U.S. patent application number 11/173020 was filed with the patent office on 2006-01-26 for microscope imaging apparatus and biological-specimen examination system.
Invention is credited to Kayuri Muraki, Yoshimasa Suzuki.
Application Number | 20060018013 11/173020 |
Document ID | / |
Family ID | 35656847 |
Filed Date | 2006-01-26 |
United States Patent
Application |
20060018013 |
Kind Code |
A1 |
Suzuki; Yoshimasa ; et
al. |
January 26, 2006 |
Microscope imaging apparatus and biological-specimen examination
system
Abstract
A microscope imaging apparatus and a biological-specimen
examination system that can accurately carry out measurement even
for an object under examination having substantial brightness
non-uniformity are provided. The microscope imaging apparatus
includes a stage that holds the object under examination, an
illumination unit that illuminates the object under examination, an
image-acquisition unit that acquires images of the object under
examination, and a motion unit that moves the stage and the
image-acquisition unit relative to each other. The
image-acquisition unit includes an imaging device capable of image
acquisition using a time delay integration method. When acquiring a
plurality of images of the object under examination, the exposure
time during which accumulated charge is produced in the imaging
device is made different for each of the acquired images, and the
plurality of images are combined into a single image.
Inventors: |
Suzuki; Yoshimasa;
(Kawasaki-shi, JP) ; Muraki; Kayuri;
(Hachioji-shi, JP) |
Correspondence
Address: |
KENYON & KENYON
1500 K STREET NW
SUITE 700
WASHINGTON
DC
20005
US
|
Family ID: |
35656847 |
Appl. No.: |
11/173020 |
Filed: |
July 5, 2005 |
Current U.S.
Class: |
359/368 |
Current CPC
Class: |
G01N 21/6458 20130101;
G01N 21/6486 20130101; G01N 21/6452 20130101; G02B 21/367 20130101;
G02B 21/002 20130101 |
Class at
Publication: |
359/368 |
International
Class: |
G02B 21/00 20060101
G02B021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 7, 2004 |
JP |
2004-200866 |
Jul 7, 2004 |
JP |
2004-200914 |
Jul 7, 2004 |
JP |
2004-200954 |
Claims
1. A microscope imaging apparatus comprising: a stage that holds an
object under examination; an illumination unit that illuminates the
object under examination; an image-acquisition unit that acquires
images of the object under examination; a motion unit that moves
the stage and the image-acquisition unit relative to each other;
and a control unit that controls the image-acquisition unit and the
motion unit, wherein the image-acquisition unit includes an imaging
device that is capable of acquiring images with the time delay
integration method, and when image acquisition of the object under
examination is carried out a plurality of times, the control unit
makes the exposure times of the imaging device different for each
image acquisition.
2. A microscope imaging apparatus according to claim 1, wherein the
control unit includes an exposure-time input unit for inputting the
exposure time and an exposure-time setting unit for setting the
length of the exposure time to be used in the subsequent image
acquisition, the exposure time to be used in the subsequent image
acquisition being determined on the basis of the image obtained by
image acquisition using the input exposure time.
3. A microscope imaging apparatus according to claim 2, wherein the
exposure time input to the exposure-time input unit defines a
maximum exposure time serving as the upper limit for the exposure
time.
4. A microscope imaging apparatus according to claim 1, wherein the
imaging device in the image-acquisition unit has an anti-smear
function.
5. A microscope imaging apparatus according to claim 1, wherein the
control unit combines images having different exposure times into a
single image by carrying out predetermined image processing.
6. A microscope imaging apparatus comprising: a stage that holds an
object under examination; an illumination unit that illuminates the
object under examination; an image-acquisition unit that acquires
images of the object under examination; a motion unit that moves
the stage and the image-acquisition unit relative to each other;
and a control unit that controls the image-acquisition unit and the
motion unit, wherein the image-acquisition unit includes an imaging
device that is capable of acquiring images with a time delay
integration method, and by performing a prescan of the object under
examination to obtain a detected intensity of the object under
examination, the control unit determines an exposure time for image
acquisition after the prescan on the basis of the detected
intensity.
7. A microscope imaging apparatus according to claim 6, wherein the
illumination unit includes a first objective lens and a second
objective lens that focus light onto the object under examination;
during the prescan, the first objective lens is disposed in a light
path of the illumination unit; during image acquisition after the
prescan, the second objective lens is disposed in the light path of
the illumination unit; and the magnification of the first objective
lens is lower than the magnification of the second objective
lens.
8. A microscope imaging apparatus according to claim 6, comprising:
a variable-power lens that can be inserted in and removed from the
light path of the illumination unit.
9. A microscope imaging apparatus according to claim 6, wherein the
control unit includes: a maximum-exposure-time input unit; and a
selection unit, wherein the maximum exposure time of the object
under examination for image acquisition after the prescan is input
to the maximum-exposure time input unit, and using the selection
unit, the maximum exposure time and the exposure time determined in
the prescan are compared, and on the basis of this comparison, the
shorter exposure time is set as the exposure time used for
measurement of the object under examination.
10. A microscope imaging apparatus according to claim 6, wherein
the object under examination has a plurality of sample located
portions formed thereon, the individual sample located portions
having substantially the same brightness, and the image-acquisition
unit acquires images of the plurality of sample located
portions.
11. A microscope imaging apparatus comprising: a stage that holds
an object under examination; an illumination unit that illuminates
the object under examination; an image-acquisition unit that
acquires images of the object under examination; a motion unit that
moves the stage and the image-acquisition unit relative to each
other; and a control unit that controls the image-acquisition unit
and the motion unit, wherein the image-acquisition unit includes an
imaging device that is capable of image acquisition using two
methods; the control unit includes: an examination-object-parameter
input unit for inputting information about the object under
examination as an examination-object parameter; a calculation unit
that calculates a time required for the relative motion on the
basis of the examination-object parameter which has been input; and
a switching unit that changes the image-acquisition method of the
imaging device on the basis of the calculation result; and the two
image-acquisition methods are a time delay integration method and a
two-dimensional imaging method in which accumulated charge is
produced by a single exposure.
12. A microscope imaging apparatus according to claim 11, wherein
the examination-object parameter is the wavelength used in the
image acquisition.
13. A microscope imaging apparatus according to claim 11, wherein
the examination-object parameter is the exposure time used in the
image acquisition.
14. A microscope imaging apparatus according to claim 11, wherein
the examination-object parameter is the density of the object under
examination.
15. A biological-specimen examination system comprising: a culture
unit for culturing a biological specimen; and a detection unit
disposed adjacent to the culture unit, wherein the detection unit
includes: a microscope imaging apparatus according to claim 1; and
a preserving unit for preserving the biological specimen in a
predetermined state.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a microscope imaging
apparatus and to a biological-specimen examination system using the
same.
[0003] 2. Description of Related Art
[0004] A known apparatus for measuring fluorescence and so on of a
biological specimen uses a microplate. Since the fluorescence
intensity of a biological specimen is extremely dark, it is
necessary to capture the fluorescence using a long exposure time.
Also, since the microplate has a shape that is larger than the
field of view of a microscope, it takes some time to examine the
entire microplate.
[0005] One method of imaging such fluorescence is a method in which
measurement is carried out with a CCD (charge coupled device) while
the microplate is repeatedly moved and stopped. A plurality of
wells are provided in the microplate, and the specimens are held in
these wells. Therefore, by repeatedly moving and stopping the
microplate, the fluorescence from the specimen in each well can be
measured. The microplate is moved by means of a moving stage.
[0006] Another known method of imaging the fluorescence is a method
using TDI (time delay integration) imaging devices (for example the
pamphlet of International Publication No. 03/014400 (FIG. 1,
etc.)). The imaging devices using the TDI method are constituted by
a plurality of optoelectronic devices. Fluorescence from the
specimen is incident on the optoelectronic devices, and a charge
corresponding to the incident fluorescence is produced in the
optoelectronic devices by optical-to-electrical conversion. This
charge is then transferred between optoelectronic devices as the
microplate moves. Since the motion of the microplate is associated
with the motion of the charge, fluorescence from the same position
on the specimen is incident again on the optoelectronic devices
after they have been moved. As a result, the charge builds up.
Thus, the charge is progressively accumulated when using the TDI
method.
[0007] The feature of the TDI method is that charge corresponding
to the fluorescence is accumulated while being transferred.
Therefore, compared to the case where a one-dimensional line sensor
is used for image acquisition, the speed at which the stage is
moved can be increased according to the number of charge transfer
lines. As a result, the measurement time can be shortened.
BRIEF SUMMARY OF THE INVENTION
[0008] A microscope imaging apparatus according to the present
invention comprises: [0009] a stage that holds an object under
examination; [0010] an illumination unit that illuminates the
object under examination; [0011] an image-acquisition unit that
acquires images of the object under examination; [0012] a motion
unit that moves the stage and the image-acquisition unit relative
to each other; and [0013] a control unit that controls the
image-acquisition unit and the motion unit, [0014] wherein the
image-acquisition unit includes an imaging device that is capable
of acquiring images with the time delay integration method, and
[0015] when image acquisition of the object under examination is
carried out a plurality of times, the control unit makes the
exposure times of the imaging device different for each image
acquisition.
[0016] Another microscope imaging apparatus according to the
present invention comprises: [0017] a stage that holds an object
under examination; [0018] an illumination unit that illuminates the
object under examination; [0019] an image-acquisition unit that
acquires images of the object under examination; [0020] a motion
unit that moves the stage and the image-acquisition unit relative
to each other; and [0021] a control unit that controls the
image-acquisition unit and the motion unit, [0022] wherein the
image-acquisition unit includes an imaging device that is capable
of acquiring images with a time delay integration method, and
[0023] by performing a prescan of the object under examination to
obtain a detected intensity of the object under examination, the
control unit determines an exposure time for image acquisition
after the prescan on the basis of the detected intensity.
[0024] Another microscope imaging apparatus according to the
present invention comprises: [0025] a stage that holds an object
under examination; [0026] an illumination unit that illuminates the
object under examination; [0027] an image-acquisition unit that
acquires images of the object under examination; [0028] a motion
unit that moves the stage and the image-acquisition unit relative
to each other; and [0029] a control unit that controls the
image-acquisition unit and the motion unit, [0030] wherein the
image-acquisition unit includes an imaging device that is capable
of image acquisition using two methods; [0031] the control unit
includes: [0032] an examination-object-parameter input unit for
inputting information about the object under examination as an
examination-object parameter; [0033] a calculation unit that
calculates a time for the relative motion on the basis of the
examination-object parameter which has been input; and [0034] a
switching unit that switches the image-acquisition method of the
imaging device on the basis of the calculation result; and [0035]
the two image-acquisition methods are a time delay integration
method and a two-dimensional imaging method in which accumulated
charge is produced by a single exposure.
[0036] A biological-specimen examination system according to the
present invention comprises: [0037] a culture unit for culturing a
biological specimen; and [0038] a detection unit disposed adjacent
to the culture unit, [0039] wherein the detection unit includes:
[0040] an above-described microscope imaging apparatus; and [0041]
a preserving unit for preserving the biological specimen in a
predetermined state.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0042] FIGS. 1A to 1C depict an image acquisition operation using
the TDI method.
[0043] FIGS. 2A to 2C depict the transfer of signal charge in the
TDI method.
[0044] FIGS. 3A to 3C show the intensity of signal charge
accumulated in horizontal lines.
[0045] FIG. 4 shows the overall configuration of a microscope
imaging apparatus.
[0046] FIGS. 5A and 5B show the structure of a specimen.
[0047] FIG. 6 depicts scanning in the TDI method.
[0048] FIG. 7 is a flowchart showing a measurement procedure
according to a first embodiment.
[0049] FIGS. 8A to 8C are graphs showing the relationship between
specimen brightness and output level.
[0050] FIG. 9 is a timing chart for an electronic shutter used in
the TDI method.
[0051] FIG. 10 shows another example of the structure of a
specimen.
[0052] FIG. 11 shows the overall configuration of a microscope
imaging apparatus according to a second embodiment.
[0053] FIG. 12 is a flowchart showing a measurement procedure in
the second embodiment.
[0054] FIG. 13 is a graph showing the relationship between the
number of saturated pixels and the exposure time used in the next
image acquisition.
[0055] FIG. 14 shows the overall configuration of a microscope
imaging apparatus according to a third embodiment.
[0056] FIG. 15 depicts an image acquired in the third
embodiment.
[0057] FIG. 16 is a flowchart showing a measurement procedure in a
fourth embodiment.
[0058] FIG. 17 is a graph showing the relationship between prescan
brightness and exposure time of the main measurement.
[0059] FIG. 18 shows another example of the microscope imaging
apparatus in the fourth embodiment.
[0060] FIG. 19 shows the overall configuration of a microscope
imaging apparatus in a fifth embodiment.
[0061] FIG. 20 shows the overall configuration of a microscope
imaging apparatus in a sixth embodiment.
[0062] FIG. 21 shows the overall configuration of a microscope
imaging apparatus in a seventh embodiment.
[0063] FIG. 22 is a flowchart showing a measurement procedure in
the seventh embodiment.
[0064] FIGS. 23A and 23B depict the structure of a specimen in an
eighth embodiment.
[0065] FIG. 24 shows the overall configuration of a microscope
imaging apparatus in a ninth embodiment.
[0066] FIGS. 25A and 25B show parameters used in estimating the
scanning time.
[0067] FIG. 26 depicts scanning in a two-dimensional imaging
method.
[0068] FIG. 27 depicts the operation of a stage in the
two-dimensional imaging method.
[0069] FIG. 28 shows a specimen in which the density of sample
located portions is asymmetric.
[0070] FIG. 29 depicts an example in which scanning is switched
between the TDI method and the two-dimensional imaging method.
[0071] FIG. 30 is a perspective view showing a biological-specimen
examination system according to a tenth embodiment of the present
invention, which is provided with a microscope imaging
apparatus.
