of the beam diameter restriction stops 1144a and 1144b
coincides with the pupil of a corresponding objective lens to be
paired. A control unit 1138 inserts one of the beam diameter
restriction stops 1144a and 1144b which has a stop diameter equal
to the pupil diameter of the objective lens on the optical path
into the optical path through a motor 1175 in accordance with
information indicating the type of objective lens on the optical
path which is detected by a revolver 1106.
[0214] In other words, the beam diameter restriction stops 1144a
and 1144b have openings with diameters coinciding with the pupils
of objective lens prepared in advance which can be placed on the
optical path. Of the beam diameter restriction stops 1144a and
1144b, a beam diameter restriction stop having an opening with the
same diameter as the pupil diameter of the objective lens on the
optical path is selectively placed on the optical path by the
control unit 1138 and motor 1175. That is, the focus detection
optical system can change the diameter of a laser light beam, and
changes the diameter of a laser light beam to a diameter equal to
the pupil diameter of the objective lens. The control unit 1138 and
motor 1175 constitute a stop control unit which selectively places
one of the beam diameter restriction stops 1144a and 1144b on the
optical path of a laser light beam from the LD 1145. The beam
diameter restriction stops 1144a and 1144b, motor 1175, and control
unit 1138 constitute a beam diameter changing unit which changes
the diameter of a laser light beam.
[0215] If the stop diameter of a beam diameter restriction stop
1144, i.e., the pupil diameter of an objective lens, is large, the
power of the LD 1145 is decreased, otherwise the power of the LD
1145 is increased. In this manner, the control unit 1138 controls
the power of laser light striking an objective lens 1105 to be
constant.
[0216] In other words, the control unit 1138 forms a power changing
unit which changes the power of the LD 1145, and changes the power
of the LD 1145 to keep the power of a laser light beam striking the
objective lens on the optical path constant regardless of the
diameter of the laser light beam.
[0217] According to conventional laser projection type focus
detection, the diameter of a laser light beam and laser power are
constant regardless of the pupil diameter of an objective lens.
When laser light reflected by a surface to which a cover glass,
slide glass, or the like adheres like a biological observation
sample is to be detected, the reflectance may be too low, and the
power of laser light striking a light-receiving element for laser
detection may be insufficient. When an objective lens having a
small pupil diameter is to be used, in particular, only part of a
projected laser light beam enters the objective lens. For this
reason, the power of laser light striking the observation sample is
too low for focus detection.
[0218] This problem can be solved by increasing the output level of
the laser light source itself. If, however, the laser power is
increased, a living cell in the observation sample is more damaged,
and the degree of risk of trouble to the skin or eyes of an
observer by laser light increases. In addition, the apparatus
requires a safety device for avoiding this, and hence increases in
size and cost. Assume that the power of a laser serving as a light
source is increased in accordance with an objective lens having a
small pupil diameter through which the power of laser light
striking a light-receiving element for laser detection becomes low.
In this case, when this objective lens is replaced with an
objective lens with a large pupil diameter or is removed, laser
light with higher power strikes the observation sample. This
further increases damage to a cell or the degree of risk of trouble
to the observer.
[0219] In this embodiment, the diameter of a laser light beam and
the laser power of the light source can be changed in accordance
with the pupil diameter of an objective lens. This makes it
possible to maintain the laser power which allows focus detection
while minimizing the power of laser light which may strike a cell
or examiner. That is, the embodiment can maintain the laser power
which allows focus detection while minimizing damage to a cell and
avoiding trouble to the examiner by laser light.
[0220] In addition, since the diameter of a laser light beam is
made equal to the pupil diameter of an objective lens, the whole
projected laser light beam is transmitted through the objective
lens without being vignetted by the objective lens regardless of
the objective lens to be used. This can prevent noise from being
superimposed on a focus signal due to flare generated by frame
reflection or the like.
