U.S. patent application number 16/592192 was filed with the patent office on 2020-01-30 for image capturing apparatus and focusing method thereof.
This patent application is currently assigned to HAMAMATSU PHOTONICS K.K.. The applicant listed for this patent is HAMAMATSU PHOTONICS K.K.. Invention is credited to Hideshi OISHI.
Application Number | 20200033564 16/592192 |
Document ID | / |
Family ID | 51209205 |
Filed Date | 2020-01-30 |
![](/patent/app/20200033564/US20200033564A1-20200130-D00000.png)
![](/patent/app/20200033564/US20200033564A1-20200130-D00001.png)
![](/patent/app/20200033564/US20200033564A1-20200130-D00002.png)
![](/patent/app/20200033564/US20200033564A1-20200130-D00003.png)
![](/patent/app/20200033564/US20200033564A1-20200130-D00004.png)
![](/patent/app/20200033564/US20200033564A1-20200130-D00005.png)
![](/patent/app/20200033564/US20200033564A1-20200130-D00006.png)
![](/patent/app/20200033564/US20200033564A1-20200130-D00007.png)
![](/patent/app/20200033564/US20200033564A1-20200130-D00008.png)
![](/patent/app/20200033564/US20200033564A1-20200130-D00009.png)
![](/patent/app/20200033564/US20200033564A1-20200130-D00010.png)
View All Diagrams
United States Patent
Application |
20200033564 |
Kind Code |
A1 |
OISHI; Hideshi |
January 30, 2020 |
IMAGE CAPTURING APPARATUS AND FOCUSING METHOD THEREOF
Abstract
In the image capturing apparatus, the optical path difference
producing member is disposed on the second optical path. Thereby,
it is possible to suppress the amount of light when an optical
image which is focused at the front of an optical image made
incident into the first imaging device (front focus) and an optical
image which is focused at the rear thereof (rear focus) are
respectively imaged at the second imaging device and also to secure
the amount of light on image pickup by the first imaging device.
Further, in the image capturing apparatus, a position of the first
imaging region and a position of the second imaging region on the
imaging area are reversed with respect to the axis P in association
with reversal of a scanning direction of the sample. Therefore,
despite the scanning direction of the sample, it is possible to
obtain a deviation direction of the focus position under the same
conditions.
Inventors: |
OISHI; Hideshi;
(Hamamatsu-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HAMAMATSU PHOTONICS K.K. |
Hamamatsu-shi |
|
JP |
|
|
Assignee: |
HAMAMATSU PHOTONICS K.K.
Hamamatsu-shi
JP
|
Family ID: |
51209205 |
Appl. No.: |
16/592192 |
Filed: |
October 3, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15889332 |
Feb 6, 2018 |
|
|
|
16592192 |
|
|
|
|
14398011 |
Oct 30, 2014 |
9921392 |
|
|
PCT/JP2013/050853 |
Jan 17, 2013 |
|
|
|
15889332 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
H04N 5/23212 20130101;
G02B 21/244 20130101; G02B 13/0055 20130101 |
International
Class: |
G02B 13/00 20060101
G02B013/00; G02B 21/24 20060101 G02B021/24; H04N 5/232 20060101
H04N005/232 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 19, 2011 |
JP |
2011-277539 |
Claims
1. An apparatus for capturing an image of a sample, the apparatus
comprising: a stage configured to support a sample; an objective
lens configured to face to the sample; a stage control unit
configured to move the stage at a moving speed; a beam splitter
optically coupled and configured to divide an optical image of at
least a portion of the sample into a first optical image and a
second optical image; a first image sensor configured to capture
the at least a portion of the first optical image; a second image
sensor configured to capture the at least a portion of the second
optical image and provide image data; a focus controller configured
to analyze the image data so as to control a focus position of the
objective lens based on the analysis result; a region control unit
of a computer system, configured to set, at an imaging area of the
second image sensor, a first imaging region and a second imaging
region so as to capture the at least the portion of the second
optical image; and wherein the region control unit is configured to
reverse a position of the first imaging region and a position of
the second imaging region on the imaging area.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/889,332, filed Feb. 6, 2018, which is a
continuation of U.S. patent application Ser. No. 14/398,011, now
U.S. Pat. No. 9,921,32, filed Oct. 30, 2014. U.S. patent
application Ser. No. 14/398,011 is a .sctn. 371 national stage
application of PCT/JP2013/050853, filed on Jan. 17, 2013, which
claims priority to Japanese Patent Application No. 2011-277539,
filed Dec. 19, 2011. The entire contents of each of the
aforementioned applications are hereby incorporated by reference in
their entirety.
TECHNICAL FIELD
[0002] The present invention relates to an image capturing
apparatus which is used for capturing images of a sample, etc., and
also relates to a focusing method thereof.
BACKGROUND ART
[0003] Image capturing apparatuses include a virtual microscope
apparatus in which, for example, an imaging region of a sample is
in advance divided into a plurality of regions to image the divided
regions at a high magnification and, thereafter, to synthesize the
regions. In capturing images by using the virtual microscope as
described above, conventionally, as conditions for picking up
images of a sample such as a biological sample, a focus map which
covers an entire region of the sample is set to capture images of
the sample, while focus control is performed based on the focus
map.
[0004] In preparation of the focus map, at first, an image
capturing apparatus equipped with a macro optical system is used to
capture an entire sample as a macro image. Next, the thus captured
macro image is used to set an image pickup range of the sample and
also the range is divided into a plurality of regions to set a
focus obtaining position for each of the divided regions. After the
focus obtaining position has been set, the sample is transferred to
the image capturing apparatus equipped with a micro optical system
to obtain a focus position at the thus set focus obtaining
position, thereby preparing a focus map with reference to the focus
position.
[0005] However, in preparation of the above-described focus maps,
there has been a problem that processing needs time. Further,
suppression of intervals and the number of focuses to be obtained
would reduce the time necessary for the processing. In this case,
however, there has been a problem of reduction in focus accuracy.