[0072] FIG. 31 is a schematic diagram showing the system
configuration of the biological-specimen examination system shown
in FIG. 30.
[0073] FIG. 32 is a perspective view of an incubator box used in
the biological-specimen examination system shown in FIG. 30.
[0074] FIG. 33 is a cross-sectional view of a chamber in the
incubator box shown in FIG. 32.
[0075] FIGS. 34A and 34B are perspective views showing other
examples of the incubator box shown in FIG. 32.
[0076] FIGS. 35A to 35D show examples of selecting the scanning
method and the detection regions in the biological-specimen
examination system.
[0077] FIG. 36 is a flowchart showing a procedure for setting
measurement parameters used in the biological-specimen examination
system.
[0078] FIGS. 37 and 38 are flowcharts showing a measurement
procedure in the biological-specimen examination system.
[0079] FIG. 39 is a flowchart showing an image processing method in
the biological-specimen examination system.
[0080] FIG. 40 is a flowchart showing a data processing procedure
in the biological-specimen examination system.
[0081] FIG. 41 is a flowchart showing a procedure for adjusting
intensity in the biological-specimen examination system.
[0082] FIG. 42 is a flowchart showing the method of supplying and
replacing culture fluid in the biological-specimen examination
system.
[0083] FIG. 43 shows a cell-tracking image representing the motion
of cells over time.
[0084] FIGS. 44 and 45 are flowcharts showing culturing and
measurement using a microplate.
[0085] FIGS. 46A and 46B are, respectively, an elevational view and
a side view of a biological-specimen examination system according
to an eleventh embodiment, in which a microscope imaging apparatus
is provided.
[0086] FIG. 47 is a plane view of a culture stage in the
biological-specimen examination system shown in FIGS. 46A and
46B.
[0087] FIG. 48 is a perspective view of the culture stage in the
biological-specimen examination system shown in FIGS. 46A and
46B.
DETAILED DESCRIPTION OF THE INVENTION
[0088] A microscope imaging apparatus will be described below with
reference to FIGS. 1A to 6.
[0089] First, an image-acquisition operation according to a time
delay integration (TDI) method will be described using FIGS. 1A to
1C.
[0090] FIGS. 1A, 1B, and 1C depict the TDI image-capturing
operation. FIGS. 2A, 2B, and 2C depict the transfer of signal
charge in the TDI method. FIG. 3A shows the level of signal charge
accumulated in a horizontal line LA, FIG. 3B shows the level of
signal charge accumulated in a horizontal line LB, and FIG. 3C
shows the level of signal charge accumulated in a horizontal line
LC.
[0091] For the sake of simplifying the explanation, a star-shaped
specimen is assumed here. However, the shape of the specimen is not
particularly limited to this shape.
[0092] The positional relationship between a specimen (subject
under examination) 20 and an imaging area S of an imaging device 63
is shown in FIG. 1A. Image-acquisition begins when the top of the
specimen 20 and the bottom of the imaging area A overlap each
other. Thus, initially, only the tip of the upward-facing point of
the star projects onto the imaging device 63. At this time, as
shown in FIG. 2A, the image of the tip of the upward-facing point
is acquired by the horizontal line LA of the imaging device 63. As
shown in FIG. 3A, signal charge with a level corresponding to the
light intensity (brightness) from the specimen 20 is accumulated in
the horizontal line LA.
[0093] Subsequently, a stage 30 is moved with a predetermined
timing. As shown in FIG. 1B, the stage 30 is moved in the positive
Y direction by a distance corresponding to one horizontal line of
the imaging device 63. By doing so, the entire upward-facing point
of the specimen 20 is imaged.
[0094] At this point, as shown in FIG. 2B, the signal charge
accumulated in the horizontal line LA is transferred to the
horizontal line LB in synchronization with the motion of the stage
30. Thus, the image of the specimen 20 is acquired by the
horizontal lines LA and LB of the imaging device 63.
[0095] Therefore, as shown in FIG. 3B, the signal charge
accumulated during this image acquisition is added to the signal
charge accumulated in the previous acquisition. In other words, the
signal charges obtained due to the previous and current image
acquisition are accumulated in the horizontal line LB. As a result,
the signal charge level is twice as high as that measured in one
line.
[0096] In this way, charge transfer is carried out from the state
shown in FIG. 2A to the state shown in FIG. 2B. The time interval
at which this charge transfer is carried out is called the TDI line
transfer rate.
[0097] The stage 30 is then moved further at the predetermined
timing. That is, as shown in FIG. 1C, the stage 30 is again moved
in the positive Y direction by an amount corresponding to one
horizontal line of the imaging device 63. By doing so,
image-acquisition of the specimen 20 proceeds.
[0098] At this point, as shown in FIG. 2C, the signal charge
accumulated in the horizontal line LB is transferred to the
horizontal line LC in synchronization with the motion of the stage
30. In addition, the signal charge accumulated in the horizontal
line LA is transferred to the horizontal line LB. Thus, the image
of the specimen 20 is acquired by the horizontal lines LA, LB, and
LC.
[0099] Therefore, as shown in FIG. 3C, the signal charge obtained
during this image-acquisition is added to the horizontal line LC.
Thus, the signal charges obtained by the previous-but-one
image-acquisition, the previous image-acquisition, and the current
image-acquisition are accumulated in the horizontal line LC. In
other words, the level of signal charge is three times higher than
that measured with a single line. The signal charge obtained in the
previous image-acquisition and the current image-acquisition are
accumulated in the horizontal line LB.
[0100] By continuing with the above-described operation, the same
number of image-acquisition operations as the number of horizontal
scanning lines is performed. Accordingly, a signal charge for the
same part of the specimen 20 is accumulated corresponding to the
number of image-acquisition operations. Thus, with the TDI method,
the image of the specimen 20 projected onto the imaging surface is
shifted along with the motion of the stage 30, and the signal
charge accumulated in the imaging device 63 is shifted in
synchronization therewith.
[0101] Next, the exposure time used when carrying out
image-acquisition in the TDI method will be discussed.
[0102] The exposure time in the TDI method is the time it takes for
the signal light returning from the specimen 20 to be accumulated
as the signal charge. In other words, the exposure time in the TDI
method can be expressed as the product of the TDI line transfer
rate and the cumulative number of pixels. Therefore, to change the
exposure time, the TDI line transfer rate can be made faster to
lengthen the exposure time or the TDI line transfer rate can be
slowed down to shorten the exposure time.
[0103] For example, when using a CCD camera having 1,000 pixels in
the Y direction as the imaging device 63, to acquire images with an
exposure time of 0.2 s, the line transfer rate is set to 5 kHz (0.2
ms transfer time) according to the calculation shown below: 0.2
s/1000=0/2 ms.
[0104] Next, the stage scanning speed used in the TDI method will
be discussed.
[0105] The stage 30 is moved in synchronization with the transfer
of the signal charge. Therefore, the speed at which the stage 30 is
moved can be expressed as the pixel size on the stage 30 divided by
the TDI line transfer rate. The pixel size on the stage 30 can be
obtained from the pixel size of the imaging device 63 (for example,
a CCD) and the projection magnification.
[0106] For example, with a projection magnification of 10, a pixel
size in the imaging device 63 of 6.45 .mu.m, and a TDI line
transfer rate of 5 kHz, the speed of the stage is 3.23 mm/s,
according to the calculation shown below: 6.45.times.10.sup.-3
mm/10.times.5.times.10.sup.3 Hz=3.23 mm/s.
[0107] Next, the structure of a microscope imaging apparatus will
be described.
[0108] FIG. 4 shows the overall configuration of a microscope
imaging apparatus 10. The microscope imaging apparatus 10 shown in
FIG. 4 is used in the first to third embodiments of the present
invention described below.
[0109] As shown in FIG. 4, the microscope imaging apparatus 10
includes the stage 30, an illumination unit 40, and an
image-acquisition unit 60. The stage 30 holds the specimen (object
under examination) 20 thereon and is moveable. The illumination
unit 40 radiates illumination light onto the specimen 20. The
image-acquisition unit 60 acquires signal light emitted from the
region irradiated with the illumination light and measures it.
[0110] The illumination unit 40 includes components capable of
Kohler illumination. Specifically, these are a lamp 41, a collector
lens 42, a reflecting mirror 43, a field stop 44, and a lens 45. A
gas discharge lamp or the like, such as a halogen lamp, xenon lamp,
or mercury lamp, is used at the light source lamp 41. The collector
lens 42 collects light emitted from the lamp 41. The reflecting
mirror 43 reflects light traveling backwards from the lamp 41 back
towards the lamp 41 again. The field stop 44 is disposed after the
collector lens 42. This field stop 44 is disposed at a conjugate
position with respect to the focal point of an objective lens 49
described below. The diameter of the opening of this field stop 44
is adjustable. A lens 45 is disposed after the field stop 44. The
lens 45 images the field stop 44 at infinity.
[0111] An aperture stop 46 is disposed after the lens 45, at the
focal plane thereof. The diameter of the opening of this aperture
stop 45 can be varied. By doing so, the size of the beam diameter
at the exit-pupil position of the objective lens 49 can be
adjusted. A mirror 47 is disposed after the aperture stop 46. A
dichroic mirror that reflects light from the lamp 41 and that
transmits return light (fluorescence) from the specimen 20 is used
as the mirror 47. A half-mirror may be used instead of a dichroic
mirror.
[0112] The objective lens 49 is disposed after the mirror 47. The
stage 30 is disposed at a position facing the objective lens
49.
[0113] A stage driving mechanism (motion unit) 31 for driving the
stage 30 is provided. The stage driving mechanism (motion unit) 31
drives the stage 30 in the X and Y directions on the basis of a
signal output from a computer described below. Known technology,
for example, a sliding motion mechanism, may be used as the stage
driving mechanism 31. It is not particularly limited to a sliding
motion mechanism, however.
[0114] An imaging lens 61 and a detector 62 are provided in the
image-acquisition unit 60. Return light (fluorescence) from the
specimen 20 is incident on the imaging lens 61, and the imaging
lens 61 focuses (images) the incident light at a predetermined
position. The detector 62 is disposed at a predetermined position
and detects the return light from the specimen 20. The imaging
device 63, which can acquire images by the TDI method, is provided
in the detector 62. As described above, the imaging device 63
accumulates signal charge in each horizontal line in response to
the movement of the stage 30.
[0115] The output of the detector 62 is connected to an image
processing unit 64, which processes the output signal from the
detector 62. A monitor 65 for displaying the processed signal is
connected to the image processing unit 64. Also, the image
processing unit 64 is connected to the computer 66. The stage
driving mechanism 31 is also connected to the computer 66.
[0116] The computer (calculating unit) 66 calculates the exposure
time in the main measurement. This calculation is carried out on
the basis of information obtained during a prescan, as described
above. This information is the brightness in each sample located
portion (described below).
[0117] FIG. 5A depicts the shape of the specimen 20. FIG. 5B is a
magnified view showing a sample located portion 22 of the specimen
20.
[0118] As shown in FIG. 5A, the specimen 20 includes a transparent
substrate 21. This transparent substrate 21 is formed of, for
example, a glass plate or a plastic plate. The sample located
portions 22 are formed in a matrix on the transparent substrate 21.
That is, a two-dimensional patterned portion 23 is formed by the
plurality of sample located portions 22.
[0119] The sample located portions 22 have a substantially circular
shape with a diameter of a few millimeters in plane view and have a
concave profile in cross-section. As shown in FIG. 5B, cells,
serving as the objects to be measured, are disposed in the sample
located portions 22. The cells are cultured inside the sample
located portions 22.
[0120] Next, stage scanning in the TDI method will be
described.
[0121] FIG. 6 depicts the scanning in the TDI method. In this
embodiment, the stage 30 is moved to perform scanning. However, in
FIG. 6, for the sake of simplifying the drawing, the imaging device
63 is depicted as moving.
[0122] As shown in FIG. 6, when measuring the specimen 20 with the
TDI method, the two-dimensional patterned portion 23 is completely
scanned. This scanning is performed regardless of whether or not
the sample located portions 22 are present.
[0123] When the stage 30 is scanned in the Y-axis direction, the
stage 30 moves at constant velocity in the -Y direction. On the
other hand, the imaging device 63 is capable of transferring charge
only in one direction. Therefore, as shown in FIG. 6, measurement
is carried out only while the stage 30 is advancing in the -Y
direction. Measurement is not carried out while the stage 30 is
advancing in the +Y direction. The motion in the +Y direction is
for returning the stage to its initial position. The time required
to return to the initial position may correspond to, for example,
the time required for storing the acquired data in a hard disk of
the computer 66.
First Embodiment
[0124] Next, a first embodiment of the present invention will be
described. Here, a measurement procedure will be described using
FIG. 7.
[0125] FIG. 7 is a flowchart showing the measurement procedure in
this embodiment. FIGS. 8A to 8C show the relationship between the
brightness of the specimen 20 and the output level from the imaging
device 63. FIG. 8A shows the relationship between the brightness
obtained in a first scan and the output level, FIG. 8B shows the
relationship between the brightness obtained in a second scan and
the output level, and FIG. 8C shows the dependency of the
brightness obtained in the first and second scans no the output
level.
[0126] First, the first scan (image acquisition) is performed to
acquire an image of the specimen 20 (step S1). This corresponds to
the prescan.
[0127] In this step, while the specimen 20 (stage 30) is moved from
position a to position b shown in FIG. 6, the specimen 20 is imaged
with a predetermined exposure time.
[0128] The image acquired in the first scan will be described in
terms of the relationship between the brightness of the image and
the output level from the imaging device 63. In this case, as shown
in FIG. 8A, there is a region where the accumulated charge is
saturated (hereinafter referred to as saturation-level region) and
a region where the accumulated charge is low (hereinafter referred
to as low-level region).
[0129] Next, preparation for the second scan is carried out (step
S2).
[0130] The output levels of the saturation-level region and the
low-level region are appropriately set. More specifically, exposure
times of suitable length are set for each of these regions.