[0221] Furthermore, the laser power of the LD 1145 is changed so as
not to change the power of laser light striking an objective lens
even with a change in the diameter of a laser light beam. This can
make the power of laser light on an observation sample constant
regardless of the objective lens to be used, and can maintain the
laser power which allows focus detection while minimizing the power
of laser light which may strike a cell or examiner. That is, this
embodiment can maintain the laser power which allows focus
detection while minimizing damage to a cell and avoiding trouble to
an examiner by laser light.
[0222] According to the above arrangement and function, since the
power of laser light striking an objective lens is kept constant
regardless of the type of objective lens, i.e., the pupil diameter,
a laser light beam is not vignetted by the frame of even an
objective lens with a small pupil diameter as compared with a case
wherein the diameter of a laser light beam is set in accordance
with an objective lens with a relatively large pupil diameter as in
the seventh embodiment. For this reason, higher laser power can be
used for focus detection, and hence the focus can be maintained
stably.
[0223] In addition, there is conceivable a method of making the
power of laser light striking an objective lens constant by
changing only the laser power in accordance with the pupil diameter
of the objective lens while keeping a beam diameter constant. In
this method, however, since the power of laser light exiting from
the objective lens mount screw holes of the revolver 1106 is not
constant, if an objective lens is erroneously removed while the
laser power is high, the human body may be damaged by the laser
power. In this embodiment, however, since the power of laser light
exiting from such a screw hole is kept always constant even if the
objective lens is removed, there is no danger as long as the laser
power is set in advance to safe power for the human body.
[0224] In this embodiment, one of the beam diameter restriction
stops 1144 is selected and inserted/withdrawn on/from the optical
path to restrict the beam diameter. However, the same function and
effect can be obtained even by using a beam diameter restriction
stop whose stop diameter can be changed.
[0225] In addition, in this embodiment, the beam diameter is
restricted by using different stop diameters to make the power of
laser light striking an objective lens constant. As in the seventh
embodiment, however, the type of objective lens placed on the
optical path may be recognized to set a beam diameter which
prevents total reflection at the boundary surface between a cover
glass 1104 and an observation object 1103 as needed. In this case
as well, the same effects as described above can be obtained by
keeping the power of laser light striking the objective lens
constant.
MODIFICATION TO EIGHTH EMBODIMENT
[0226] A modification to the eighth embodiment will be described
next.
[0227] In the eighth embodiment, the stop diameter of a beam
diameter restriction stop can be changed in accordance with the
pupil diameter of an objective lens. In this modification, however,
the stop diameter of a beam diameter restriction stop is set in
accordance with one of objective lenses used in combination which
has the smallest pupil diameter. That is, the diameter of a laser
light beam is equal to the pupil diameter of one of objective
lenses prepared in advance which is smallest.
[0228] As a consequence, the power of laser light striking an
objective lens is constant regardless of the pupil diameter of the
objective lens, and the same effects as those in the eighth
embodiment can be obtained. In addition, even if the objective lens
is removed, the laser power remains constant, and the same effects
as describe above can be obtained. In addition, even if the
objective lens is removed, the laser power remains constant and the
beam diameter restriction stop and the power of an LD 1145 remain
unchanged. This improves the reliability and provides safety for
the human body. In the case of an objective lens with a low
magnification and large pupil diameter, since a laser light beam
does not coincide with the pupil, the depth of focus increases, and
the focus detection precision slightly decreases. However, the
focus drift of a low-magnification objective lens due to a change
in room temperature is small. In addition, in time lapse
observation, since a high-magnification objective lens with a small
pupil diameter is mainly used, no practical problems arise.
NINTH EMBODIMENT
[0229] The ninth embodiment of the present invention will be
described next with reference to FIGS. 17A and 17B. This embodiment
is directed to a sensor head which can replace the sensor head in
the seventh embodiment. FIG. 17A is a plan view of the sensor head
according to the ninth embodiment of the present invention. FIG.
17B is a front view of the sensor head shown in FIG. 17A.