Therefore, development of dynamic focus for capturing images of a
sample at a high magnification, with a focus position being
obtained, is now underway. The dynamic focus is a method in which a
present direction of the focus position deviating from the height
of an objective lens is detected based on a difference in light
intensity or a difference in contrast between an optical image
which is focused at the front of an optical image made incident
into an imaging device for capturing an image (front focus) and an
optical image which is focused at the rear thereof (rear focus),
thereby allowing the objective lens to move in a direction at which
the deviation is cancelled to capture an image.
[0006] A microscope system disclosed, for example, in Patent
Document 1, is provided with a second imaging unit which images a
region at the front of a region imaged by a first imaging unit, an
automatic focusing control unit which adjusts a focusing position
of an objective lens at an imaging position of the first imaging
unit based on an image picked up by the second imaging unit, and a
timing control unit which synchronizes timing at which a divided
region moves from an imaging position of the second imaging unit to
the imaging position of the first imaging unit with timing at which
an image forming position of the divided region imaged by the
second imaging unit is positioned at an imaging area of the first
imaging unit depending on a distance between the divided regions
and a speed at which a sample moves. Further, in a microscope
apparatus disclosed, for example, in Patent Document 2 or Patent
Document 3, a glass member is used to make a difference in optical
path length inside a light guiding optical system for focus
control.
CITATION LIST
Patent Literature
[Patent Document 1] Japanese Patent Application Laid-Open No.
2011-081211
[0007] [Patent Document 2] Japanese Patent Publication No.
WO2005/114287 [Patent Document 3] Japanese Patent Publication No.
WO2005/114293
SUMMARY OF INVENTION
Technical Problem
[0008] In the microscope system described in Patent Document 1, a
half mirror and a mirror are used to form an optical path
difference optical system, by which light different in optical path
length is made incident into each of two imaging regions of the
second imaging unit. In the conventional microscope system, for
example, a line sensor is used to constitute a first imaging unit
and a second imaging unit. In the line sensor, it is important to
secure an amount of light for capturing a clear image due to short
exposure time. However, in the conventional microscope system,
light is divided by the optical path difference optical system.
Thus, there is posed such a problem that it is difficult to secure
an amount of light.
Technical Problem
[0009] Further, in the conventional microscope system, an optical
surface of an optical path dividing unit is inclined, by which a
region imaged by the second imaging unit is adjusted so as to be on
the front side of the sample in a scanning direction with respect
to a region imaged by the first imaging unit, thereby capturing in
advance a direction at which the focus position will deviate.
However, the above-described configuration has difficulty in
adjusting the optical surface, which poses a problem. In
particular, where the sample changes in scanning direction, it is
necessary to adjust the optical surface before or after the change
in scanning direction, which may result in complicated adjustment
work. Further, the half mirror and the mirror are used to
constitute the optical path difference optical system. Thus, there
has also been a problem that light which has passed through the
optical path difference optical system is less likely to gather on
an imaging area of the second imaging unit.
[0010] The present invention has been made in order to solve the
above-described problems, an object of which is to provide an image
capturing apparatus which is simple in configuration and capable of
obtaining a direction at which a focus position deviates under the
same conditions, despite a scanning direction of a sample, and also
to provide a focusing method thereof.
Solution to Problem
[0011] In order to solve the above-described problems, the image
capturing apparatus of the present invention is characterized by
having a stage on which a sample is placed, a stage control unit
which scans the stage at a predetermined speed, a light source
which radiates light to the sample, a light guiding optical system
including a light dividing unit which divides an optical image of
the sample into a first optical path for capturing an image and a
second optical path for focus control, a first imaging unit which
captures a first image by a first optical image divided into the
first optical path, a second imaging unit which captures a second
image by a second optical image divided into the second optical
path, a focus control unit which analyzes the second image to
control a focus position of the image pickup by the first imaging
unit based on the analysis result, a region control unit which sets
a first imaging region and a second imaging region for capturing a
partial image of the second optical image on an imaging area of the
second imaging unit, and an optical path difference producing
member which is disposed on the second optical path to give an
optical path difference to the second optical image along an
in-plane direction of the imaging area, in which the optical path
difference producing member is disposed in such a manner that an
optical path difference of the second optical image is symmetrical
with respect to an axis of the imaging area orthogonal to a
direction at which the second optical image moves in association
with scanning of the sample, and the region control unit reverses a
position of the first imaging region and a position of the second
imaging region on the imaging area with respect to the axis in
association with reversal of the scanning direction of the
sample.
[0012] In the image capturing apparatus, the optical path
difference producing member is disposed on the second optical path.
Thereby, at the first imaging region and the second imaging region
of the second imaging unit, it is possible to image respectively an
optical image which is focused at the front of an optical image
made incident into the first imaging unit (front focus) and an
optical image which is focused at the rear thereof (rear focus). In
the image capturing apparatus, it is possible to make a difference
in optical path length without dividing light on the second optical
path for focus control. Therefore, an amount of light at the second
optical path necessary for obtaining information on a focus
position can be suppressed to secure an amount of light on the
image pickup by the first imaging unit. Further, in the image
capturing apparatus, it is possible to reverse the position of the
first imaging region and the position of the second imaging region
on the imaging area with respect to the axis of the imaging area in
association with reversal of the scanning direction of the sample.
Therefore, despite the scanning direction of the sample, it is
possible to obtain a direction at which the focus position deviates
under the same conditions.
[0013] It is also preferable that a region into which an optical
image is made incident which is conjugate to the first optical
image made incident into the first imaging unit is substantially in
alignment with the axis of the imaging area on the imaging area.
Thereby, even where the sample is reversed in scanning direction,
it is possible to keep a positional relationship between the first
imaging region, the second imaging region and the region into which
an optical image is made incident which is conjugate to the first
optical image on the imaging area.
[0014] It is also preferable that the axis of the imaging area is
substantially in alignment with a central axis of the imaging area
orthogonal to a direction at which the second optical image moves
in association with scanning of the sample. In this case, it is
possible to reliably reverse the position of the first imaging
region and the position of the second imaging region on the imaging
area in association with reversal of the scanning direction of the
sample.