[0131] That is, when the saturation-level region is to be
eliminated, the exposure time is set to be shorter than the
exposure time in the first scan. At this time, the output level is
set to utilize the entire dynamic range of the imaging device 63.
When the low-level region is to be eliminated, the exposure time is
set to be longer than the exposure time in the first scan. In this
case too, the output level is set to utilize the entire dynamic
range of the imaging device 63.
[0132] Simultaneously, the specimen 20 (stage 30) is moved from
position b to position a.
[0133] This embodiment is described in terms of an example in which
the saturation-level region is eliminated.
[0134] Next, a second scan (image acquisition) is performed to
acquire an image of the specimen 20 again (step S3).
[0135] In this image acquisition, the exposure time determined in
step S2 is used to acquire an image of the specimen 20 in the same
way as in step S1.
[0136] The image acquired in the second scan is a graph indicating
the relationship between the brightness of the acquired image and
the output level of the imaging device 63. The image acquired in
the second scan is shown in FIG. 8B. As shown in this figure, the
saturation in the saturation-level region in the previous scan is
eliminated, whereas the output level is increased in the low-level
region.
[0137] Next, the images acquired in the first and second scans are
combined into a single image (step S4). A predetermined image
processing operation is carried out during this image
combining.
[0138] In this step, the images acquired in steps S1 and S3 are
combined. By doing so, a single image with wider dynamic range can
be obtained.
[0139] Thus, in this embodiment, as shown in FIG. 8C, the image
acquired in step S1 with the long exposure time and the image
acquired in step S3 with the short exposure time are combined to
form a single image.
[0140] Thereafter, the specimen 20 (stage 30) is moved from
position b to position c, and the process proceeds to preparation
for measuring the next line.
[0141] This embodiment has been described in terms of an example in
which scanning of the same position is repeated twice. However, the
number of scans is not limited to two. For example, scanning may be
repeated at the same position three or more times. In such a case,
between the image acquisitions performed in each scan, preparation
for the next scan is carried out, and the acquired images are
combined at the end.
[0142] Instead of the imaging device 63 described above, an imaging
device 63a having an electronic shutter may be used. Use of such an
imaging device 63a is preferable since it can reduce the exposure
time.
[0143] The electronic shutter is the type of shutter normally used
in CCD digital cameras and mobile telephones with built-in cameras.
Such an electronic shutter uses a phenomenon whereby charge is not
accumulated if the CCD is not operating, even if exposed to light.
Accordingly, the CCD function itself is one form of shuttering.
[0144] On the other hand, in a mechanical shutter used in a
silver-halide camera, an opaque plate is placed between the lens
and the film, and exposure is performed by opening and closing
(moving) this plate. Thus, the electronic shutter is different from
the mechanical shutter. The electronic shutter controls the
exposure time without using a mechanical shutter.
[0145] FIG. 9 is a timing chart for the electronic shutter in the
TDI method.
[0146] As shown in FIG. 9, for example, when the maximum transfer
rate in the TDI method is t1 (seconds), the charge is transferred
to a neighboring horizontal line every t1 seconds. The timing of
the transfers is indicated by the arrows in the figure.
[0147] The electronic shutter is open for a period of t2 seconds
from each charge transfer, and charge accumulation is performed. In
other words, the charge accumulation of the CCD is carried out for
t2 seconds after every charge transfer. On the other hand, the
exposure time when not using the electronic shutter is t1.
Therefore, the ratio of the exposure time when using the electronic
shutter to the exposure time when not using the electronic shutter
is t2/t1.
[0148] For example, assume that the imaging device 63a has 1000
pixels in the Y direction and a maximum transfer rate (t1) of 10
kHZ (0.1 ms). When using this imaging device 63a to acquire images
with an exposure time of 1 ms, the open time of the electronic
shutter (t2) should be set to 0.001 ms, according to the
calculation shown below: 1000.times.0.1 (ms).times.(t2/0.1)=1 (ms)
t2=0.001 ms
[0149] When the electronic shutter is not used, the shortest
exposure time is determined by the product of the maximum TDI
transfer rate and the cumulative number of pixels. Accordingly, by
using an electronic shutter of the type described above, it is
possible to increase the range of exposure times for which
measurement is possible. Therefore, even a high-brightness specimen
20 can be accurately measured.
[0150] With the configuration described above, two images obtained
by two scans with different exposure times in steps S1 and S3 are
combined into a single image (S4). By doing so, it is possible to
acquire an image with a wider dynamic range compared to an image
acquired with a single exposure time. Therefore, it is possible to
accurately carry out measurement even for a specimen 20 whose
brightness variation is large.
[0151] As shown in FIGS. 5A and 5B, the specimen 20 may include
only the sample located portions 22 on the transparent substrate
21. Alternatively, as shown in FIG. 10, a reference line BL may
also be formed in a region close to the left-hand side of the
transparent substrate 21.
[0152] If the reference line BL is formed, it can serve as a
reference when combining the images acquired in each scan.
Accordingly, it is possible to simplify the image combining
operation.
[0153] In this embodiment, since fluorescence examination is
carried out, it is preferable that the reference line BL be formed
of a material that fluoresces.
Second Embodiment
[0154] Next, a second embodiment of the present invention will be
described with reference to FIGS. 11 to 13.
[0155] The basic structure of the microscope imaging apparatus of
this embodiment is the same as that of the first embodiment, but
the method of measuring the specimen is different from that in the
first embodiment. Therefore, in this embodiment, only the method of
measuring the specimen shall be described, using FIGS. 11 to 13,
and a description of the TDI method and so on shall be omitted.
[0156] FIG. 11 depicts the overall configuration of a microscope
imaging apparatus 110 of this embodiment.
[0157] As shown in FIG. 11, the microscope imaging apparatus 110
includes a stage 30, an illumination unit 40, and an
image-acquisition unit 160. The stage 30 holds a specimen 20 and is
moveable. The illumination unit 40 irradiates the specimen 20 with
illumination light. The image-acquisition unit 160 acquires signal
light emitted from the region irradiated with illumination and
measures it.
[0158] An imaging lens 61 and a detector 62 are provided in the
image-acquisition unit 160. Return light from the specimen 20 is
incident on the imaging lens 61, and the detector 62 detects the
return light.
[0159] The detector 62 is connected to an image processing unit 64,
which processes the output of the detector 62. The image processing
unit 64, a monitor 65 for displaying the processed signal, and a
stage driving mechanism 31 are connected to a computer 166.
[0160] An exposure-time input unit 167 and a setting unit 168 are
provided in the computer 166. A maximum exposure time Tmax is input
using the exposure-time input unit 167. This maximum exposure time
Tmax is related to measurement of the specimen 20 and is set by the
user. The setting unit 168 is used to set an exposure time Tmeasure
used for image acquisition and measurement.
[0161] Next, the measurement procedure will be described.
[0162] FIG. 12 is a flowchart showing the measurement procedure in
this embodiment.
[0163] First, the user sets the maximum exposure time Tmax (step
S10) by inputting the maximum exposure time Tmax to the
exposure-time input unit 167.
[0164] Next, a first scan (image acquisition) is performed to
acquire an image of the specimen 20.
[0165] In this step, the maximum exposure time Tmax input to the
setting unit 168 is set as the exposure time Tmeasure used for
image acquisition and measurement. Therefore, an image of the
specimen 20 is acquired with the maximum exposure time Tmax.
[0166] Next, preparation is arried out for a second scan (step
S12).
[0167] In this step, the setting unit 168 sets the exposure time
Tmeasure for the second scan on the basis of the magnitude (number
of saturated pixels) in the saturation-level region.
[0168] More specifically, as shown in FIG. 13, the reltionship
(table) between the number of saturated pixels and the exposure
time in the next image acquisition is stored in the setting unit
168. On the basis of this table, the computer 66 sets the exposure
time Tmeasure for the second scan. Thus, as the number of saturated
pixels, for example, in each sample located portion 22 increases,
the exposure time for the second scan decreases.
[0169] Saturated pixels shall now be explained. For example, if a
12-bit CCD is used as the imaging device 63, the maximum number of
gradation levels is 4095 (=2.sup.12, the maximum value that can be
represented by 12 bits). Therefore, a saturated pixel is a pixel
for which the imaging device outputs a value of 4095, in other
words, a pixel whose output value reaches the upper limit.
[0170] Next, a second scan (image acquisition) is performed to
acquire an image of the specimen 20 (step S13), and the images
acquired in the first and second scans are combined to form a
single image (step S14).
[0171] The measurement procedure after step S13 is the same as in
the first embodiment, and therefore it is merely shown in FIG. 12,
but a description thereof is omitted.
[0172] With the configuration described above, the user inputs the
maximum exposure time Tmax in advance to the exposure-time input
unit 167. By doing so, the exposure time can be reduced. Thus,
image acquisition can be made more efficient, and it is possible to
efficiently acquire images with a wider dynamic range.
[0173] This embodiment has been described in terms of an example in
which the user sets the maximum exposure time. However, the time
input to the exposure-time input unit 167 may be just the actual
exposure time. In other words, it is not limited to the maximum
exposure time.
Third Embodiment
[0174] Next, a third embodiment will be described with reference to
FIGS. 14 and 15.
[0175] The basic configuration of the microscope imaging apparatus
of this embodiment is the same as that in the first embodiment, but
the configuration of the image-acquisition unit is different from
the first embodiment. Therefore, in this embodiment only the
image-acquisition unit will be described using FIGS. 14 and 15, and
a description of the illumination unit and so on shall be
omitted.
[0176] FIG. 14 shows the overall configuration of a microscope
imaging apparatus 210 in this embodiment.
[0177] As shown in FIG. 14, the microscope imaging apparatus 210
includes a stage 30, an illumination unit 40, and an
image-acquisition unit 260. The stage 30 holds a specimen 20 and is
moveable. The illumination unit 40 irradiates the specimen 20 with
illumination light. The image-acquisition unit 260 acquires signal
light emitted from the region irradiated with the illumination
light and measures it.
[0178] An imaging lens 61 and a detector 262 are provided in the
image-acquisition unit 260. Return light from the specimen 20 is
incident on the imaging lens 61, and the detector 262 detects the
return light from the specimen 20. An imaging device 263 is
disposed in the detector 262. The imaging device 263 has an
anti-smear function and is capable of acquiring images on the basis
of the TDI method. As described above, the TDI method is a
technique in which, according to the motion of the stage 30, the
signal charge in each horizontal line is accumulated in the imaging
device 63.
[0179] Smearing is a phenomenon occurring in photoelectric
conversion devices like CCDs (charge coupled devices). When an
excessive signal above a certain level is input to a photoelectric
conversion device, this phenomenon produces a false signal as a
result of completely changing the signal in the longitudinal or
transverse direction during signal processing.
[0180] That is, smearing is a phenomenon whereby, when a highly
bright spot of light, for example, is incident on a photoelectric
conversion device, a bright band of light extending in the
longitudinal or transverse direction is produced.
[0181] It is possible to prevent the occurrence of smearing with
the configuration described above. For example, as shown in FIG.
15, a cell BC of high brightness and a cell DC of low brightness
are adjacent to each other. In this case, if the exposure time is
long enough for the low-brightness cell (biological specimen) DC,
smearing occurs in the image of the high-brightness cell BC.
However, with the configuration described above, even if image
acquisition is carried out with a long exposure time, which is
suitable for the low-brightness cell DC, it is possible to prevent
the occurrence of smearing. Therefore, it is possible to accurately
measure both the high-brightness cell BC and the low-brightness
cell DC.
[0182] When using an imaging device not having the anti-smear
function, as described above, there is a risk of causing smearing
at the low-brightness cell DC due to the high-brightness cell BC.
As a result, it may not be possible to accurately measure the
low-brightness cell DC.
[0183] With the microscope imaging apparatus of this embodiment,
when acquiring a plurality of images of the object under
examination, it is possible to acquire images with different
exposure times. Then, these images are combined into a single
image. By doing so, it is possible to accurately perform
measurement even for an examination object having significant
brightness variation.
Fourth Embodiment
[0184] Next, a fourth embodiment will be described. The microscope
imaging apparatus shown in FIG. 4 is also used in this embodiment.
The measurement procedure shall be described using FIG. 16.
[0185] FIG. 16 is a flowchart showing the measurement procedure in
this embodiment.
[0186] First, when measurement commences, prescan preparation is
carried out (step S201). In this step, the stage 30 moves to a
measurement starting point (the point where the imaging device 63
and the specimen 20 have the positional relationship shown in FIG.
6).
[0187] Next, the prescan is carried out (step S202). Fluorescence,
for example, is emitted from each of the sample located portions
22, and the prescan is performed to acquire the brightness
distribution of this fluorescence. The exposure time used in the
prescan is set to be sufficiently short so that the signal charge
accumulated in the imaging device 63 is not saturated. As described
above, the movement speed of the stage 30 is set according to the
TDI line transfer rate.
[0188] FIG. 17 is graph showing the relationship between the
prescan brightness and the exposure time in the main
measurement.
[0189] When the prescan is completed, preparation for the main
measurement of the specimen 20 is carried out (step S203). In this
step, the stage 30 is moved back to the measurement starting
point.
[0190] The brightness in each sample located portion 22 has already
been acquired in the prescan (step S202). Therefore, from among the
acquired brightnesses, the maximum value, that is, the maximum
intensity value (count), is found. This maximum intensity value is
the digital value output from the imaging device 63.
[0191] Then, on the basis of the maximum intensity value, the
exposure time for each sample located portion 22 in the current
measurement is determined. The exposure time is determined on the
basis of the relationship between the prescan brightness and the
exposure time for the main measurement, as shown in FIG. 17. The
relationship shown in FIG. 17 is obtained in advance before
measurement. (For example, the relationship shown in FIG. 17 is
obtained by measurement or simulation.)
[0192] Once the exposure time has been determined, the main
measurement (image acquisition) of the specimen 20 is carried out
(step S204). In this step, the image of each sample located portion
22 is acquired on the basis of the exposure time found in the
main-measurement preparation step (step S203). As described above,
the exposure time is expressed by the TDI line transfer rate and
the cumulative number of charged pixels. Also, the stage 30 is
moved at a speed in synchronization with the TDI line transfer
rate.