[0230] A microscope and an optical path switching unit on the
microscope side of a microscope focus maintaining device in this
embodiment are the same as those in the seventh embodiment shown in
FIG. 13, and a sensor head 1137 differs from that in the seventh
embodiment. Therefore, only the sensor head 1137 will be described
below. With regard to other arrangements, the same reference
numerals as in the ninth embodiment denote the same parts in the
seventh embodiment, and a detailed description thereof will be
omitted.
[0231] The sensor head 1137 in this embodiment is based on a scheme
of achieving an in-focus state by detecting the contrast of an
image unlike the laser projection type microscope focus maintaining
devices according to the seventh and eighth embodiments. Referring
to FIG. 17A, an imaging lens 1139 is identical to the imaging lens
1120 incorporated in the microscope in the seventh embodiment.
[0232] Of transmitted illumination observation light or
fluorescence observation light exiting from an objective lens 1105,
light with wavelengths of 800 nm or more which is reflected by a
dichroic mirror 1134 is condensed by the imaging lens 1139 and
reflected by a mirror 1151. Part of this light is transmitted
through a beam splitter 1152 to form an objective lens primary
image 1155a at a position slightly away from a CCD line sensor
1154. On the other hand, the light reflected by the beam splitter
1152 is reflected by a mirror 1153 to form an objective lens
primary image 1155b at a position slightly before the CCD line
sensor 1154.
[0233] A focus detection method in this arrangement will be
described briefly. The focus detection method is disclosed in, for
example, Jpn. Pat. Appln. KOKAI Publication No. 6-78112.
[0234] The CCD line sensor 1154 is roughly divided into two regions
with the center of the interval between the optical axis of the
objective lens primary image 1155a and the optical axis of the
objective lens primary image 1155b being a boundary. The objective
lens primary image 1155a side will be referred to as near focus;
and the objective lens primary image 1155b side, far focus. A
control unit 1138 can compute a contrast by calculating the
luminance difference between adjacent pixels in each of the
near-focus and far-focus regions of the CCD line sensor 1154.
[0235] Since the objective lens primary images 1155a and 1155b are
formed at equal distances from the CCD line sensor 1154, focus
detection is performed by determining, as an in-focus position, a
position where the contrast at the near focus becomes equal to that
at the far focus. In practice, the control unit 1138 controls focus
driving by a motor 1123 to allow a CCD 1136 to image and store a
time lapse image in an in-focus state.
[0236] If the observation wavelength is shorter than 800 nm in the
case of fluorescence observation, when an in-focus state is
achieved upon focus detection with transmitted illumination
observation light, imaging may be performed by the CCD 1136 upon
switching to fluorescence observation by inserting a fluorescence
filter in an optical path. Alternatively, even when fluorescence
observation is performed with a wavelength of 800 nm or more, the
above operation may be done if no excitation light is to be applied
to a cell in an observation object 1103 to reduce damage to the
cell.
[0237] In this embodiment, with the above arrangement and function,
the same effects as those in the seventh embodiment can be obtained
even with the optical path difference contrast scheme. The above
combination is effective for an observation sample or experiment
for which it is convenient to determine a position where the
contrast is high as an in-focus position.
10TH EMBODIMENT
[0238] The 10th embodiment of the present invention will be
described next with reference to FIGS. 18, 19A, and 19B. This
embodiment is directed to a microscope including a microscope focus
maintaining device. FIG. 18 shows the overall arrangement of the
microscope according to the 10th embodiment of the present
invention. Referring to FIG. 18, a side surface of the microscope
is shown on the left side, and portions around the objective lens
of the microscope are shown on the right side. FIG. 19A is a plan
view of a sensor head shown on the right side of FIG. 18. FIG. 19B
is a front view of the sensor head shown in FIG. 19A.
[0239] An optical path switching unit on the microscope side of a
microscope focus maintaining device is the same as that in the
seventh embodiment, and the microscope is also the same as that of
the seventh embodiment except that transmitted illumination
observation is limited to differential interference observation. A
sensor head 1137 differs only partly from that in the seventh
embodiment. Therefore, only different portions will be described
below. With regard to other arrangements, the same reference
numerals as in the 10th embodiment denote the same parts in the
seventh embodiment, and a detailed description thereof will be
omitted.