[0015] Still further, it is preferable that the optical path
difference producing member is a flat plate member which is
disposed so as to overlap at least on a part of the imaging area
and that the region control unit sets the first imaging region and
the second imaging region respectively to give a region which will
overlap on the flat plate member and a region which will not
overlap on the flat plate member in order to avoid a shadow of the
second optical image by an edge part of the flat plate member. In
this case, use of the flat plate member enables the optical path
difference producing member to be simple in configuration. Further,
the edge part of the flat plate member forms the shadow of the
second optical image at the imaging area of the second imaging
device. Therefore, the first imaging region and the second imaging
region are set so as to avoid the shadow, thus making it possible
to secure accurate control of the focus position.
[0016] It is also preferable that the optical path difference
producing member is a member having a part which undergoes a
continuous change in thickness along an in-plane direction of the
imaging area and that the region control unit sets the first
imaging region and the second imaging region so as to overlap on
the part of the optical path difference producing member which is
different in thickness. In this case, adjustment of a position of
the first imaging region and that of the second imaging region
makes it possible to adjust freely an interval between the front
focus and the rear focus. Thereby, it is possible to detect a focus
position of the sample at high accuracy.
[0017] It is also preferable that there are provided an objective
lens which faces to a sample and an objective lens control unit
which controls a position of the objective lens relatively with
respect to the sample based on control by the focus control unit,
in which the objective lens control unit will not actuate the
objective lens during analysis of the focus position which is being
performed by the focus control unit and will allow the objective
lens to move with respect to the sample in one direction during
analysis of the focus position which is not being performed by the
focus control unit. In this case, since no change in positional
relationship will take place between the objective lens and the
sample during analysis of the focus position, it is possible to
secure analysis accuracy of the focus position.
[0018] It is also preferable that the region control unit sets
waiting time from image pickup at the first imaging region to image
pickup at the second imaging region based on a scanning speed of
the stage and an interval between the first imaging region and the
second imaging region. Therefore, since light from the same
position of the sample is made incident into the first imaging
region and the second imaging region, it is possible to control a
focus position at high accuracy.
[0019] Further, the focusing method of the image capturing
apparatus in the present invention is a focusing method of an image
capturing apparatus which is characterized by having a light source
which radiates light to a sample, a light conductive optical system
including a light dividing unit which divides an optical image of
the sample into a first optical path for capturing an image and a
second optical path for focus control, a first imaging unit which
captures a first image by the first optical image divided into the
first optical path, a second imaging unit which captures a second
image by the second optical image divided into the second optical
path, and a focus control unit which analyzes the second image to
control a focus position of the image by the first imaging unit
based on an analysis result thereof, in which the first imaging
region and the second imaging region for capturing a partial image
of the second optical image are set on the imaging area of the
second imaging unit, an optical path difference producing member
which gives an optical path difference to the second optical image
along an in-plane direction of the imaging area is disposed on the
second optical path in such a manner that the optical path
difference of the second optical image is symmetrical with respect
to the axis of the imaging area orthogonal to a direction at which
the second optical image moves in association with scanning of the
sample, and the region control unit reverses a position of the
first imaging region and a position of the second imaging region on
the imaging area with respect to the axis of the imaging area in
association with reversal of the scanning direction of the
sample.
[0020] In the focusing method of the image capturing apparatus, the
optical path difference producing member is disposed on the second
optical path. Thereby, at the first imaging region and the second
imaging region of the second imaging unit, it is possible to image
respectively an optical image which is focused at the front of an
optical image made incident into the first imaging unit (front
focus) and an optical image which is focused at the rear thereof
(rear focus). In the focusing method, it is possible to make a
difference in optical path length without dividing light on the
second optical path for focus control. Therefore, the amount of
light at the second optical path necessary for obtaining
information on a focus position can be suppressed to secure the
amount of light on image pickup by the first imaging unit. Further,
in the focusing method, the position of the first imaging region
and the position of the second imaging region on the imaging area
are reversed with respect to the axis in association with reversal
of the scanning direction of the sample. Therefore, despite the
scanning direction of the sample, it is possible to obtain a
deviation direction of the focus position under the same
conditions.
[0021] It is also preferable that a region into which an optical
image is made incident which is conjugate to the first optical
image made incident into the first imaging unit on the imaging area
is made substantially in alignment with the axis of the imaging
area. Thereby, even where the sample is reversed in scanning
direction, it is possible to keep a positional relationship between
the first imaging region, the second imaging region and the region
into which an optical image is made incident which is conjugate to
the first optical image on the imaging area.
[0022] It is preferable that the axis of the imaging area is made
substantially in alignment with the central axis of the imaging
area orthogonal to a direction at which the second optical image
moves in association with scanning of the sample. In this case, it
is possible to reliably reverse the position of the first imaging
region and the position of the second imaging region on the imaging
area in association with reversal of the scanning direction of the
sample.
[0023] It is also preferable that as the optical path difference
producing member, there is used a flat plate member which is
disposed so as to overlap at least on a part of the imaging area
and in order to avoid a shadow of the second optical image by an
edge part of the flat plate member, the first imaging region and
the second imaging region are set by the region control unit
respectively so as to give a region which will overlap on the flat
plate member and a region which will not overlap on the flat plate
member. In this case, use of the flat plate member enables the
optical path difference producing member to be made simple in
configuration. Further, the edge part of the flat plate member
forms the shadow of the second optical image at an imaging area of
the second imaging device. Therefore, the first imaging region and
the second imaging region are set so as to avoid the shadow, thus
making it possible to secure accurate control of the focus
position.
[0024] It is also preferable that as the optical path difference
producing member, there is used a member which has a part
undergoing a continuous change in thickness along an in-plane
direction of the imaging area and that the first imaging region and
the second imaging region are set by the region control unit so as
to overlap on the part of the optical path difference producing
member which is different in thickness. In this case, adjustment of
a position of the first imaging region and that of the second
imaging region makes it possible to freely adjust an interval
between the front focus and the rear focus. It is, thereby,
possible to detect a focus position of the sample at high
accuracy.