[0193] In this embodiment too, instead of the imaging device 63
described above, an imaging device 63a having an electronic shutter
may be used. Use of such an imaging device 6.3a is beneficial in
that it is possible to reduce the exposure time. The electronic
shutter and the mechanical shutter are as described previously.
[0194] The TDI method is also used in this embodiment. The timing
chart for the electronic shutter used in this TDI method is as
shown in FIG. 9.
[0195] With the configuration described above, it is possible to
obtain a suitable exposure time for measurement of the specimen 20
on the basis of the prescan performed before measurement of the
specimen 20. Accordingly, on the basis of the prescan before
measurement, a suitable exposure time can be determined even when
measuring a specimen 20 whose brightness changes with time. As a
result, the specimen can be accurately measured.
[0196] More concretely, setting an exposure time longer than the
appropriate time can prevent the problem of saturation of the
accumulated charge. Also, setting an exposure time shorter than the
appropriate time can prevent the problem of insufficient charge
accumulation (only accumulating the noise level).
[0197] FIG. 18 shows an example of the configuration of another
microscope imaging apparatus 200. As shown in FIG. 18, for example,
a light control member 70 may be disposed between the imaging lens
61 and the detector 62 in the image-acquisition unit 60. For
example, a neutral density (ND) filter or an electro-micro mirror
device whose transmittance is varied by application of an electric
potential may be used as the light control member 70.
[0198] With the configuration described above, the transmittance of
the light control member 70 can be adjusted according to the
exposure time obtained in the prescan. Thus, the range of possible
exposure times for measurement can be increased. As a result, it is
possible to accurately carry out measurement even for a specimen 20
having high brightness or low brightness.
Fifth Embodiment
[0199] Next, a fifth embodiment will be described with reference to
FIG. 19.
[0200] The basic configuration of the microscope imaging apparatus
of this embodiment is the same as that of the fourth embodiment,
but the prescan method is different from that in the fourth
embodiment. Therefore, in this embodiment, only the prescan method
is described using FIG. 19, and a description of the main
measurement method and so on is omitted.
[0201] FIG. 19 shows the overall configuration of a microscope
imaging apparatus 210 of this embodiment.
[0202] As shown in FIG. 19, the microscope imaging apparatus 210
includes a stage 30, an illumination unit 140, and an
image-acquisition unit 60. The stage 30 holds a specimen 20 and is
moveable. The illumination unit 140 irradiates the specimen 20 with
illumination light. The image-acquisition unit 60 acquires signal
light emitted from the region irradiated with the illumination
light and measures it.
[0203] A light source lamp 41, a collector lens 42, a reflecting
mirror 43, a field stop 44, a lens 45, an aperture stop 46, and a
mirror 47 are provided in the illumination unit 140. The collector
lens 42 collects light emitted from the lamp 41. The reflecting
mirror 43 reflects backward-propagating light back to the lamp 41.
The diameter of the opening of the field stop 44 can be adjusted.
The lens 45 images the field stop 44 at infinity. The diameter of
the opening of the aperture stop 46 can be adjusted. The mirror 47
selectively transmits light. An objective lens 149 forms an image
of the specimen 20; however, since the objective lens 149 focuses
the light from the lamp 41 on the specimen 20, it can also be
considered part of the illumination unit 140.
[0204] A low-magnification objective lens 149A used in the prescan
and a high-magnification objective lens 149B used in the main
measurement are used as the objective lens 149. These objective
lenses 149A and 149B are retained by a known switching mechanism
such as a revolver or the like. Thus, it is possible to change the
objective lens as required.
[0205] With the configuration described above, since it is possible
to acquire a wide-field image during the prescan, the prescan time
can be shortened.
[0206] For example, the high-magnification objective lens 149B used
in the main measurement is a 20.times. lens, and the
low-magnification objective lens 149A used in the prescan is a
4.times. lens. Thus, because the pixel pitch on the stage 30 is
five times larger, the stage speed can be increased by a factor of
five. Furthermore, because the field is five times wider, the
number of line scans can be reduced to one fifth. Therefore,
compared to the case where the prescan and the main measurement are
carried out with the same magnification, the prescan time required
for the entire surface of the specimen 20 can be reduced to
1/25.
[0207] Also, the light (brightness) from the specimen may vary over
time, depending on the specimen. In such a case, the appropriate
exposure time also changes over time. Therefore, if the time
required for the prescan can be reduced, as in this embodiment, it
is possible to reduce the time interval from the prescan to the
main measurement. As a result, it is possible to carry out
measurement with the appropriate exposure time.
Sixth Embodiment
[0208] Next, a sixth embodiment will be described with reference to
FIG. 20.
[0209] The basic configuration of the microscope imaging apparatus
of this embodiment is the same as that of the fourth embodiment,
but the image-acquisition unit is different from that in the fourth
embodiment. Therefore, in this embodiment, only the
image-acquisition unit is described using FIG. 20, and a
description of the illumination unit and so on is omitted.
[0210] FIG. 20 shows the overall configuration of a microscope
imaging apparatus 220 of this embodiment.
[0211] As shown in FIG. 20, the microscope imaging apparatus 220
includes a stage 30, an illumination unit 40, and an
image-acquisition unit 260. The stage 30 holds a specimen 20 and is
moveable. The illumination unit 40 irradiates the specimen 20 with
illumination light. The image-acquisition unit 260 acquires signal
light emitted from the region irradiates with the illumination
light and measures it.
[0212] An imaging lens 61, a detector 62, and a scaling lens 261
are provided in the image-acquisition unit 260. Return light from
the specimen 20 is incident on the imaging lens 61. The detector 62
detects the return light from the specimen 20. The scaling lens 261
can acquire images over a wide field.
[0213] The scaling lens 261 is inserted into the light path between
the objective lens 49 and the detector 62. It may be capable of
being inserted in and removed from the light path. Also, the
scaling lens 261 may be retained by a known switching mechanism
such as a revolver. By doing so, it is possible to switch between
scaling lenses with different magnifications, as required.
[0214] With the configuration described above, by inserting the
scaling lens 261 into the light path between the objective lens 49
and the detector 62 during the prescan, it is possible to acquire
images over a wide field of view. Accordingly, the prescan time can
be reduced.
[0215] Furthermore, since it is possible to reduce the time
interval from the prescan to the main measurement, even for a
specimen 20 whose brightness varies with time, it is possible to
carry out measurement with the appropriate exposure time.
Seventh Embodiment
[0216] Next, a seventh embodiment will be described with reference
to FIGS. 21 and 22.
[0217] The basic configuration of the microscope imaging apparatus
of this embodiment is the same as that of the fourth embodiment,
but the method of controlling the exposure time is different from
that in the fourth embodiment. Therefore, only the method of
controlling the exposure time is described using FIGS. 21 and 22,
and a description of the structure and so on is omitted.
[0218] FIG. 21 shows the overall configuration of a microscope
imaging apparatus 230 in this embodiment. FIG. 22 is a flowchart
showing the measurement procedure in this embodiment.
[0219] As shown in FIG. 21, the microscope imaging apparatus 230
includes a stage 30, an illumination unit 40, and an
image-acquisition unit 360. The stage 30 holds a specimen 20 and is
moveable. The illumination unit 40 irradiates the specimen 20 with
illumination light. The image-acquisition unit 360 acquires signal
light emitted from the region irradiated with the illumination
light and measures it.
[0220] An imaging lens 61 and a detector 62 are provided in the
image-acquisition unit 360. Return light from the specimen 20 is
incident on the imaging lens 61. The detector 62 detects the return
light from the specimen 20.
[0221] The detector 62 is connected to an image processing unit 64,
which processes the output from the detector 62. A monitor 65 for
displaying the processed signal is connected to the image
processing unit 64. A stage driving mechanism 31 is connected to a
computer 366.
[0222] The computer 366 includes a maximum-exposure-time input unit
367, a calculating unit 368, and a selecting unit 369. A maximum
exposure time Tmax for measuring the specimen 20 is input to the
maximum-exposure-time input unit 367. The maximum exposure time
Tmax is set by the user. The calculating unit 368 calculates an
exposure time Tpre on the basis of information obtained in a
prescan. The selecting unit 369 carries out comparison and
selection of the exposure time Tpre and the maximum exposure time
Tmax.
[0223] Next, the measurement procedure will be described.
[0224] As shown in FIG. 22, first, the user inputs the maximum
exposure time Tmax for the main measurment (step S211). Then,
measurement commences.
[0225] Next, prescan preparation is carried out (step S212). In
this step, the stage 30 moves to a measurement starting point (the
point where the imaging device 63 and the specimen 20 have the
positional relationship shown in FIG. 6).
[0226] Next, the prescan is performed (step S213). Fluorescence,
for example, is emitted from each sample located portion 22, and
the prescan is performed to acquire brightness distribution
information of this fluorescence. The exposure time in the prescan
is set to a sufficiently short duration so that, for example, the
signal charge accumulated in the imaging device 63 is not
saturated. Also, as described above, the movement speed of the
stage 30 is set according to the TDI line transfer rate.
[0227] Once the prescan has been completed, preparation for the
main measurement of the specimen 20 is carried out (step S214). In
this step, the stage 30 is moved back to the measurement starting
point.
[0228] The brightness in each sample located portion 22 has already
been acquired in the prescan (S213). Thus, a maximum value, that
is, a maximum intensity value (count), is found from among the
acquired brightnesses. This maximum intensity value is a digital
value output from the imaging device 63.
[0229] Then, the exposure time Tpre for each sample located portion
22 is determined on the basis of the maximum intensity value found.
The exposure time Tpre is determined on the basis of the
relationship between the prescan brightness and the exposure time
in the main measurement, as shown in FIG. 17. The relationship
shown in FIG. 17 is determined in advance before the measurement.
(For example, the relationship shown in FIG. 17 is determined by
measurement or simulation.)
[0230] Here, the maximum exposure time Tmax and the exposure time
Tpre are compared. On the basis of the comparison result, if the
exposure time Tpre is shorter than the maximum exposure time Tmax,
the exposure time Tpre is used as the exposure time for the main
measurement. On the other hand, if the exposure time Tpre is longer
than the maximum exposure time Tmax, the maximum exposure time Tmax
is used as the exposure time in the main measurement.
[0231] When the exposure time has been determined, the main
measurement (image acquisition) of the specimen 20 is carried out
(S215). In this step, an image of each sample located portion 22 is
acquired on the basis of the exposure time determined in the main
measurement preparation (step S214). As described above, the
exposure time is given by the TDI line transfer rate and the
cumulative number of charged pixels. Also, the stage 30 is moved at
a speed in synchronization with the TDI line transfer rate.
[0232] By setting the exposure time as described above, it is
possible to prevent the exposure time from becoming too long when
carrying out the main measurement of a specimen 20 having low
brightness. Moreover, it is possible to measure a specimen 20 whose
brightness varies over time using the appropriate exposure
time.
Eighth Embodiment
[0233] Next, an eighth embodiment will be described with reference
to FIGS. 23A and 23B.
[0234] The basic configuration of the microscope imaging apparatus
of this embodiment is the same as that in the fourth embodiment,
but the specimen is different from that in the fourth embodiment.
Therefore, only the specimen is described using FIGS. 23A and 23B,
and a description of the illumination unit and so on is
omitted.
[0235] FIGS. 23A and 23B show the configuration of a specimen 20 in
this embodiment.
[0236] As shown in FIG. 23A, the specimen 20 is disposed so that
the brightness in the sample located portions 22 arrayed in the X
direction (the sample located portions 22 enclosed by the ellipses
in FIG. 23A) is substantially uniform.
[0237] With the configuration described above, by performing the
prescan in one row in the Y direction in FIG. 23A, it is possible
to obtain an appropriate exposure time. Accordingly, the time
required for the prescan can be reduced.
[0238] The specimen 20 may be arranged as shown in FIG. 23A. Also,
as shown in FIG. 23B, sample located portions 22 having
substantially uniform brightness may be arranged so as to be
grouped in regions formed of two or three rows in the X direction
and two rows in the Y direction (the region enclosed by the
ellipses in FIG. 23B).
[0239] By adopting such an arrangement, as shown in FIG. 23B, it is
possible to achieve the appropriate exposure time by performing the
prescan in two rows. Therefore, the time required for the prescan
can be reduced.
[0240] The arrangement of the sample located portions 22 having
substantially uniform brightness is not particularly limited; any
arrangement is possible so long as the number of prescans can be
reduced.
[0241] With the microscope imaging apparatus of this embodiment, it
is possible to achieve an appropriate exposure time for measuring
the object under examination on the basis of the prescan performed
before the main measurement. Therefore, a specimen whose brightness
varies over time can be accurately measured.
Ninth Embodiment
[0242] A ninth embodiment will now be described. FIG. 24 shows the
overall configuration of a microscope imaging apparatus 310 of this
embodiment. Components that are the same as those in FIG. 4 are
assigned the same reference numeral, and a description thereof
shall be omitted.
[0243] With the microscope imaging apparatus 310 of this
embodiment, it is possible to acquire images by the TDI method and
by a two-dimensional imaging method.
[0244] An imaging lens 61 and a detector 62 are provided in an
image-acquisition unit 60. Return light (fluorescence) from the
specimen 20 is incident on the imaging lens 61. The imaging lens 61
focuses (images) the incident light at a predetermined position.
The detector 62 is disposed at the predetermined position and
detects the return light from the specimen 20. An imaging device 63
that is capable of the TDI method and the two-dimensional imaging
method is provided in the detector 62.
[0245] The output from the detector 62 is connected to an image
processing unit 64 that processes the signal output from the
detector 62. A monitor 65 for displaying the processed signal is
connected to the image processing unit 64. The image processing
unit 64 is connected to a computer 66. A mirror driving mechanism
48 and a stage driving mechanism 31 are also connected to the
computer 66.