[0240] The arrangement and function of differential interference
observation will be described with reference to FIG. 18.
[0241] In addition to the arrangement of the seventh embodiment
described above, on a transmitted illumination optical axis 1101, a
polarizer 1156 is placed above a condenser lens 1113, and an
illumination-side DIC prism 1157 is placed in the condenser lens
1113, whereas on an observation optical axis 1108, an
observation-side DIC prism 1158 is placed in a revolver 1106, and
an analyzer 1159 is placed such that its vibration direction is
perpendicular to the polarizer 1156. The observation-side DIC prism
1158 is held by the revolver 1106 so as to be inserted/withdrawn
on/from the observation optical axis 1108, and can be moved in a
direction perpendicular to the observation optical axis 1108 by
rotating a contrast adjustment knob 1160. This makes it possible to
adjust the contrast of a DIC image by changing the retardation.
That is, the observation-side DIC prism 1158 forms a retardation
changing element for differential interference observation. In
addition, insertion/withdrawal of the observation-side DIC prism
1158 on/from the observation optical axis 1108 and a change in
retardation by the contrast adjustment knob 1160 are detected by
sensors 1161 and 1162 and can be recognized by a control unit
1138.
[0242] In this arrangement, the illumination light emitted from a
light source 1111 of a transmitted illumination pillar 1112 is
converted into linearly polarized light by the polarizer 1156, made
to cause a predetermined retardation by the observation-side DIC
prism 1158, and split into ordinary light and extraordinary light
to illuminate an observation object 1103 and cover glass 1104. An
image from the observation object 1103 is projected at infinity by
an objective lens 1105, and is made to cause a predetermined
retardation by the observation-side DIC prism 1158. The ordinary
light and extraordinary light are combined and transmitted through
the analyzer 1159. Subsequently, this light can be observed with
the CCD 1136, the eyes, or the like as in the case of transmitted
illumination observation in the seventh embodiment.
[0243] The arrangement of the sensor head 1137 will be described
next with reference to FIGS. 19A and 19B.
[0244] The 10th embodiment differs from the seventh embodiment in
that a beam diameter restriction stop 1144 is fixed for the sake of
simplicity, the beam splitter 1141 is replaced with a polarizing
beam splitter (PBS) 1141b, and a .lamda./4 plate 1163 is placed
closer to the imaging lens side than the PBS 1141b. The .lamda./4
plate 1163 can be rotated and controlled by a motor 1164 under the
control of the control unit 1138. The .lamda./4 plate 1163, motor
1164, and control unit 1138 constitute a retardation correction
device which generates a retardation for canceling out the
retardation caused by the observation-side DIC prism 1158. The PBS
1141b has the property of transmitting linearly polarized light in
the direction indicated by the arrow which is parallel to the
drawing surface of FIG. 19A and reflects linearly polarized light
in a direction perpendicular to the drawing surface.
[0245] The function of the sensor head with this arrangement will
be described in association with a case wherein no differential
interference observation is performed.
[0246] A laser light beam exiting from an LD 1145 is linearly
polarized light in the direction indicated by the arrow in FIG.
19A. This laser light beam is transmitted through the beam diameter
restriction stop 1144, a pupil division stop 1142, and the PBS
1141b and reflected by a mirror 1140. The light beam is then
transmitted through the .lamda./4 plate 1163 to be circularly
polarized, and is collimated by an imaging lens 1139 to exit to the
objective lens 1105. In this case, if no differential interference
observation is performed, the control unit 1138 recognizes on the
basis of the sensor 1162 that the observation-side DIC prism 1158
is not placed on the optical path, and causes the motor 1164 to set
the .lamda./4 plate 1163 at a position where its optical axis tilts
at 45.degree. with respect to the polarization direction of the LD
1145.