[0025] It is also preferable that the image capturing apparatus is
provided with an objective lens which faces to a sample and an
objective lens control unit which controls a position of the
objective lens relatively with respect to the sample based on
control by the focus control unit, in which the objective lens
control unit will not drive the objective lens during analysis of
the focus position which is being performed by the focus control
unit and will allow the objective lens to move with respect to the
sample in one direction during analysis of the focus position which
is not being performed by the focus control unit. Since no change
in positional relationship will take place between the objective
lens and the sample during analysis of the focus position, it is
possible to secure analysis accuracy of the focus position.
[0026] It is also preferable that waiting time from image pickup at
the first imaging region to image pickup at the second imaging
region is set by the region control unit based on a scanning speed
of the stage and an interval between the first imaging region and
the second imaging region. Therefore, since light from the same
position of the sample is made incident into the first imaging
region and the second imaging region, it is possible to control the
focus position at high accuracy.
Advantageous Effects of Invention
[0027] The present invention is able to obtain a direction at which
the focus position deviates by a simple configuration according to
the scanning direction of the sample.
BRIEF DESCRIPTION OF DRAWINGS
[0028] FIG. 1 is a drawing which shows one embodiment of a macro
image capturing device which constitutes an image capturing
apparatus of the present invention.
[0029] FIG. 2 is a drawing which shows one embodiment of a micro
image capturing device which constitutes the image capturing
apparatus of the present invention.
[0030] FIG. 3 is a block diagram which shows functional components
of the image capturing apparatus.
[0031] FIG. 4A and FIG. 4B are drawings which show an analysis
result of a contrast value where a distance to the surface of a
sample is in agreement with the focal length of an objective
lens.
[0032] FIG. 5A and FIG. 5B are drawings which show an analysis
result of a contrast value where a distance to the surface of the
sample is longer than the focal length of the objective lens.
[0033] FIG. 6A and FIG. 6B are drawings which show an analysis
result of a contrast value where a distance to the surface of the
sample is shorter than the focal length of the objective lens.
[0034] FIG. 7A and FIG. 7B are drawings which show one example of
an optical path difference producing member and a second imaging
device (forward direction).
[0035] FIG. 8A and FIG. 8B are drawings which show one example of
the optical path difference producing member and the second imaging
device (reverse direction).
[0036] FIG. 9A and FIG. 9B are drawings which show another example
of the optical path difference producing member and the second
imaging device (forward direction).
[0037] FIG. 10A and FIG. 10B are drawings which show another
example of the optical path difference producing member and the
second imaging device (reverse direction).
[0038] FIG. 11A and FIG. 11B are drawings which show a modified
example of the optical path difference producing member.
[0039] FIG. 12 is a drawing which shows a relationship of the
distance between the objective lens and the surface of the sample
with respect to scanning time of a stage.
[0040] FIG. 13 is a drawing which shows control of a scanning
direction of the stage by a stage control portion.
[0041] FIG. 14A and FIG. 14B are drawings which show control of a
scanning speed of the stage by the stage control portion.
[0042] FIG. 15 is a flow chart which shows motions of the image
capturing apparatus.
DESCRIPTION OF EMBODIMENTS
[0043] Hereinafter, a description will be given in detail of
preferred embodiments of the image capturing apparatus and the
focusing method of the image capturing apparatus in the present
invention with reference to drawings.
[0044] FIG. 1 is a drawing which shows one embodiment of the macro
image capturing device which constitutes the image capturing
apparatus of the present invention. FIG. 2 is a drawing which shows
one embodiment of the micro image capturing device which
constitutes the image capturing apparatus of the present invention.
As shown in FIG. 1 and FIG. 2, an image capturing apparatus M is
constituted with a macro image capturing device M1 for capturing a
macro image of a sample S and a micro image capturing device M2 for
capturing a micro image of the sample S. The image capturing
apparatus M is an apparatus which sets, for example, a plurality of
line-shaped divided regions 40 with respect to the macro image
captured by the macro image capturing device M1 (refer to FIG. 13)
and produces a virtual micro image by capturing and synthesizing
each of the divided regions 40 by the micro image capturing device
M2 at a high magnification.
[0045] As shown in FIG. 1, the macro image capturing device M1 is
provided with a stage 1 which supports the sample S. The stage 1 is
an XY stage which is actuated in a horizontal direction by a motor
or an actuator such as a stepping motor (pulse motor) or a piezo
actuator, for example. The sample S which is observed by using the
image capturing apparatus M is, for example, a biological sample
such as cells and placed on the stage 1 in a state of being sealed
on a slide glass. The stage 1 is actuated inside the XY plane, by
which an imaging position with respect to the sample S is allowed
to move.
[0046] The stage 1 is able to move back and forth between the macro
image capturing device M1 and the micro image capturing device M2
and provided with functions to deliver the sample S between the
devices. It is acceptable that when a macro image is captured, an
entire image of the sample S is picked up at one time or the sample
S is divided into a plurality of regions to pick up each of the
images. It is also acceptable that the stage 1 is installed both on
the macro image capturing device M1 and on the micro image
capturing device M2.
[0047] A light source 2 which radiates light to the sample S and a
condensing lens 3 which concentrates light from the light source 2
at the sample S are disposed on a bottom of the stage 1. It is
acceptable that the light source 2 is disposed so as to radiate
light obliquely to the sample S. Further, a light guiding optical
system 14 which guides an optical image from the sample S and an
imaging device 5 which images the optical image of the sample S are
disposed on an upper face of the stage 1. The light guiding optical
system 14 is provided with an image forming lens 6 which forms the
optical image from the sample S at an imaging area of the imaging
device 5. Still further, the imaging device 5 is an area sensor
which is capable of capturing, for example, a two-dimensional
image. The imaging device 5 captures an entire image of the optical
image of the sample S made incident into the imaging area via the
light guiding optical system 14 and is housed at a virtual micro
image housing portion 39 to be described later.
[0048] As shown in FIG. 2, the micro image capturing device M2 is
provided on the bottom of the stage 1 with a light source 12 and a
condensing lens 13, as with the macro image capturing device M1.