[0246] The computer 66 is provided with a specimen-parameter input
unit (examination-object-parameter input unit) 67, a calculating
unit 68, and a switching unit 69. The specimen-parameter input unit
67 obtains and inputs information on the specimen 20 in advance.
The calculation unit 68 calculates the scanning time for the stage
30 on the basis of the specimen parameters output from the
specimen-parameter input unit 67. The switching unit 69 switches
between the TDI method and the two-dimensional imaging method on
the basis of the calculation results from the calculation unit 68,
and output a switching signal to the detector 62.
[0247] The calculation unit 68 calculates the scanning time for the
stage 30 in the TDI method and the two-dimensional imaging method.
Then, by comparing the durations of the scanning times used in each
method, it selects the method having the shorter scanning time.
[0248] In this way, the measurement carried out by the microscope
imaging apparatus 310 is performed while scanning the plurality of
sample located portions 22, that is, while scanning the cells in
the two-dimensional patterned portion 23. Accordingly, when
measuring the two-dimensional patterned portion 23 with the TDI
method or the two-dimensional imaging method, the shorter time
required for scanning, that is, the shorter measurement time, is
employed. The specimen 20 used here has the configuration shown in
FIGS. 5A and 5B.
[0249] The method of estimating the respective scanning times of
the specimen 20 when the specimen 20 is imaged with the TDI method
and the two-dimensional imaging method shall be described next.
[0250] First, the parameters used in estimating the scanning time
of the specimen 20 will be described.
[0251] FIG. 25A shows the parameters for the specimen. FIG. 25B
shows the parameters for the imaging device 63.
[0252] As shown in FIG. 25A, a measurement substrate 21 has a
rectangular outer shape. The length in the X direction is A (mm),
and the length in the Y direction is B (mm). Also, the sample
located portions 22 are arranged in a CxD array, where C is the
number in the X direction and D is the number in the Y direction.
Therefore, the total number of sample located portions 22 is
C.times.D.
[0253] The exposure time for each sample located portion is E (s),
and the number of measurement wavelengths is F. For example, when
observing two different fluorescence dyes (fluorescence
wavelengths), the number of measurement wavelengths F is two.
[0254] As shown in FIG. 25B, the pixel pitch of the imaging device
63 is G (mm). The number of pixels disposed in the X direction is H
(pixels), and the number of pixels disposed in the Y direction is I
(pixels). Furthermore, the magnification of the objective lens is
J1, and the projection magnification is J2.
[0255] When measuring the specimen 20 with the TDI method, as shown
in FIG. 6, the entire surface of the two-dimensional patterned
portion 23 is scanned regardless of whether or not the sample
located portions 22 exist.
[0256] When measuring fluorescence with a plurality of wavelengths,
the number of wavelengths that can be observed in one scan is only
one. Therefore, the stage must be moved back and forth
corresponding to the number of wavelengths. For example, when
measuring fluorescence with two wavelengths, to acquire an image of
one line, the stage 30 must be moved back and forth twice.
[0257] The calculation for estimating the measurement time in the
TDI method is described below.
[0258] The product of the TDI line transfer rate and the number of
pixels in the imaging device 63 in the transfer direction gives the
exposure time. Therefore, the transfer rate (Tshift) is given by:
Tshift=E/I (seconds).
[0259] The stage speed (Vstage) can be expressed by the ratio of
the pixel size on the examination surface to the transfer rate.
Therefore, the stage speed is expressed by:
Vstage=(G/(J1.times.J2))/Tshift (mm/sec).
[0260] The time required for a one-way scan of the measurement
substrate 21 (Tsingle) is given by: Tsingle=B/Vstage (seconds).
[0261] Detection is not performed when returning the stage 30, but
it is assumed that the same time is required as for the one-way
scan of the stage 30. In such a case, the time required for one
reciprocation of the stage 30 (Tdouble) is expressed as:
Tdouble=2.times.Tsingle (seconds).
[0262] When measuring fluorescence with a plurality of wavelengths,
the stage is reciprocated according to the number of wavelengths
measured. Therefore, the time required to complete acquisition of
one line (Tline) is expressed as: Tline=F.times.Tdouble
(seconds).
[0263] The number of scans (N) can be expressed as the ratio of the
size of the measurement substrate 21 in the X direction to the size
of the imaging device 63 in the X direction, on the surface of the
object under examination. Therefore, the number of scans is given
by: N=[A/(H.times.G/J)]+1 (repetitions) [0264] where the square
brackets indicate Gauss' symbol, that is, the greatest integer that
is not over that value, for example, [2.34]=2.
[0265] Therefore, the scanning time in the TDI method (Ttdi) is:
Ttdi=C.times.Tline (seconds).
[0266] Instead of the imaging device 63 described above, an imaging
device 63a having an electronic shutter may be used in this
embodiment too. Using such an imaging device 63a is advantageous in
that the exposure time can be reduced. The electronic shutter and
the mechanical shutter are as described previously.
[0267] The timing chart of the electronic shutter in the TDI method
is as shown in FIG. 9.
[0268] When determining the scanning time of the specimen 20, the
maximum TDI transfer rate (t1) is Tshift.
[0269] Next, the method of estimating the measurement time in the
two-dimensional imaging method will be described.
[0270] FIG. 26 depicts scanning in the two-dimensional imaging
method. In FIG. 26, for the sake of simplifying the illustration,
the imaging device 63 is depicted as moving.
[0271] As shown in FIG. 26, when carrying out measurement of the
specimen 20 with the two-dimensional imaging method, the stage 30
is moved while performing measurement. By moving the stage 30, the
measurement point sequentially moves to the next sample located
portion 22 in the examination region.
[0272] FIG. 27 is a graph depicting the operation of the stage 30
in the two-dimensional imaging method.
[0273] As shown in FIG. 27, the stage 30 is driven while
alternating between acceleration and deceleration. The motion of
the sample located portions 22 advances in this way. The stage 30
accelerates during the period T1 to T2, moves at constant speed
during period T2 to T3, decelerates during period T3 to T4, and
stops during period T4 to T5.
[0274] Thus, measurement of the sample located portion 22 is
carried out during period T4 to T5 where the stage 30 stops.
[0275] In the two-dimensional imaging method, charge transfer in
the imaging device 63 does not occur, unlike the TDI method.
Therefore, in the two-dimensional imaging method, image acquisition
can be carried out regardless of the direction of motion of the
stage 30.
[0276] Next, the method of estimating the measurement time in the
two-dimensional imaging method will be described.
[0277] Here, the time to move between the sample located portion 22
is K (seconds). In addition, when measuring a plurality of
wavelengths, the dichroic mirror in the microscope is changed.
Therefore, the time required to change the dichroic mirror is L
(seconds). In the microscope, the dichroic mirrors are normally
held in a turret, which is rotated by a driving mechanism such as a
motor or the like. Thus, changing of the dichroic mirror is carried
out electrically.
[0278] The measurement time Tmeasure for one sample located portion
22 depends on the exposure time E (seconds), the time for changing
the dichroic mirror L (seconds), and the number of measurement
wavelengths F, and is given by: Tmeasure=F.times.E+(F-1).times.L
(seconds).
[0279] Considering the period for moving between the sample located
portions 22, the measurement interval Twell between one sample
located portion 22 and another sample located portion 22 is given
by: Twell=Tmeasure+K (seconds).
[0280] Also, because the sample located portions 22 are arranged in
a C.times.D matrix, the scanning time for the two-dimensional
imaging method (T2D) is: T2D=C.times.D.times.Twell (seconds).
[0281] Next, an example of an actual estimation is shown in Tables
1 to 3.
[0282] Table 1 shows values of parameters used in the estimation.
The values of parameters C, D, E, and F in Table 1 are shown in
Table 2. Table 2 shows the estimated results of the measurement
time corresponding to the parameters C, D, E, and F. Tables 3 and 4
show intermediate results of the estimation for each parameter.
TABLE-US-00001 TABLE I Switching time for Measurement No. measure-
Magnification two-dimensional substrate size No. wells ment wave-
No. pixels Objective Projection imaging method X Y X Y Exposure
lengths Pixel X Y lens (magnifi- lens (magnifi- Moving Switching
(mm) (mm) (no.) (no.) time (s) Wavelength size (mm) pixels pixels
cation) cation) time (s) time (s) A B C D E F G H I J1 J2 K L 26 76
parameters 0.0065 1300 1000 20 0.5 4 1
[0283] TABLE-US-00002 TABLE 2 Measurement time(excluding
Measurement conditions image transfer time) No. sample Exposure No.
TDI Two-dimensional located portions time wavelengths method
imaging method C*D (no.) E (s) F Ttdi (s) T2D (s) Method used 1000
0.1 1 725 4100 TDI method 100 0.1 1 725 410 Two-dimensional imaging
method 1000 0.1 2 1450 5200 TDI method 100 0.1 2 1450 520
Two-dimensional imaging method
[0284] TABLE-US-00003 TABLE 3 1 2 3 4 Tshift (s) 1.00E-04 1.00E-04
1.00E-04 1.00E-04 Vstage (s) 6.5 6.5 6.5 6.5 Tstage (s) 11.7 11.7
11.7 11.7 Tdouble (s) 23.4 23.4 23.4 23.4 Tline (s) 23.4 23.4 46.8
46.8 N (repetitions) 31 31 31 31 Ttdi (s) 725 725 1450 1450
[0285] TABLE-US-00004 TABLE 4 5 6 7 8 Tmeasure (s) 0.1 0.1 1.2 1.2
Twell (s) 4.1 4.1 5.2 5.2 T2D (s) 4100 410 5200 520
[0286] FIG. 28 shows an arrangement in which the density of sample
located portions 22 on the measurement substrate 21 changes
depending on position (that is, the density is non-uniform). FIG.
29 depicts an example where the scanning method switches between
the TDI method and the two-dimensional imaging method.
[0287] As shown in FIG. 28, when the density of the sample located
portions 22 is non-uniform, two imaging methods are used.
Specifically, as shown in FIG. 29, the region where the density of
specimen location portions 22 is high (the region at the left in
FIGS. 28 and 29) is measured with the TDI method, and the region
where the density is low (the region at the right in FIGS. 28 and
29) is measured with the two-dimensional imaging method. In this
way, by switching between the TDI method and the two-dimensional
imaging method depending on the distribution of sample located
portions 22, it is possible to select the method having the higher
scanning speed for carrying out measurement. As a result, the
measurement time can be shortened.
[0288] In the measurement described above, measurement of the
specimen 20 is carried out right after the measurement preparation
has been completed. However, a prescan may be carried out to
measure the density of sample located portions 22 in the specimen
20 before measuring the specimen 20. Doing so allows the main
measurement to be carried out after selecting the TDI method or the
two-dimensional imaging method for measuring the specimen 20 on the
basis of information about the density obtained in the prescan.
[0289] When performing the prescan, it is preferable that the
objective lens 49 have low magnification. By doing so, the field of
view of the microscope is widened, and therefore, it is possible to
complete the prescan in a shorter period of time. Also, user input
is not required beforehand.
[0290] Furthermore, when the density of sample located portions 22
is non-uniform, as shown in FIG. 28, it requires a substantial
effort for the user to input information for changing the imaging
method. Conversely, since the imaging method can be changed
automatically on the basis of the prescan, no such effort is
required.
[0291] With the configuration described above, the method having
the shorter scanning time among the TDI method and the
two-dimensional imaging method is selected as the method used in
the measurement. Therefore, compared to the case where the method
is not selected, the scanning in the measurement can be carried out
more quickly. Also, being able to select the method having the
higher scanning speed allows the time required for measuring the
specimen 20 to be reduced.
[0292] Moreover, it is possible to automatically select either the
TDI method or the two-dimensional imaging method, whichever is most
appropriate, on the basis of the number of measurement wavelengths,
the exposure time, and the density of the specimen 20. Therefore,
it is easy to speed up the scanning procedure, which allows the
measurement time to be easily reduced.
[0293] With the microscope imaging apparatus of this embodiment,
the method for measuring the object under examination can be
selected from either the TDI method or the two-dimensional imaging
method on the basis of the scanning time described above, and it is
therefore possible to select the method having the shorter scanning
time.
[0294] Accordingly, compared to a case where the imaging method is
not selected, the scanning during measurement of the object under
examination can be made faster, and the time required for
measurement can thus be reduced.
Tenth Embodiment
[0295] Next, a tenth embodiment will be described with reference to
FIGS. 30 to 45. The tenth embodiment is a biological-specimen
examination system using one of the microscope imaging apparatuses
according to the first to ninth embodiments described above.
[0296] FIG. 30 is a perspective view showing an overview of a
biological-specimen examination system 410 according to this
embodiment. FIG. 31 is a schematic diagram showing the system
configuration of the biological-specimen examination system.
[0297] As shown in FIGS. 30 and 31, the biological-specimen
examination system 410 includes a detection unit 420 and a culture
unit 470. The detection unit 420 and the culture unit 470 are
preferably disposed adjacent to each other, and more preferably,
the units 420 and 470 are disposed in contact with each other.
[0298] As shown in FIGS. 30 and 31, the detection unit 420 includes
an insulated compartment 421 that contains biological specimens and
a detection section (microscope imaging apparatus) 440 that
measures cells CE serving as the biological specimens.
[0299] The insulated compartment 421 includes a heater 421H for
keeping the interior of the insulated compartment 421 at a
predetermined temperature; a stage 422 that holds an incubator box
500 (described later); a transmission light source (illuminating
unit) 423 that irradiates the cells CE with light; a fan 424 to
make the temperature inside the insulated compartment 421 uniform;
a UV lamp 425 for sterilizing the interior of the insulated
compartment 421; a carrier 426 that covers a culture-fluid
circulating tube 477 (described later), a culture-gas supply tube
497, and so on; a door 427 used when putting the incubator box 500
or the like in the insulated compartment 421 or when removing it
therefrom; and a main power-supply switch 428 for turning on and
off the main power supply of the detection unit 420.