[0247] The laser light beam which has returned from the objective
lens 1105 is condensed by the imaging lens 1139, is transmitted
through the .lamda./4 plate 1163, and the circularly polarized
light is converted into linearly polarized light perpendicular to
the polarization direction of the LD 1145. This light is reflected
by the PBS 1141b, transmitted through the pupil division stop 1147,
and strikes a PD 1149, thereby determining an in-focus position
upon focus detection in the same manner as in the seventh
embodiment. As described above, when no differential interference
observation is performed, since the laser projection optical path
is polarization-split from the detection optical path, both the
optical paths are transmitted through the PBS 1141b without any
loss. This improves the utilization efficiency of reflected laser
power.
[0248] A function in the case of differential interference
observation will be described next.
[0249] Since the internal arrangement of the sensor head 1137 is
the same as that in the case of differential interference
observation except for the rotating direction of the .lamda./4
plate 1163, a description thereof will be omitted. A laser light
beam 1146b exiting from the sensor head 1137 is reflected by a
dichroic mirror 1134, transmitted through the observation-side DIC
prism 1158, and condensed onto the observation object 1103 by the
objective lens 1105. This light beam is reflected by the boundary
surface between the cover glass 1104 and the observation object
1103 and projected at infinity by the objective lens 1105. In
addition, the light beam is transmitted through the
observation-side DIC prism 1158 and returns to the sensor head
1137.
[0250] In this case, since the light beam is transmitted through
the observation-side DIC prism 1158 twice, a retardation also
occurs here. As a consequence, if the optical axis of the .lamda./4
plate 1163 remains at 45.degree. with respect to the linearly
polarized light exiting from the PBS 1141b, when the linearly
polarized laser light beam exiting from the PBS 1141b is
transmitted through the .lamda./4 plate 1163 and observation-side
DIC prism twice and returns to the PBS 1141b, the light is not
polarized in a direction perpendicular to the linearly polarized
light exiting from the PBS 1141b. This causes a loss of laser power
when the light is reflected by the PBS 1141b.
[0251] The control unit 1138 therefore causes the motor 1164 to
rotate the .lamda./4 plate 1163 to generate a retardation which
cancels out the retardation caused by the observation-side DIC
prism 1158 and detected by the sensor 1161 of the contrast
adjustment knob 1160. As a result, the laser light beam returning
to the PBS 1141b becomes polarized light perpendicular to the
linearly polarized light exiting from the PBS 1141b. This light
beam is then reflected by the PBS 1141b without any loss and guided
to the PD 1149. Obviously, in the case of differential interference
observation, the above operation is executed after the control unit
1138 recognizes through the sensor 1162 that the observation-side
DIC prism 1158 is placed on the optical path.
[0252] In this embodiment, in order to generate a retardation which
cancels out the retardation caused upon contrast adjustment, the
.lamda./4 plate 1163 is rotatably placed between the polarization
beam splitter 1141b and the observation sample. This can prevent a
loss of the power of laser light striking a light-receiving element
for focus detection. In addition, the generated retardation is
detected by the sensors 1161 and 1162, and the retardation
generated by the .lamda./4 plate 1163 is automatically corrected by
the control unit 1138 and motor 1164 on the basis of the detected
retardation. This can prevent variations in laser power and can
properly maintain a focus capture range.
[0253] According to the above arrangement and function, since no
loss of laser power occurs on the PD regardless of whether a DIC
prism is inserted/withdrawn or contrast is adjusted, the focus can
be stably maintained.
[0254] In this embodiment, a retardation is canceled out by
rotating the .lamda./4 plate prepared in advance. However, another
.lamda./4 plate or a retardation generating element may be
used.
[0255] In this embodiment, the retardation caused by the
observation-side DIC prism 1158 is detected by the sensor 1161 of
the contrast adjustment knob 1160, and a retardation which cancels
out the detected retardation is generated. However, a retardation
may be generated such that the output of the PD 1149 is maximized.
This also generates a retardation which cancels out the retardation
caused by the observation-side DIC prism 1158.
[0256] In addition, in this embodiment, the power of laser light
striking the PD 1149 is not varied by the observation-side DIC
prism. However, a parameter for focus detection, e.g., a threshold,
may be changed in accordance with insertion/withdrawal of the
observation-side DIC prism or the power of laser light striking the
PD which is changed by a retardation.