Further, a light guiding optical system 14 which guides an optical
image from the sample S is disposed on the upper face of the stage
1. The optical system which radiates light from the light source 12
to the samples may include an excitation light radiating optical
system which radiates excitation light to the sample S and a
dark-field illuminating optical system which captures a dark-field
image of the sample S.
[0049] The light guiding optical system 14 is provided with an
objective lens 15 disposed so as to face to the sample S and a beam
splitter (light dividing unit) 16 disposed at a rear stage of the
objective lens 15. The objective lens 15 is provided with a motor
and an actuator such as a stepping motor (pulse motor) and a piezo
actuator for actuating the objective lens 15 in a Z direction
orthogonal to a face on which the stage 1 is placed. A position of
the objective lens 15 in the Z direction is changed by these
actuation units, thus making it possible to adjust a focus position
of image pickup when an image of the sample S is captured. It is
acceptable that the focus position is adjusted by changing a
position of the stage 1 in the Z direction or by changing positions
of both the objective lens 15 and the stage 1 in the Z
direction.
[0050] The beam splitter 16 is a portion which divides an optical
image of the sample S into a first optical path L1 for capturing an
image and a second optical path L2 for focus control. The beam
splitter 16 is disposed at an angle of approximately 45 degrees
with respect to an optical axis from the light source 12. In FIG.
2, an optical path passing through the beam splitter 16 is given as
the first optical path L1, while an optical path reflected at the
beam splitter 16 is given as the second optical path.
[0051] On the first optical path L1, there are disposed an image
forming lens 17 which forms an optical image of the sample S (first
optical image) which has passed through the beam splitter 16 and a
first imaging device (first imaging unit) 18 in which an imaging
area is disposed at an image forming position of the image forming
lens 17. The first imaging device 18 is a device which is capable
of capturing a one-dimensional image (first image) by the first
optical image of the sample S, including, for example, a
two-dimensional CCD sensor which is capable of realizing, TDI (time
delay integration) actuation. The first imaging device 18 has a
structure formed by combining, for example, two TDI sensors
different in transfer direction and in which light receiving
portions, each of which has plural stages of light receiving
arrays, are positioned at the center of the imaging area. It is
noted that the first imaging device 18 may be a line sensor.
[0052] Further, in a method which captures sequentially images of
the sample S, with the stage 1 controlled at a constant speed, the
first imaging device 18 may be a device such as a CMOS sensor or a
CCD sensor which is capable of capturing a two-dimensional image.
First images picked up by the first imaging device 18 are
sequentially stored at a temporary storage memory such as a lane
buffer, thereafter, compressed and output at an image producing
portion 38 to be described later.
[0053] On the other hand, on the second optical path L2, there are
disposed a view-field adjusting lens 19 which contracts an optical
image of a sample reflected by the beam splitter 16 (second optical
image) and a second imaging device (second imaging unit) 20.
Further, at a front stage of the second imaging device 20, there is
disposed an optical path difference producing member 21 which gives
an optical path difference to the second optical image. It is
preferable that the view-field adjusting lens 19 is constituted in
such a manner that the second optical image is formed at the second
imaging device 20 in a dimension similar to that of the first
optical image.
[0054] The second imaging device 20 is a device which is capable of
capturing a two-dimensional image (second image) by the second
optical image of the sample S, including, for example, sensors such
as a CMOS (complementary metal oxide semiconductor) and a CCD
(charge coupled device). It is also acceptable that a line sensor
is used.
[0055] The imaging area 20a of the second imaging device 20 is
disposed so as to be substantially in alignment with an XZ plane
orthogonal to the second optical path L2. The second optical image
is projected on the imaging area 20a. The second optical image is
such that where the sample S is scanned by the stage 1 in the
forward direction, the imaging area 20a will move in a +Z direction
and where the sample S is scanned in the reverse direction, the
imaging area 20a will move in a -Z direction. The first imaging
region 22A and the second imaging region 22B, each of which
captures a partial image of the second optical image, are set on
the imaging area 20a (for example, refer to FIG. 7A and FIG. 7B).
The first imaging region 22A and the second imaging region 22B are
set in a direction perpendicular to a direction (scanning
direction: Z direction) at which the second optical image moves on
the imaging area 20a in association with scanning of the sample S.
Further, the first imaging region 22A and the second imaging region
22B are set, with a predetermined interval kept, both of which
captures a part of the second optical image in a line shape.
Thereby, an optical image which is at the same region as that of
the first optical image of the sample S captured by the first
imaging device 18 can be captured as the second optical image at
the first imaging region 22A and the second imaging region 22B.
[0056] Thereby, the second imaging device 20 is able to capture an
optical image which is focused at the front of a first optical
image made incident into the first imaging device 18 (front focus)
and an optical image which is focused at the rear thereof (rear
focus) based on the position of the first imaging region 22A and
that of the second imaging region 22B. A focus difference between
the front focus and the rear focus is dependent on a difference
between a thickness and an index of refraction of the optical path
difference producing member 21 through which the second optical
image made incident into the first imaging region 22A passes and a
thickness and an index of refraction of the optical path difference
producing member 21 through which the second optical image made
incident into the second imaging region 22B passes.
[0057] The optical path difference producing member 21 is a glass
member which gives an optical path difference to the second optical
image along an in-plane direction of the imaging area 20a. The
optical path difference producing member 21 is disposed in such a
manner that an optical path difference of the second optical image
is symmetrical with respect to the axis P of the imaging area 20a
orthogonal to a direction at which the second optical image moves
in association with scanning of the sample S. Further, it is
preferable that the optical path difference producing member 21 is
disposed in such a manner that a face which faces to the second
imaging device 20 is parallel with the imaging area (light
receiving face) 20a of the second imaging device. Thereby, it is
possible to reduce deflection of light by the face which faces to
the second imaging device 20 and also to secure the amount of light
which is received by the second imaging device 20. There will be
described later a shape of the optical path difference producing
member 21 and an example in which the first imaging region 22A and
the second imaging region 22B are disposed on the imaging area
20a.