[0300] The stage 422 includes an X-axis motion stage 422.times. and
a Y-axis motion stage 422Y that move relative to each other in
mutually orthogonal directions, and scanning of the stage 422 is
controlled by a stage scanning unit (motion unit) 429.
[0301] The stage scanning unit 429 is formed of an X-axis
coordinate detection unit 430 that detects the X-axis coordinate
value of the X-axis motion stage 422X, an X-axis scanning
controller 431 that controls the motion (scanning) of the X-axis
motion stage 422X, a Y-axis coordinate detection unit 432 that
detects the Y-axis coordinate value of the Y-axis motion stage
422Y, and a Y-axis scanning controller 433 that controls the motion
(scanning) of the Y-axis motion stage 422Y.
[0302] The X-axis coordinate detection unit 430 and the Y-axis
coordinate detection unit 432 are configured so as to output the
detected X-coordinate of the X-axis motion stage 422X and the
Y-coordinate of the Y-axis motion stage 422Y, respectively, to a
computer PC. The X-axis scanning controller 431 and the Y-axis
scanning controller 433 are configured so as to control the
scanning of the X-axis motion stage 422X and the scanning of the
Y-axis motion stage 422Y on the basis of respective instructions
from the computer PC.
[0303] The mechanism for driving the X-axis motion stage 422X and
the Y-axis motion stage 422Y may be, for example, a combination of
a motor and a ball screw.
[0304] As described above, the computer PC controls the scanning of
the X-axis motion stage 422X and the Y-axis motion stage 422Y, and
in addition, as described later, it also controls the detection
system of the cells CE, performs analysis of the images of the
cells CE, and so on and controls the X-axis motion stage 422X, the
Y-axis motion stage 422Y, the detection system, and the analysis
system in a coordinated fashion.
[0305] A condenser lens 434 that focuses the light emitted from the
transmission light source 423 onto the cells CE is disposed between
the transmission light source 423 and the incubator box 500.
[0306] A shutter 435 may be disposed between the condenser lens 434
and the incubator box 500, or the shutter 435 may be omitted.
[0307] The fan 424 is disposed on the wall of the insulated
compartment 421. Operating this fan 424 causes air convection
inside the insulated compartment 421, which enables the temperature
inside the insulated compartment 421 to be easily kept uniform and
constant.
[0308] The UV lamp 425 is connected to a UV-lamp switch 436
disposed on the wall of the detection unit 420, and a control timer
437 that periodically operates the UV lamp 425 is disposed between
the UV lamp 425 and the UV-lamp switch 436. Furthermore, a
sterilization-in-progress indicator lamp (not shown) for indicating
that the UV lamp 425 is turned on is provided.
[0309] For example, if the UV-lamp switch 436 is pressed when not
measuring the cells CE, the timer 437 commences counting and power
is supplied to the UV lamp 425 to irradiate the interior of the
insulated compartment 421 with UV light (ultraviolet light). At the
same time, the sterilization-in-progress indicator lamp is
illuminated. Then, after a predetermined amount of time (for
example, 30 minutes), the timer 437 finishes counting, the timer
437 stops supplying power to the UV lamp 425, and the UV
irradiation is stopped. Also, the sterilization-in-progress lamp is
turned off.
[0310] The UV lamp 425 can be controlled independently of the main
power-supply switch 428 so that it can be operated even if the main
power-supply is off.
[0311] The illumination time of the UV lamp 425 may be 30 minutes,
as described above. Alternatively, an illumination time less than
30 minutes or more than 30 minutes may also be used so long as
contamination and so forth inside the insulated compartment 421 can
be completely killed.
[0312] The door 427 is formed of a metal such as aluminum or the
like that has been subjected to anodizing, or it may be formed of a
translucent resin with high opacity.
[0313] The door 427 may have a double-layer construction with an
air gap, or the inside may be formed of metal and the outside
formed of resin. Using resin on the outside of the door 427 can
prevent heat from inside the insulated compartment 421 from
escaping through the door 427. Also, forming the inside of the door
427 of anodized metal can prevent deterioration of the lifetime of
the door 427 due to the UV lamp 425.
[0314] If the door 427 has a double-layer construction of metal or
metal and resin, since light is completely blocked, it is
preferable to provide an inspection window at a position where the
incubator box 500 can be viewed. The inspection window is
preferably formed of transparent resin or glass, and an
openable/closable cover is preferably disposed at the outer side
thereof.
[0315] As shown in FIGS. 30 and 31, a detection section 440
includes a heater 440H for keeping the interior of the detection
section 440 at a predetermined temperature; incident light sources
441A and 441B that irradiate the cells CE from the detection
section 440 side; a light-path switching unit 442 that switches the
light path from the incident light sources 441A and 441B; a
light-intensity adjusting mechanism 443 that adjusts the intensity
of the irradiated light; a lens system 444 that focuses the
irradiated light towards the cells CE; a filter unit 445 that
controls the wavelength of the irradiated light and the wavelength
of the detection light; an autofocus (AF) unit 446 that performs a
focusing operation with respect to the cells CE; a revolver 447
provided with a plurality of objective lenses 448 having different
magnifications and properties; a detector (imaging unit) 449 that
detects detection light from the cells CE; a light-intensity
monitor 450 that measures the intensity of the detection light; a
fan 451 that makes the temperature inside the detection section 440
uniform; and a cooling fan 452 that cools the interior of the
detection section 440.
[0316] The incident light sources (illumination unit) 441A and
441B, which are formed of mercury lamps, for example, are disposed
outside the detection section 440 and are connected to a power
supply 453 that supplies power thereto.
[0317] Normally, a single incident light source, for example, the
incident light source 441A, is used; however, if the intensity of
the incident light source 441A falls below a certain prescribed
value, light is irradiated from the other incident light source
441B and the power supply to the first incident light source 441A
is turned off.
[0318] The light-path switching unit 442 is configured to guide
illumination light from either the incident light source 441A or
the incident light source 441B to the light-intensity adjusting
mechanism 443. Also, the light-path switching unit 442 is provided
with a light-path control unit 454, which is connected to the
computer PC (described later) for controlling the light-path
switching unit 442 on the basis of an instruction from the computer
PC.
[0319] At the emitting side of the light-path switching unit 442
where the illumination light is emitted, a shutter 442S that
controls the transmission and blocking of the illumination light is
provided.
[0320] The light-intensity adjusting mechanism 443, which is
disposed at the emission side of the shutter 442S where the
illumination light is emitted, adjusts the intensity of the
illumination light passing through the shutter 442S. A known
aperture mechanism, for example, may be used, or any other known
mechanism or technique that can adjust the light intensity may be
used.
[0321] The light-intensity adjusting mechanism 443 is provided with
a light-intensity control unit 455, which is connected to the
computer PC (described later) for controlling the light-intensity
adjusting mechanism 443 on the basis of an instruction from the
computer PC.
[0322] The lens system 444 is disposed at the emission side of the
light-intensity adjusting mechanism 443 where the illumination
light is emitted. The lens system 444 includes a pair of lenses
444A and 444B and a stop 444C disposed between the lens 444A and
the lens 444B.
[0323] The filter unit 445 includes an excitation filter 456, a
dichroic mirror 457, and an absorption filter 458. The excitation
filter 456 is a filter that transmits wavelengths which contribute
to the generation of fluorescence in the cells CE (excitation
light) from among the illumination light and is disposed so that
the illumination light emitted from the lens system 444 is incident
on the excitation filter 456. The dichroic mirror 457 is an optical
element that splits excitation light and fluorescence. More
specifically, the dichroic mirror 457 is disposed so as to reflect
excitation light transmitted through the excitation filter 456
towards the cells CE and to transmit fluorescence from the cells
CE. The absorption filter 458 is an optical element that separates
fluorescence from the cells CE from other unwanted scattered light.
The absorption filter 458 is disposed so that light transmitted
through the dichroic mirror 457 is incident thereon.
[0324] The filter unit 445 is provided with a filter control unit
446C that controls the wavelengths of the excitation light emitted
from the filter unit 445 and the detection light (fluorescence) on
the basis of instructions from the computer PC (described
later).
[0325] One excitation filter 456, one dichroic mirror 457, and one
absorption filter 458 may be used, or alternatively, a plurality of
each may be used.
[0326] The AF unit 446 is disposed at the emission side of the
filter unit 445 where the excitation light is emitted and is
disposed so that the excitation light is focused onto the cells CE
via one of the objective lenses 448, on the basis of an instruction
from the computer PC (described later).
[0327] The revolver 447 is disposed at the emission side of the AF
unit 446 where the excitation light is emitted and is provided with
the plurality of objective lenses 448 having different
magnifications. The revolver 447 is provided with an objective-lens
control unit 459 which selects and controls the objective lens 448
on which the excitation light is incident, on the basis of an
instruction from the computer PC (described later).
[0328] The objective lenses 448 are configured to allow
examination, from the detection section 440, of the interior of the
incubator box inside the insulated compartment 421 via holes
provided in the X-axis motion stage 422.times. and the Y-axis
motion stage 422Y.
[0329] The holes in the X-axis motion stage 422.times. and the
Y-axis motion stage 422Y are large enough to allow viewing over the
operating region of the stage, with some additional margin.
[0330] Therefore, although the ambient air inside the insulated
compartment 421 should be kept at a humidity suitable for culturing
cells, the ambient air may escape to the detection section 440
through the holes, which makes it impossible to maintain the
temperature suitable for culturing cells, and therefore, there is a
risk of bringing about a reduction in cell activity.
[0331] Thus, a containment mechanism 449 for suppressing the
passage of such ambient air, which is at a temperature suitable for
cell culturing, between the insulated compartment 421 and the
detection section 440.
[0332] The containment mechanism 449 should be capable of
suppressing the flow of air while not interfering with the
operation of the revolver 447 and the objective lenses 448. For
example, it may be a sheet-like mechanism in which, for example, a
sheet formed of a flexible material, such as a film or transparent
sheet, is attached to the perimeter of a hole provided at the
boundary between the insulated compartment 421 and the detection
section 440 and in such a manner that it is draped around the
perimeter of the revolver.
[0333] A focusing lens 460 that focuses the detection light onto
the detector 449 and the light-intensity monitor 450 is provided at
the emission side of the filter unit 445 where the detection light
is emitted.
[0334] A half-mirror 461 that reflects some of the detection light
towards the detector 449 and that transmits the remaining detection
light towards the light-intensity monitor 450 is provided at the
emission side of the focusing lens 460 where the detection light is
emitted.
[0335] The detector 449 is disposed at a position where the
detection light reflected from the half mirror 461 is incident
thereon. Also, a detector calculation unit 462 that calculates a
detection signal from the detector 449 and outputs it to the
computer PC (described later) is connected to the detector 449.
[0336] The detector 449 is not particularly limited and may use a
line sensor, an area sensor, or both a line sensor and an area
sensor.
[0337] The light-intensity monitor 450 measures the detection light
transmitted through the half-mirror 461 and is configured so as to
output the measured value to the computer PC.
[0338] The intensity of the detection light may be measured using
the light-intensity monitor 450, as described above, or
alternatively, the intensity of the detection light may be measured
using an illuminance meter or a power meter.
[0339] The heater 440H controls the temperature inside the
detection section 440 to be from 30.degree. C. to 37.degree. C. The
fan 451 is disposed to cause air convection inside the detection
section 440 to make the temperature inside the detection section
440 uniform. Therefore, the temperature inside the detection
section 440 can be maintained close to the temperature in the
insulated compartment 421, and the temperature in the insulated
compartment 421 can thus be more easily stabilized.
[0340] The cooling fan 452 is operated to reduce the temperature
inside the detection section 440 on the basis of the output from a
temperature sensor (not shown) provided inside the detection
section 440. Therefore, it is possible to prevent an abnormal rise
in temperature inside the detection section 440 due to heating by,
for example, the motors and so forth.
[0341] FIG. 32 is a perspective view of the incubator box according
to this embodiment, and FIG. 33 is a cross-sectional view of a
chamber according to this embodiment.
[0342] As shown in FIGS. 32 and 33, the incubator box 500 includes
a frame 501, containing a chamber (object under examination) 510,
and a cover 502 that forms a sealed space together with the frame
501. The frame 501 and the cover 502 are subjected to
magnetic-shielding treatment for blocking external magnetic fields
and anti-static treatment for eliminating the build-up of static
electricity in the incubator box 500.
[0343] The frame 501 is formed of a base plate 503 and side walls
504, and a region corresponding to the measurement area of the base
plate 503 is formed of a transparent material, such as glass. The
other regions of the base plate 503 and the side walls 504 are
preferably formed of an anti-corrosive material having high
opacity, such as anodized aluminum or stainless steel, like SUS316.
More preferably, from the viewpoint of maintaining the temperature,
a material having a low thermal conductivity may be selected.
[0344] An adaptor 505 for holding the chamber 510 and a temperature
sensor 506 for measuring the temperature of the chamber 510 are
provided on the base plate 503. The chamber 510 may be held using
the adaptor 505, as described above, or the chamber 510 may be held
without using the adaptor 505.
[0345] The output from the temperature sensor 506 is input to the
computer PC via an incubator-temperature detection unit 506S and is
also input to a temperature-display unit 507 disposed on the wall
of the detection unit 420. The computer PC controls the heater 421H
and so on via an incubator-temperature control unit 506C shown in
FIG. 31 to control the temperature inside the incubator box 500 in
order to keep it constant.
[0346] The cover 502 includes a glass plate 517 that transmits the
illumination light and a support portion 517A that supports the
glass plate 517. An anti-reflection film may be formed on both
sides of the glass plate 517 in a region corresponding to the
measurement area. Forming such an anti-reflection film on both
sides allows prevention of reflection by the glass plate 517 during
transmission examination and incidence examination.
[0347] The area of the glass plate 517 may be substantially the
same as that of the base plate 503 of the incubator box 500, or it
may be the minimum necessary area that does not cause any problem
during measurement.