11TH EMBODIMENT
[0257] The 11th embodiment of the present invention will be
described next with reference to FIGS. 20, 21A, and 21B. This
embodiment is directed to a microscope comprising a microscope
focus maintaining device. FIG. 20 shows the overall arrangement of
the microscope according to the 11th embodiment of the present
invention. FIG. 20 shows a side surface of the microscope on the
left side, together with portions around the objective lens of the
microscope on the right side. FIG. 21A is a plan view of a sensor
head shown on the right side in FIG. 20. FIG. 21B is a front view
of the sensor head shown in FIG. 21A.
[0258] The microscope and the optical path switching unit on the
microscope side of the microscope focus maintaining device shown in
FIG. 20 are the same as those in the seventh embodiment except for
a sensor head 1137. The sensor head 1137 differs only partly from
that in the seventh embodiment. Therefore, only different portions
will be described below. With regard to other arrangements, the
same reference numerals as in the 11th embodiment denote the same
parts in the seventh embodiment, and a detailed description thereof
will be omitted.
[0259] In the seventh embodiment, both the observation optical path
based on the CCD 1136 and the focus detection optical path use
objective lens primary images formed by the imaging lens. With this
arrangement, however, the magnification of the observation optical
path cannot be changed unless the objective lens is exchanged with
another objective lens.
[0260] In this embodiment, as shown in FIG. 21A, a variable power
lens 1165 can be inserted/withdrawn on/from the optical path in the
sensor head 1137 unlike in the seventh embodiment. In other words,
the sensor head 1137 comprises the variable power lens 1165, which
can be inserted/withdrawn on/from the optical path, in addition to
an imaging lens 1139. The variable power lens 1165 is
inserted/withdrawn on/from the optical path by a motor 1177
controlled by a control unit 1138. Referring to FIG. 21A, a beam
diameter restriction stop 1144 is fixed for the sake of descriptive
convenience. As shown in FIG. 20, a variable power lens 1165
identical to that in the sensor head 1137 is mounted in advance on
the objective lens side of the CCD 1136. The variable power lens
1165 is attached/detached in accordance with insertion/withdrawal
of the variable power lens 1165 in the sensor head 1137.
[0261] In this embodiment, both a focus detection optical system
and an observation optical system include variable power lenses
which can be inserted/withdrawn on/from the optical path. These
variable power lenses have the same magnification. This makes it
possible for the focus detection optical system and observation
optical system to change their magnifications to the same
magnification. Therefore, no focus drift difference occurs due to
changes in temperature of the focus detection optical system and
observation optical system, and the current magnification can be
changed to a desired observation magnification. This makes it
possible to stably maintain the focus and change the current
magnification to a desired observation magnification.
[0262] Since an LD 1145 and PD 1149 are kept at positions conjugate
to an image plane even when the variable power lens 1165 in the
sensor head 1137 is inserted/withdrawn on/from the optical path,
other arrangements and functions are almost the same as those in
the seventh embodiment. However, when the variable power lens 1165
is inserted/withdrawn, the NA of a laser light beam on the PD 1149
changes, and the total magnification changes. It is therefore
necessary to change parameters for focus detection. For this
reason, the control unit 1138 detects the insertion/withdrawal of
the variable power lens 1165 by the motor 1177 through a sensor
(not shown), and automatically sets optimal parameters for focus
detection. Even if the current magnification is changed to a
desired observation magnification, optimal focus detection can be
done.
[0263] According to the above arrangement and function, the
observation magnification can be changed without changing the
objective lens, and the focus can be maintained stably.
12TH EMBODIMENT
[0264] The 12th embodiment of the present invention will be
described next with reference to FIGS. 22, 23A, and 23B. This
embodiment is directed to a microscope comprising a microscope
focus maintaining device. FIG. 22 shows the overall arrangement of
the microscope according to the 12th embodiment of the present
invention. FIG. 22 shows a side surface of the microscope on the
left side, together with portions around the objective lens of the
microscope on the right side. FIG. 23A is a plan view of a sensor
head shown on the right side in FIG. 22. FIG. 23B is a front view
of the sensor head shown in FIG. 23A.