[0058] FIG. 3 is a block diagram which shows functional components
of the image capturing apparatus. As shown in the diagram, the
image capturing apparatus M is provided with a computer system
having housing portions such as a CPU, a memory, a communication
interface and a hard disk, an operation portion 31 such as a
keyboard, a monitor 32 etc. The functional components of the
control portion 33 include a focus control portion 34, a region
control portion 35, an objective lens control portion 36, a stage
control portion 37, an image producing portion 38 and a virtual
micro image housing portion 39.
[0059] The focus control portion 34 is a portion which analyzes a
second image captured by the second imaging device 20 to control a
focus position of an image picked up by the first imaging device 18
based on the analysis result. More specifically, the focus control
portion 34 first determines a difference between a contrast value
of the image captured at the first imaging region 22A and a
contrast value captured at the second imaging region 22B in the
second imaging device 20.
[0060] Here, as shown in FIG. 4A and FIG. 4B, where a focus
position of the objective lens 15 is in alignment with the surface
of the sample S, an image contrast value of the front focus
captured at the first imaging region 22A is substantially in
agreement with an image contrast value of the rear focus captured
at the second imaging region 22B. Thereby, a difference value
between them is almost zero.
[0061] On the other hand, as shown in FIG. 5A and FIG. 5B, where a
distance to the surface of the sample S is longer than a focal
length of the objective lens 15, an image contrast value of the
rear focus captured at the second imaging region 22B is greater
than an image contrast value of the front focus captured at the
first imaging region 22A. Therefore, a difference value between
them is a positive value. In this case, the focus control portion
34 outputs instruction information to the objective lens control
portion 36 so as to be actuated in a direction at which the
objective lens 15 is brought closer to the sample S.
[0062] Further, as shown in FIG. 6A and FIG. 6B, where a distance
to the surface of the samples is shorter than a focal length of the
objective lens 15, an image contrast value of the rear focus
captured at the second imaging region 22B is smaller than an image
contrast value of the front focus captured at the first imaging
region 22A. Therefore, a difference value between them is a
negative value. In this case, the focus control portion 34 outputs
instruction information to the objective lens control portion 36 so
as to be actuated in a direction at which the objective lens 15 is
brought away from the sample S.
[0063] The region control portion 35 is a portion which controls a
position of the first imaging region 22A and a position of the
second imaging region 22B at the imaging area 20a of the second
imaging device 20. The region control portion 35 sets at first the
first imaging region 22A at a predetermined position based on
operation from the operation portion 31 and releases the setting of
the first imaging region 22A after image pickup at the first
imaging region 22A. Then, the region control portion 35 sets the
second imaging region 22B, with a predetermined interval kept in
the Z direction from the first imaging region 22A, and releases the
setting of the second imaging region 22B after image pickup at the
second imaging region 22B.
[0064] At this time, waiting time W from image pickup at the first
imaging region 22A to image pickup at the second imaging region 22B
is set based on an interval d between the first imaging region 22A
and the second imaging region 22B and a scanning velocity v of the
stage 1. For example, where the waiting time W is given as time W1
from the start of image pickup at the first imaging region 22A to
the start of image pickup at the second imaging region 22B, it is
possible to determine the waiting time with reference to a formula
of W1=d/v-e1-st, with consideration given to exposure time e1 of
image pickup at the first imaging region 22A and time st from
release of the setting of the first imaging region 22A to the
setting of the second imaging region 22B.
[0065] Further, where waiting time W is given as waiting time W2
from completion of image pickup at the first imaging region 22A to
start of image pickup at the second imaging region 22B, it is
possible to determine the waiting time with reference to a formula
of W2=d/v-st, with consideration given to time st from release of
setting of the first imaging region 22A to setting of the second
imaging region 22B. Still further, an interval d between the first
imaging region 22A and the second imaging region 22B is set based
on a difference in optical path length given by the optical path
difference producing member 21. However, the interval d actually
corresponds to a distance of the sample S on a slide glass.
Eventually, it is necessary to convert the interval d to the number
of pixels of the second imaging region 22B. Where a pixel size of
the second imaging device 20 is expressed in terms of AFpsz and
magnification is expressed in terms of AFmag, the number of pixels
dpix corresponding to the interval d can be determined with
reference to a formula of dpix=d/(AFpsz/AFmag).
[0066] Further, the region control portion 35 reverses the position
of the first imaging region 22A and the position of the second
imaging region 22B on the imaging area 20a so as to give line
symmetry to the axis P of the imaging area 20a in association of
reversal of the scanning direction of the sample S. This setting is
carried out, whenever necessary, depending on the shape and
arrangement of the optical path difference producing member 21. For
example, in the examples shown in FIG. 7A and FIG. 7B, and FIG. 8A
and FIG. 8B, the optical path difference producing member 21A which
is formed with a flat plate-shaped glass member is disposed so as
to be in alignment with the axis P of the imaging area 20a. A
region 22C into which an optical image is made incident which is
conjugate to the first optical image made incident into the first
imaging device 18 is positioned so as to be in alignment with the
axis P of the imaging area 20a. In the present embodiment, the axis
P of the imaging area 20a is kept in alignment with the central
axis of the imaging area 20a orthogonal to a direction at which the
second optical image moves in association with scanning of the
sample S.
[0067] Then, as shown in FIG. 7A and FIG. 7B, where the sample S is
scanned in the forward direction (+Z direction), at an upper half
region of the imaging area 20, the first imaging region 22A is set
at a region which will not overlap on the optical path difference
producing member 21A, and the second imaging region 22B is set at a
region which will overlap on the optical path difference producing
member 21A. Further, as shown in FIG. 8A and FIG. 8B, where the
sample S is scanned in the reverse direction (-Z direction), at a
lower half region of the imaging area 20a, the first imaging region
22A is set at a region which will not overlap on the optical path
difference producing member 21A, and the second imaging region 22B
is set at a region which will overlap on the optical path
difference producing member 21A.