[0348] As shown in FIG. 33, the chamber 510 is formed of a lower
glass member 511 for observation with the objective lens 448, an
upper glass member 512 for transmitting light from the transmission
light source 423, and a frame member 513 that supports the lower
glass member 511 and the upper glass member 512.
[0349] Joints 514 having channels formed therein for circulating
culture fluid are formed at opposing sides of the frame member 513.
A culture-fluid circulating tube 477 (described later) is connected
to the joints 514, for allowing culture fluid to circulate between
the culture unit 470 and the detection unit 420.
[0350] A pair of flow smoothers 515 for making the flow of culture
fluid uniform are disposed in the frame member 513 so as to be
substantially orthogonal to the flow of culture fluid. The flow
smoothers 515 are formed of sheet members in which, for example,
small holes are formed in a matrix, and by splitting the culture
fluid and flowing it through the plurality of small holes, the flow
becomes uniform. A slide glass 516 on which the cells CE are
disposed is provided between the two flow smoothers 515.
[0351] As described above, the incubator box 500 may be provided
with the chamber 510 inside, or, as shown in FIG. 34A, a microplate
(object under examination) 520 (or a well plate) may be disposed
inside.
[0352] In this configuration, as shown in FIG. 34A, the frame 501
of an incubator box 500a is provided with a square-shaped reservoir
521 surrounding the microplate 520, an internal fan 522 disposed at
the inner side of the reservoir 521, a connector 523 for supplying
culture gas, and a culture-gas concentration sensor 524 for
detecting the concentration of carbon dioxide in the culture
gas.
[0353] The temperature sensor 506 is disposed so as to measure the
temperature of the microplate 520. The microplate temperature input
to the computer PC from the temperature sensor 506 is collected in
the form of text data in a memory and can be subjected to data
processing in the computer PC.
[0354] The culture-gas concentration sensor 524 outputs the
carbon-dioxide concentration to the computer PC and to a
culture-gas concentration display unit 524D.
[0355] The height of side walls 521W of the reservoir 521 is formed
to be lower than the height of the side walls of the frame 501.
Also, the positional relationship with respect to the connector 523
is adjusted so that the supplied culture gas blows against the side
walls 521W. Sterilized water is stored in the reservoir 521, and
the humidity inside the incubator box 500a is regulated at about
100%.
[0356] The internal fan 522 is disposed so that the microplate 520
is not positioned in the blowing direction thereof and so that it
blows along the side walls 521W of the reservoir 521.
[0357] The culture-gas concentration sensor 524 may be disposed on
the inner surface of one of the side walls 521W of the reservoir
521. Alternatively, the tubes from the incubator box 500a may be
disposed outside and the culture gas inside the incubator box 500a
may be evacuated with a suction pump to detect the concentration
thereof with the culture-gas concentration sensor 524.
[0358] When using this kind of incubator box 500a, the destination
of the culture gas supplied from a culture-gas mixing tank 491
(described later) is changed from a culture fluid vessel 472 to the
incubator box 500a, and because it is not necessary to supply
culture fluid from the culture unit 470, the operation of a
culture-fluid pump 480 or the like can be stopped.
[0359] With such a construction, since the humidity environment and
culture-gas concentration in the incubator box 500a are maintained
so that little damage is caused to the cells CE by changes and
non-uniformity in the humidity and culture-gas concentration,
damage to the cells CE can be reduced compared to a thermal
environment.
[0360] Also, because the humidity and culture-gas concentration in
the incubator box 500a, which is not directly in contact with the
detection section 440, are maintained, it is possible to prevent
contamination during examination.
[0361] Furthermore, since it is not necessary to maintain the
proper humidity and culture-gas concentration when culturing the
cells CE in the insulated compartment 421, the performance of the
objective lenses 448 and so on disposed in the insulated
compartment 421 can be prevented from deteriorating. Thus, a
reduction in lifetime of the objective lenses 448 and so on can be
prevented.
[0362] The chamber 510 may be a sealed enclosure, as described
above, or it may be an open chamber that is not sealed. Such an
open chamber is formed with the same construction as the chamber
510 except for the provision of the upper glass member 512.
[0363] When using such an open chamber, using the incubator box 500
described above, the incubator box 500a is filled with the culture
gas and the open chamber is supplied with culture liquid.
[0364] As shown in FIG. 34B, the chamber 510 described above may
also be used in the incubator box 500a. In such a case, the
connector 523 for supplying culture gas is blocked off, and the
culture-gas concentration sensor 524 is not used. If the size of
the chamber 510 is different from that of the microplate 520, the
chamber 510 may be placed in the incubator box 500a using an
adaptor 505. Sterilized water need not be placed in the reservoir
521; in fact, the reservoir 521 itself may be eliminated from the
incubator box 500a. Also, the temperature sensor 506 measures the
temperature in the chamber 510.
[0365] The connector 523 may be blocked off, as described above, or
it may be left connected to the culture-gas supply tube 497 and the
supply of culture gas to the incubator box 500a simply stopped.
[0366] As shown in FIGS. 30 and 31, the culture unit 470 includes a
sterilized compartment 471 containing the culture fluid and a
mixing section 490 for producing the culture gas.
[0367] The sterile compartment 471 includes a heater 471H for
keeping the interior of the sterile compartment 471 at a
predetermined temperature; a culture-fluid vessel 472 for storing
the culture fluid; an auxiliary tank 473 for storing spare culture
fluid; a waste tank 474 into which used culture fluid is
discharged; a UV lamp 425 for sterilizing the interior of the
sterile compartment 471; a door 475 used when putting the
culture-liquid vessel 472 into the sterile compartment 471 and when
removing it therefrom; and a main power-supply switch 476 for
turning on and off the main power supply for the culture unit
470.
[0368] The culture-fluid vessel 472 is provided with a
culture-fluid circulating tube 477 for circulating culture fluid
between the culture-fluid vessel 472 and the incubator box 500; a
supply tube 478 for supplying spare culture fluid from the
auxiliary tank 473; and a waste tube 479 for discharging used
culture fluid from the culture-fluid vessel 472 to the waste tank
474.
[0369] A culture-fluid pump 480 for delivering culture fluid from
the culture-fluid vessel 472 to the incubator box 500 and
circulating the culture fluid is provided for the culture-fluid
circulating tube 477. Using the culture-fluid pump 480, it is
possible to replace the culture fluid in the chamber 510 with fresh
culture fluid, and therefore, the cells CE can be cultured for a
longer period of time compared to a case where the culture fluid is
not replaced.
[0370] A supply pump 481 for transferring culture fluid from the
auxiliary tank 473 to the culture-fluid vessel 472 is provided for
the supply tube 478. In addition, a waste pump 482 for transferring
the used culture fluid from the culture-fluid vessel 472 to the
waste tank 474 is provided for the waste tube 479.
[0371] As described above, the waste tank 474 for storing the used
culture fluid may be used. Alternatively, instead of using the
waste tank 474, a discharge port for directly discharging the used
culture fluid may be provided.
[0372] A culture-fluid temperature sensor (not shown) for detecting
the culture-fluid temperature is provided in the culture-fluid
vessel 472, and the output from the culture-fluid temperature
sensor is input to the computer PC via a culture-fluid temperature
detector 483. Data concerning the culture-fluid temperature input
to the computer PC is collected in a memory in the form of text
data and is used when comparing and verifying the detection results
of the cells CE.
[0373] The heater 471H is provided with a culture-fluid temperature
controller 484 that controls the temperature of the culture fluid
via the temperature inside the sterilized compartment 471, on the
basis of an instruction from the computer PC. The temperature of
the culture fluid supplied from the culture-fluid vessel 472 is
held at about 37.degree. C. by the culture-fluid temperature
controller 484, which prevents the activity of the cells CE from
dropping due to temperature changes of the culture fluid. Also, a
temperature display unit 485 for displaying the culture-fluid
temperature detected by the culture-fluid temperature sensor is
provided on the wall of the culture unit 470.
[0374] The culture-fluid pump 480 is provided with a
culture-fluid-pump controller 486 for controlling the circulation
of the culture fluid on the basis of an instruction from the
computer PC. The operation of the supply pump 481 and the waste
pump 482 is also controlled on the basis of instructions from the
computer PC.
[0375] The UV lamp 425 is connected to a UV-lamp switch 436
disposed on the wall of the culture unit 470, and a timer 437 for
periodically controlling the operation of the UV lamp 425 is
provided between the UV lamp 425 and the UV-lamp switch 436.
Furthermore, a sterilization-in-progress indicator lamp (not shown)
for indicating that the UV lamp 425 is illuminated is also
provided.
[0376] The UV lamp 425 is controlled independently of the main
power-supply switch 476 and can be operated even when the main
power-supply switch 476 is off.
[0377] As shown in FIGS. 30 and 31, the mixing section 490 includes
a heater (not shown) for keeping the interior of the mixing section
490 at a predetermined temperature; a culture-gas mixing tank 491
for adjusting the carbon-dioxide concentration in the culture gas
supplied to the incubator box 500; and a CO.sub.2-pump 493 for
supplying carbon dioxide from a CO.sub.2 tank 492 provided outside
the culture unit 470 to the culture-gas mixing tank 491.
[0378] A CO.sub.2-concentration detector 494 is provided in the
culture-gas mixing tank 491 for detecting the concentration of
carbon dioxide therein, and the output from the
CO.sub.2-concentration detector 494 is input to the computer PC.
The CO.sub.2 pump 493 is provided with a CO.sub.2-concentration
controller 495 for controlling the amount of carbon dioxide
supplied to the culture-gas mixing tank 491 on the basis of an
instruction from the computer PC. Also, a CO.sub.2-concentration
display unit 496 for displaying the carbon-dioxide concentration
inside the culture-gas mixing tank 491, which is detected by the
CO.sub.2-concentration detector 494, is provided on the wall of the
culture unit 470.
[0379] Furthermore, a culture-gas supply tube 497 is provided
between the culture-gas mixing tank 491 and the culture-fluid
vessel 472. Accordingly, culture gas is supplied to the culture
fluid via the culture-gas supply tube 497, which allows a
sufficient level of culture gas to be dissolved in the culture
fluid. In this way, by producing culture fluid in which culture gas
having a 5% concentration of carbon dioxide is dissolved inside the
culture-fluid vessel 472, culture fluid including culture gas and
nutrients necessary for nourishing the cells CE is supplied to the
chamber 510. Also, by dissolving the culture gas in the culture
fluid, the pH and so forth of the culture fluid can be
regulated.
[0380] The carbon-dioxide concentration input to the computer PC
from the CO.sub.2-concentration detector 494 is collected in the
memory in the form of text data, and data processing can be carried
out in the computer PC.
[0381] Next, an examination method used in the biological-specimen
examination system 410 having the above-described configuration
will be described.
[0382] First, the scanning method and selection of a detection
region in this embodiment will be described with reference to FIGS.
35A to 35D.
[0383] FIGS. 35A to 35D depict examples of the scanning method and
selection of the detection areas in this embodiment.
[0384] In the example shown in FIG. 35A, a measurement region M
(the region surrounded by the dashed line in the figure) is set by
specifying an upper-left point a and a lower-right point b defining
the measurement region M in the displayed image. More concretely,
the measurement region M may be set by dragging a device like a
mouse from point a to point b, or it may be specified by inputting
the coordinates of the points a and b.
[0385] As indicated by the arrows in the figure, regarding the part
to be measured by the detector 449, the specified measurement
region M is scanned from left to right. That is, when scanning from
the left to the right in the figure, scanning is performed parallel
to the X direction, and when scanning from the right to the left,
scanning is performed downward and to the left at an angle. While
scanning from left to right, image acquisition of the cells CE is
carried out.
[0386] FIG. 35B is an example in which two measurement regions M
are specified by the method described above. First, the two
measurement regions MA and MB are specified using the method
described above. The measurement region MA and the measurement
region MB are arranged with a certain gap therebetween in the X
direction in the figure, and they are disposed so as to completely
overlap in the Y direction.
[0387] As indicated by the arrows, the part to be measured by the
detector 449 in this example is scanned so as to measure the
measurement regions MA and MB in parallel. That is, when scanning
from the left to the right in the figure, scanning is performed
from the measurement region MA to the measurement region MB, and
when scanning from the right to the left, scanning is performed
from the measurement region MB to the measurement region MA.
[0388] FIG. 35C is an example in which two measurement regions are
set using the method described above. The two measurement regions
MA and MB are disposed at different positions. Here, the
measurement region MA and the measurement region MB are arranged
with a gap therebetween in the X direction in the figure, and they
are disposed so that they partially overlap in the Y direction in
the figure.
[0389] As indicated by the arrows in the figure, regarding the
parts to be measured by the detector 449 in this example, only the
portions of the measurement regions MA and MB that overlap in the Y
direction are sequentially scanned. That is, first the
non-overlapping portion of the measurement region MA is scanned.
Next, the overlapping portions of the measurement regions MA and MB
are sequentially scanned. Then, the non-overlapping portion of the
measurement region MB is scanned.
[0390] FIG. 35D is an example in which two measurement regions M
are set by the method described above. In this example, the two
measurement regions MA and MB are the same as those in FIG. 35B,
but the scanning method is different.
[0391] As indicated by the arrows in the figure, regarding the
parts to be measured by the detector 449 in this example, the
measurement regions MA and MB are scanned independently. That is,
after first scanning the entire measurement region MA, the entire
measurement region MB is scanned.
[0392] Among the scanning methods shown in FIGS. 35A to 35D
described above, the method with the shortest total distance moved
or shortest scanning time is automatically selected by the computer
PC on the basis of specified parameters and a measurement mode,
which are described later.
[0393] When acquiring images of the region where the cells are
cultured, it is possible to carry out image acquisition only in the
required parts, as necessary, if that region is formed of a
plurality of divided regions (detection regions) by, for example,
specifying measurement regions M that can be imaged.
[0394] For example, if the settings of the computer PC are changed
to alternately scan the entire region and predetermined parts of
the object to be scanned, it is possible to observe phenomena
unique to biological specimens which occur only for a short time.