[0265] Referring to FIG. 22, the microscope portion has an
arrangement equivalent to that of the seventh embodiment except
that the revolver, the stage, the raising members for the
transmitted illumination pillar, and the dichroic mirror switching
unit are removed. That is, other arrangements and functions are
almost the same as those of the seventh embodiment.
[0266] Note, however, this embodiment differs from the seventh
embodiment in that a CCD 1136 is not directly mounted on the
microscope body but is mounted through a sensor head 1137.
[0267] The sensor head 1137 in this embodiment differs from that in
the seventh embodiment in the following points. The sensor head
itself guides light from an optical path closer to the image side
than an imaging lens 1120 incorporated in the microscope. The
imaging lens 1120 therefore also has a function of collimating a
laser light beam, and no imaging lens exists in the sensor head
1137. In addition, the CCD 1136 is mounted on a sensor head optical
axis 1170, and a mirror 1140b is a dichroic mirror which reflects
Laser light on the sensor head side and transmits observation light
toward the CCD 1136. For the sake of descriptive convenience, a
beam diameter restriction stop 1144 is fixed. Other arrangements
and functions are the same as those in the seventh embodiment.
[0268] According to the arrangement and function described above,
no raising members such as a stage are required, and an imaging
lens is shared for focus detection and observation. Therefore, the
above device can be easily formed at low cost and can be easily
mounted on the microscope afterward.
13TH EMBODIMENT
[0269] The 13th embodiment of the present invention will be
described next with reference to FIG. 24. This embodiment is
directed to a microscope comprising a microscope focus maintaining
device. FIG. 24 shows the overall arrangement of the microscope
according to the 13th embodiment of the present invention. FIG. 24
shows a side surface of the microscope on the left side, together
with portions around the objective lens of the microscope on the
right side.
[0270] The microscope portion shown in FIG. 24 in this embodiment
differs from that in the 12th embodiment in the following points. A
fluorescence filter cassette 1119 can be switched by a motor 1168.
Light from a sensor head 1137 is guided from the right side through
a dichroic mirror 1166 between the fluorescence filter cassette
1119 and an imaging lens 1120. In addition, the dichroic mirror
1166 can be switched by a motor 1167. A CCD 1136 is directly
mounted on a microscope body 1169. Other arrangements and functions
are the same as those in the 12th embodiment. Note that the sensor
head 1137 is identical to that in the seventh embodiment which is
shown in FIGS. 15A and 15B.
[0271] According to this embodiment, in the above arrangement, a
control unit 1138 inserts the dichroic mirror 1166 on the optical
path and withdraws the fluorescence filter from the optical path
when focus detection is to be performed. When exposure on the CCD
1136 is to be performed, the control unit 1138 withdraws the
dichroic mirror 1166 from the optical path and inserts the
fluorescence filter necessary for observation on the optical
path.
[0272] In a conventional focus maintaining device, a light guide
element which guides focus detection light to an observation
optical path is always located on the optical path. The light guide
element is unnecessary in period other than a focus detection
period, and causes a loss of observation light. In fluorescence
observation, in particular, observation light is weak, and hence
even a slight loss of observation light needs to be avoided in
order to realize observation with as high contrast as possible. In
addition, when fluorescence photography is to be performed with a
CCD or the like, a loss of observation light will prolong the
exposure time, resulting in quick deterioration in the color of a
fluorescent dye. This also increases damage to a cell if it is
living.
[0273] In this embodiment, the dichroic mirror 1166 is located on
the optical path only when focus detection is to be performed. This
reduces a loss of observation light, and hence allows observation
with high contrast.
[0274] It is more preferable to allow selection of whether to
enable/disable the operation of locating the dichroic mirror 1166
on the optical path only at the time of focus detection. This makes
it possible to select whether to sacrifice vibrations or a loss of
time caused by insertion/withdrawal of the dichroic mirror 1166 or
to sacrifice a loss of observation light due to the dichroic mirror
1166. This selection should be made in consideration of the purpose
of an experiment.