[0068] There is a fear that an edge part E of the optical path
difference producing member 21A may form a shadow 23 of the second
optical image on the imaging area 20a. Therefore, it is preferable
that the interval d between the first imaging region 22A and the
second imaging region 22B is made wider than the width of the
shadow 23 and that the first imaging region 22A and the second
imaging region 22B are set at a position so as to avoid the shadow
23.
[0069] Further, in the examples shown in FIG. 9A and FIG. 9B, and
FIG. 10A and FIG. 10B, an optical path difference producing member
21B composed of a prism-shaped glass member which undergoes a
continuous change in thickness toward the center thereof in the Z
direction is disposed so as to be in alignment with the axis P of
the imaging area 20a. The optical path difference producing member
21B changes in thickness so as to give a symmetric shape at the
center of the axis P of the imaging area in which the thickness is
switched from an increase to a decrease along the Z direction. The
region 22C into which an optical image is made incident which is
conjugate to the first optical image made incident into the first
imaging device 18 is positioned so as to be in alignment with the
center of the imaging area 20a in the Z direction. In the present
embodiment as well, the axis P of the imaging area 20a is kept in
alignment with the central axis of the imaging area 20a orthogonal
to a direction at which the second optical image moves in
association with scanning of the sample S.
[0070] Moreover, as shown in FIG. 9A and FIG. 9B, where the sample
S is scanned in the forward direction (+Z direction), at an upper
half region of the imaging area 20a, the first imaging region 22A
and the second imaging region 22B are set respectively so as to be
on the upper side and on the lower side. Further, as shown in FIG.
10A and FIG. 10B, where the sample S is scanned in the reverse
direction (-Z direction), at a lower half region of the imaging
area 20a, the first imaging region 22A and the second imaging
region 22B are set respectively so as to be on the lower side and
on the upper side.
[0071] Moreover, it is acceptable that, as the optical path
difference producing member 21, for example, as shown in FIG. 11A,
there is used the optical path difference producing member 21C
whose central part in the Z direction is constant in thickness and
which decreases in thickness moving towards the outside the both
ends thereof in the Z direction. For example, as shown in FIG. 11B,
it is also acceptable that an optical path difference producing
member 21D composed of two glass members, each of which has a
triangular prism-shaped cross section, is disposed so as to
decrease in thickness moving towards the center of the imaging area
20a from both ends thereof in the Z direction.
[0072] Where there is set a position of the region 22C into which
an optical image is made incident which is conjugate to the first
optical image made incident into the first imaging device 18 on the
imaging area 20a, a distance between the stage 1 and the objective
lens 15 is at first fixed to adjust a position of the stage 1 in
such a manner that a cross line of a calibration slide is
positioned at the center of a visual field of the first imaging
device 18. Then, the second imaging device 20 is adjusted for its
back focus in such a manner that the cross line of the calibration
slide comes into a visual field of the second imaging device 20.
Finally, an in-plane direction position of the second imaging
device 20 is adjusted in such a manner that the cross line of the
calibration slide is positioned at a desired site of the imaging
area 20a of the second imaging device 20.
[0073] The objective lens control portion 36 is a portion which
controls actuation of the objective lens 15. Upon receiving
instruction information output from the focus control portion 34,
the objective lens control portion 36 actuates the objective lens
15 in the Z direction in accordance with contents of the
instruction information. It is, thereby, possible to adjust a focus
position of the objective lens 15 with respect to the sample S.
[0074] The objective lens control portion 36 will not actuate the
objective lens 15 during analysis of the focus position which is
being performed by the focus control portion 34 and will actuate
the objective lens 15 only in one direction along the Z direction
until start of analysis of a next focus position. FIG. 12 is a
drawing which shows a relationship of the distance between the
objective lens and the surface of the sample with respect to
scanning time of the stage. As shown in the drawing, during
scanning of the sample S, there will take place alternately an
analysis period A of the focus position and an objective lens
actuation period B based on an analysis result thereof. As
described so far, no change in positional relationship takes place
between the objective lens 15 and the sample S during analysis of
the focus position, thus making it possible to secure analysis
accuracy of the focus position.
[0075] The stage control portion 37 is a portion which controls
actuation of the stage 1. More specifically, the stage control
portion 37 allows the stage 1 on which the sample S is placed to be
scanned at a predetermined speed based on operation by the
operation portion 31. The stage 1 is scanned, by which an imaging
field of the sample S moves relatively and sequentially at the
first imaging device 18 and the second imaging device 20. Scanning
of the stage 1 is, as shown in FIG. 13, for example, bi-directional
scanning in which after completion of scanning of one divided
region 40, the stage 1 is allowed to move in a direction orthogonal
to the scanning direction and a next divided region 40 is scanned
in the opposite direction.
[0076] Although the stage 1 is scanned at a constant speed while
images are captured, actually, immediately after the start of
scanning, there is a period during which the scanning speed is
unstable due to influences of vibrations of the stage 1 etc. Thus,
as shown in FIG. 14A and FIG. 14B, it is preferable that there is
set a scanning width longer than the divided region 40 and an
acceleration period C for accelerating the stage 1, a stabilization
period D for stabilizing a scanning speed of the stage 1 and a
slowing-down period F for slowing down the stage 1 are allowed to
take place individually when scanning is performed outside the
divided region 40. It is, thereby, possible to capture an image in
synchronization with a constant speed period E during which the
stage 1 is scanned at a constant speed. It is acceptable that image
pickup is started during the stabilization period D and a data part
obtained during the stabilization period D is deleted after the
image has been captured. The above-described method is desirable
when used for an imaging device which requires void reading of
data.
[0077] The image producing portion 38 is a portion at which a
captured image is synthesized to produce a virtual micro image. The
image producing portion 38 receives sequentially first images
output from the first imaging device 18, that is, images of
individual divided regions 40, synthesizing these images to produce
an entire image of the sample S. Then, based on the thus
synthesized image, prepared is an image, the resolution of which is
lower than that of the synthesized image, and housed in a virtual
micro image housing portion 39 by associating a high resolution
image with a low resolution image. It is acceptable that an image
captured by the macro image capturing device M1 is also associated
at the virtual micro image housing portion 39. It is also
acceptable that the virtual micro image is housed as one image or
plurally divided images.