[0275] It is further preferable to automatically determine whether
to enable/disable the operation of locating the dichroic mirror
1166 on the optical path only at the time of focus detection, upon
switching of objective lenses and microscopic examination methods,
on the basis of the types of objective lens and microscopic
examination method. When, for example, transmitted observation is
to be performed with a high-magnification objective lens, since the
brightness of an observation image is sufficient, priority may be
given to the elimination of the influences of a loss of time and
vibrations on a deterioration in image quality. When fluorescence
observation is to be performed with a low-magnification objective
lens, since the influence of vibrations is small because of the low
magnification and an observation image is dark, priority may be
given to the minimization of a loss of fluorescence. This
eliminates the necessity to change settings during an experiment,
and hence achieves labor saving in the experiment.
[0276] According to the above arrangement and function, since the
dichroic mirror 1166 is withdrawn from the optical path during CCD
exposure, even a slight loss of observation light due to the
dichroic mirror can be prevented. In addition, when IR light with a
wavelength of 800 nm or more is to be observed, observation can be
done without reflection of light by a dichroic mirror. Furthermore,
since the fluorescence filter is withdrawn from the optical path
during focus detection, a loss of laser light due to the
fluorescence filter can be minimized. When a filter which does not
transmit laser light with a wavelength of 800 nm, e.g., a filter
for IR fluorescence observation or bandpass barrier filter, is
used, since the fluorescence filter is withdrawn from the optical
path, there is no possibility that any laser light is cut.
[0277] Note that when a laser microscope device or disk scan device
is mounted in place of an epi-fluorescence emitting tube 1115 in
this embodiment, a mirror is mounted in place of a fluorescence
filter. In this case as well, the same effects can be obtained
owing to a function similar to the fluorescence filter. In
addition, when a laser microscope device is mounted on a portion
where the CCD 1136 is mounted, and two-photon excitation is to be
performed, excitation light with a wavelength of about 800 nm to
1,100 nm is used. If the dichroic mirror 1166 is withdrawn from the
optical path while a laser light beam is scanned to acquire an
image with the laser microscope as in the case of exposure on the
CCD 1136, there is no possibility that any excitation light is cut
by the dichroic mirror 1166.
14TH EMBODIMENT
[0278] The 14th embodiment of the present invention will be
described next with reference to FIGS. 25, 26A, and 26B. This
embodiment is directed to a microscope comprising a microscope
focus maintaining device. FIG. 25 shows the overall arrangement of
the microscope according to the 14th embodiment of the present
invention. FIG. 25 shows a side surface of the microscope on the
left side, together with portions around the objective lens of the
microscope on the right side. FIG. 26A is a side view of a sensor
head shown on the right side of FIG. 25. FIG. 26B is a front view
of the sensor head shown in FIG. 26A.
[0279] The microscope portion shown in FIG. 25 in this embodiment
differs from that in the 13th embodiment in the following points. A
sensor head 1137 from which light is guided from between a
fluorescence filter and an imaging lens is mounted on a bottom
board below a mirror 1122 on an observation optical axis 1108. This
makes it possible to omit a dichroic mirror between the
fluorescence filter and the imaging lens and a switching mechanism
for the mirror. Instead, the mirror 1122 is switched between an
optical path on the sensor head 1137 side and an observation
optical path for the eyes by a motor (not shown). For the sake of
descriptive convenience, a fluorescence mirror cassette is operated
manually instead of being motor-driven. As shown in FIGS. 26A and
26B, the sensor head 1137 is the same as that in the 12th
embodiment except that the CCD 1136 is removed and a general mirror
1140 is used in place of the mirror 1140b which is a dichroic
mirror.
[0280] According to the above arrangement and function, since the
sensor head 1137 is mounted on the bottom board, other peripheral
devices of the microscope can be freely arranged without being
hindered by the sensor head unlike in the seventh to 13th
embodiments.
[0281] 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.
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