[0078] Next, a description will be given of motions of the
above-described image capturing apparatus M.
[0079] FIG. 15 is a flow chart which shows motions of the image
capturing apparatus M. As shown in the flow chart, at the image
capturing apparatus M, at first, a macro image of the sample S is
captured by the macro image capturing device M1 (Step S01). The
thus captured macro image is subjected to binarization by using,
for example, a predetermined threshold value and, thereafter,
displayed on a monitor 32. A scope for capturing micro images from
macro images is set by automatic setting based on a predetermined
program or manual setting by an operator (Step S02).
[0080] Then, the sample S is transferred to the micro image
capturing device M2 and focusing conditions are set (Step S03).
Here, as described above, based on a scanning velocity v of the
stage 1 and an interval d between the first imaging region 22A and
the second imaging region 22B, a waiting time W is set up to the
start of image pickup at the second imaging region 22B. It is more
preferable that consideration is given to exposure time e1 of image
pickup at the first imaging region 22A, time st from release of
setting of the first imaging region 22A to setting of the second
imaging region 22B etc.
[0081] After the focusing conditions have been set, scanning of the
stage 1 is started to capture a micro image for each of the divided
regions 40 of the sample S by the micro image capturing device M2
(Step S04). In capturing the micro image by the first imaging
device 18, at the second imaging device 20, a deviating direction
of the objective lens 15 with respect to the sample S is analyzed
based on a difference in contrast value between the front focus and
the rear focus by the first imaging region 22A and the second
imaging region 22B, thereby adjusting a position of the objective
lens 15 in real time. After micro images have been captured
completely for all the divided regions 40, the thus captured micro
images are synthesized to produce a virtual micro image (Step
S05).
[0082] As described so far, in the image capturing apparatus M, the
optical path difference producing members 21 are disposed on the
second optical path L2. Thereby, at the first imaging region 22A
and the second imaging region 22B of the second imaging device 20,
it is possible to image respectively an optical image which is
focused at the front of an optical image made incident into the
first imaging device 18 (front focus) and an optical image which is
focused at the rear thereof (rear focus). In the image capturing
apparatus M, a difference in optical path length can be made
without dividing light on the second optical path L2 for focus
control. Therefore, it is possible to suppress the amount of light
at the second optical path L2 necessary for obtaining information
on a focus position and to secure the amount of light on image
pickup at the first imaging device 18. Further, in the image
capturing apparatus M, a position of the first imaging region 22A
and a position of the second imaging region 22B on the imaging area
20a are reversed so as to give line symmetry with respect to the
axis P in association with reversal of the scanning direction of
the sample S. Therefore, despite the scanning direction of the
sample S, it is possible to obtain a deviation direction of the
focus position under the same conditions.
[0083] Further, in the image capturing apparatus M, based on a
scanning velocity v of the stage and an interval d between the
first imaging region 22A and the second imaging region 22B, a
waiting time W is set from image pickup at the first imaging region
22A to image pickup at the second imaging region 22B. As a result,
light from the same position of the sample S is made incident into
the first imaging region 22A and the second imaging region 22B.
Thus, it is possible to control a focus position of the objective
lens 15 at high accuracy.
[0084] Where, as the optical path difference producing member of
the present embodiment, there are used optical path difference
producing members 21 (21B to 21D) composed of a glass member which
has a part changing in thickness along an in-plane direction of the
imaging area 20a at the imaging device 20, the region control
portion 35 is used to adjust a position of the first imaging region
22A and a position of the second imaging region 22B. Thereby, it is
possible to freely adjust an interval between the front focus and
the rear focus. Accordingly, for example, where a plural number of
contrast peaks are found in an image picked up by the second
imaging device 20 or where a peak is flat in shape, a focus
difference between the front focus and the rear focus is adjusted,
thus making it possible to detect a focus position of the sample S
at high accuracy.
[0085] Further, where, as the optical path difference producing
member of the present embodiment, there are used optical path
difference producing members 21 (21A) composed of a flat-plate like
glass member, the optical path difference producing member 21 can
be made simple in structure. In this case, an edge part E of the
flat plate member forms a shadow 23 of the second optical image at
the imaging area 20a of the second imaging device 20. Therefore,
the first imaging region 22A and the second imaging region 22B are
set so as to avoid the shadow 23, by which it is possible to secure
a focus position of the objective lens 15 at high accuracy.
[0086] Further, in the above-described embodiment, the first
imaging region 22A and the second imaging region 22B are set so as
to be positioned on the front side of the sample S in the scanning
direction on the imaging area 20a with respect to the region 22C
into which an optical image is made incident which is conjugate to
the first optical image made incident into the first imaging device
18. Thereby, it is possible to obtain in advance a deviation
direction of the focus position in relation to an imaging position
of the first imaging device 18. The present invention shall not be
limited to the above-described mode but may include a mode in which
analysis is made for a deviation direction of the focus position at
an imaging position of the first imaging device 18.
[0087] In the above-described embodiment, there is exemplified an
apparatus for producing a virtual micro image. The image capturing
apparatus of the present invention is, however, applicable to
various types of apparatuses, as long as the apparatuses are those
in which an image is captured by scanning a sample at a
predetermined speed by a stage etc.
REFERENCE SIGNS LIST
[0088] 1 . . . stage, 12 . . . light source, 14 . . . light guiding
optical system, 15 . . . objective lens, 16 . . . beam splitter
(light dividing unit), 18 . . . first imaging device (first imaging
unit), 20 . . . second imaging device (second imaging unit), 20a .
. . imaging area, 21 (21A to 21D) . . . optical path difference
producing member, 22A . . . first imaging region, 22B . . . second
imaging region, 34 . . . focus control portion (focus control
unit), 35 . . . region control portion (region control unit), 36 .
. . objective lens control portion (objective lens control unit), E
. . . edge part, L1 . . . first optical path, L2 . . . second
optical path, M . . . image capturing apparatus, M1 . . . macro
image capturing device, M2 . . . micro image capturing device, P .
. . axis, S . . . sample.
* * * * *