As one example, in a case where scanning of the entire region of an
object is normally performed every 30 minutes, so long as scanning
is performed in a predetermined measurement region M where cells of
interest exist, it is possible to determine the occurrence of a
specific phenomenon that is exhibited only every 15 minutes in
those cells of interest.
[0395] In order to scan the desired measurement region M in the
required time, the scanning time can be reduced, and the time
required to irradiate other cells with light can be reduced.
[0396] Next, the procedure for measuring the cells CE will be
described using a flowchart.
[0397] First, before measuring the cells CE, measurement parameters
are set. Therefore, the procedure for setting the measurement
parameters will be described with reference to FIG. 36.
[0398] FIG. 36 is a flowchart showing the procedure for setting the
measurement parameters.
[0399] First, the measurement parameters are set (step S21).
[0400] Then, default conditions are set (step S22). Here, the
conditions set are the culturing conditions and the measurement
conditions, for example, a CO.sub.2 concentration of 5%, a
temperature of 37.degree. C., and so forth. These conditions can be
changed to predetermined conditions by the user.
[0401] Next, the measurement object is selected (step S23).
Measurement object means the container of the cells CE, for
example, the microplate 520 or the slide glass 516.
[0402] Next, the measurement mode is selected (step S24). Possible
measurement modes include an area acquisition mode, a line
acquisition mode, an automatic mode, and so on. In the automatic
mode, the measurement mode having the shortest measurement time is
automatically selected from among the other modes.
[0403] Next, the measurement magnification is selected (step S25),
and after that, the detection wavelength is selected (step S26).
The measurement magnification and the detection wavelength can each
be automatically selected from among two or more options.
[0404] Here, as the method of selecting the detection wavelength, a
list of fluorescent proteins used, for example, GFP, HC-Red, and so
on, is stored in advance in the computer PC, and one of them is
selected from the stored list. The computer PC automatically
selects the most appropriate excitation filter 456, absorption
filter 458, and so on for examination, on the basis of the selected
fluorescent protein. In this way, specific fluorescence from the
cells CE can be detected.
[0405] The excitation filter 456, the absorption filter 458, the
objective lens 448 and so on used for measurement are automatically
changed in synchronization with the driving of the X-axis motion
stage 422X and the Y-axis motion stage 422Y.
[0406] Next, the measurement interval is set (step S27).
[0407] Then, a preview image is acquired (step S28), and the
preview image is displayed on the monitor (step S29). In the latter
step, the preview image is displayed on the monitor when the user
issues an instruction using a preview button or the like for
instructing display of the preview image on the monitor. Thus, the
user can confirm the preview image displayed on the monitor.
[0408] Next, the measurement region is selected (step S30). After
selecting the measurement region, the preview image may be
displayed on the monitor again to confirm whether the measurement
region has been correctly selected.
[0409] Next, a predetermined measurement interval is selected from
a plurality of specified measurement intervals (step S31).
[0410] Then, upon pressing a start-measurement switch (not shown;
step S32), measurement of the cells CE commences (step S33). If the
start-measurement switch is not pressed, the process stands by
until the start-measurement switch is pressed (step S32).
[0411] If the start-measurement switch is not pressed in step S32,
the process may jump back to any predetermined step to allow the
measurement parameters to be set again.
[0412] After the measurement parameters have been set, measurement
of the cells CE is carried out. Therefore, the procedure for
measuring the cells CE will be described with reference to FIGS. 37
and 38.
[0413] FIGS. 37 and 38 are flowcharts showing the measurement
procedure.
[0414] First, when measurement starts, the measurement region is
retrieved (step S41). Then, the magnification is retrieved (step
S42), and the detection wavelength is retrieved (step S43).
[0415] Next, the measurement mode is retrieved (step S44). Here,
the appropriate stage scanning method is determined on the basis of
the retrieved measurement region, magnification, detection
wavelength (fluorescence wavelength), and so on. If the measurement
mode is set to the automatic mode, the image acquisition mode is
also determined at this point.
[0416] Next, the method of operating the X-axis motion stage 422X
and the Y-axis motion stage 422Y in accordance with the determined
stage scanning mode is analyzed (step S45), and data for the
analyzed operating method (operating data) is saved in a table in
the computer PC (step S46).
[0417] Thereafter, measurement is carried out using a different
measurement method depending on whether or not the area sensor mode
is selected (step S47).
[0418] First, a description will be given in the case where the
area sensor mode is selected.
[0419] When the start-measurement switch is pressed, the X-axis
motion stage 422X and the Y-axis motion stage 422Y are moved to a
measurement starting position (S50). In this step, the computer PC
retrieves the measurement starting position which has been input
and moves the X-axis motion stage 422X and the Y-axis motion stage
422Y to the measurement starting position, and the cells CE are
thus moved to a position within the imaging field of the objective
lens 448.
[0420] Then, the shutter 435 is opened (step S51) and the objective
lens 448 is selected (step S52). Here, the computer PC drives the
revolver 447 to select an objective lens 448 having a predetermined
magnification on the basis of the specified measurement
magnification.
[0421] Next, the filter unit 445 is selected (step S53). Here, the
filter control unit 446C selects the excitation filter 456, the
absorption filter 458, and so on that are most appropriate for the
measurement on the basis of the fluorescent protein specified in
the computer PC.
[0422] The operations carried out from when the start-measurement
switch described above is pressed up to this point (steps S50 to
S53) are automatically selected and executed according to the
measurement mode.
[0423] After that, the focus position is detected (step S54), and
then image acquisition is performed and the image data is output to
an image memory in the computer PC (step S55).
[0424] Then, if the required image acquisition has not yet been
completed, the operations from selection of the objective lens 448
(step S52) to image acquisition and output of the image data to the
image memory in the computer PC (step S55) are repeated until the
required image acquisition is completed (step S56).
[0425] When the required image acquisition has been completed, the
X-axis motion stage 422X or the Y-axis motion stage 422Y is driven
by one step (step S57). Then, if the position to which the X-axis
motion stage 422X or the Y-axis motion stage 422Y has been moved is
within the measurement region, the operations from selection of the
objective lens 448 (step S52) to driving the motion stage by one
step (step S57) are repeated. These operations are repeated until
the position to which the X-axis motion stage 422X or the Y-axis
motion stage 422Y has been moved is outside the measurement region
(step S58).
[0426] When the position of the X-axis motion stage 422X or the
Y-axis motion stage 422Y is outside the measurement region, the
shutter 435 is closed (step S59).
[0427] Thereafter, once the predetermined measurement time interval
is over, the operations from opening of the shutter 435 (step S51)
to closing of the shutter 435 (step S59) are repeated until the end
of the measurement time (step S60).
[0428] Next, a case where the area sensor mode is not selected
shall be described.
[0429] When the start-measurement switch is pressed, the x-axis
motion stage 422X and the Y-axis motion stage 422Y are moved to the
measurement starting position (step S70). Here, the computer
retrieves the measurement start position that was input and moves
the X-axis motion stage 422X and the Y-axis motion stage 422Y to
the measurement starting position to move the cells CE to a
position within the imaging field of the objective lens 448.
[0430] Then, the shutter 435 is opened (step S71), and the focus
position is detected (step S72).
[0431] Next, the objective lens 448 is selected (step S73). Here,
the computer PC drives the revolver 447 to select an objective lens
448 having a predetermined magnification, on the basis of the
specified measurement magnification.
[0432] Next, the filter unit 445 is selected (step S74). Here, the
filter control unit 446C selects the excitation filter 456, the
absorption filter 458, and so on that are most appropriate for the
measurement on the basis of the fluorescent protein specified in
the computer PC. The operations carried out from when the
start-measurement switch is pressed up to this point (steps S70 to
S74) are automatically selected and executed according to the
measurement mode.
[0433] After that, driving of the X-axis motion stage 422X and the
Y-axis stage 422Y commences (step S75), and then image acquisition
is performed and the image data is output to an image memory in the
computer PC (step S76).
[0434] Then, if the required image acquisition has not yet been
completed, the operations from selection of the objective lens 448
(step S73) to image acquisition and output of the image data to the
memory in the computer PC (step S76) are repeated until the
required image acquisition is completed (step S77).
[0435] When the required image acquisition has been completed, the
shutter 435 is closed (step S78).
[0436] Thereafter, once the predetermined measurement time interval
is over, the operations from opening of the shutter 435 (step S71)
to closing of the shutter 435 (step S78) are repeated until the end
of the measurement time (step S79).
[0437] When image acquisition of the cells CE has been completed,
the acquired images are then processed. Therefore, the method of
processing the acquired images will be explained with reference to
FIG. 39.
[0438] FIG. 39 is a flowchart showing the image processing
method.
[0439] First, the image processing unit of the computer PC extracts
the background image from the acquired images collected in the
memory (step S91), and removes the background image from the
acquired images (step S92).
[0440] Next, the maximum brightness region of the image, which can
be enhanced, is read out (step S93) and multiplied by, for example,
a predetermined coefficient to enhance the image (step S94). With
this processing, the image is enhanced so that the individual cells
CE can easily be recognized as spots in the image from which the
background is removed.
[0441] Then, by extracting portions having a brightness higher
than, for example, a predetermined threshold from the enhanced
image, the bright cells CE can be clearly recognized as individual
spots (step S95).
[0442] Next, geometrical features, such as the center of gravity
and the area, chemical features, and optical features, such as the
fluorescence intensity, of the cells CE are more accurately
determined, and positional information of the cells CE is
determined and extracted (step S96). Extracting these features
allows the individual cells CE to be distinguished.
[0443] After extracting the features of the cells CE, correction
(step S97) of the enhancement (S94) carried out for recognizing the
cells CE is carried out. With this correction, the effect of the
predetermined coefficient used for enhancing the image is
removed.
[0444] Next, after correction, the features are output to a file,
for example, and are stored in that file (step S98).
[0445] Accordingly, the image processing unit of the computer PC
can convert the fluorescence distribution of the cells CE at each
position on the entire surface of the slide glass, microplate, or
the like into an image. Also, since the image processing unit can
accurately track the individual cells CE, it can target a
predetermined number of cells CE, which allows localized, long-term
measurement of the fluorescence distribution of the cells CE while
performing culturing. Furthermore, while culturing the cells CE,
the entire surface of the slide glass, microplate, or the like is
measured at fixed time intervals, for example, and the fluorescence
intensity of the cells CE over time can be automatically
measured.
[0446] Next, data processing carried out after extracting data
about the features of the cells CE from the acquired images will be
described with reference to FIG. 40.
[0447] FIG. 40 is a flowchart depicting the data processing
procedure.
[0448] Here, processing of the cell data (features) stored in the
file is carried performed by a data processing unit of the computer
PC.
[0449] First, the data processing unit reads out (step S101) raw
data (features) of the cells CE, which is stored in the file, and
sorts the data to arrange it time-sequentially for each cell CE
(step S102). When the data has been sorted, the data processing
unit graphs the variation in brightness of each cell CE, that is,
the level of expression, with time (step S103).
[0450] When the graphing has been completed, the data processing
unit displays a preview of the graph (step S104), and outputs the
graph data to a file (step S105).
[0451] By performing this processing, when cells CE are cultured
for an extended period of time, the variation of a single cell with
time can be easily examined. Therefore, during culturing, the
variation in the level of expression of the cells CE with time can
be accurately and easily measured.
[0452] Next, adjustment of the irradiation intensity carried out
during measurement of the cells CE will be explained with reference
to FIG. 41.
[0453] FIG. 41 is a flowchart showing the procedure for adjusting
the intensity.
[0454] First, the intensity of light irradiated onto the cells CE
is measured (step S111). The irradiation intensity may be
calculated from the output of the light-intensity monitor 450, by
providing an irradiance meter for measuring the intensity, or by
providing a power meter and calculating the intensity from the
output of the power meter.
[0455] If the measured irradiation intensity is within a
permissible range, the process returns to measurement of the
irradiation intensity (step S111) and repeats this until the
irradiation intensity is outside the permissible range (step
S112).
[0456] Once the irradiation intensity is outside the permissible
range, the ND filter (not shown) included in the light-intensity
adjusting mechanism 443 is changed (step S113) to adjust the
irradiation intensity so that it falls within the permissible
range. Thereafter, the process returns to measurement of the
irradiation intensity (step S111), and repeats the adjustment of
the irradiation intensity.
[0457] Next, a control method for supplying and replacing the
culture fluid in the chamber 510 is described with reference to
FIG. 42.
[0458] FIG. 42 is a flowchart showing the method for supplying and
replacing the culture fluid.
[0459] First, the background level of the acquired image is
analyzed (step S121). The autofluorescence of the culture fluid in
the background of the acquired image is acquired, and the
brightness of this autofluorescence is analyzed.
[0460] Here, since the brightness of this autofluorescence
increases as the culture fluid ages, the point at which the culture
fluid should be replaced can be detected by measuring the
brightness of this autofluorescence.
[0461] Then, if the temporal variation in the analyzed background
level is within a predetermined level, the process returns to
analysis of the background level (step S121) and repeats this until
the temporal variation of the background level exceeds the
predetermined level (step S122).
[0462] Once the temporal variation of the background level exceeds
the predetermined value, the waste pump 482 for the culture fluid
is operated (step S123), and then, the supply pump 481 for the
culture fluid is operated (step S124).
[0463] The intervals at which the culture fluid should be supplied
and replaced may be determined on the basis of the autofluorescence
of the culture fluid, as described above. Alternatively, the
culture fluid may be supplied/replaced continuously, or
automatically at intervals specified in advance by the user.
Alternatively, the point at which the culture fluid should be
replaced can be specified at will by selecting the type of cells CE
from a table that is registered in advance. In addition, the amount
of culture fluid to be replaced may be specified by the user or may
be determined on the basis of the autofluorescence of the culture
fluid. Alternatively, the amount of culture fluid to be replaced
can be specified at will by selecting the type of cells CE from a
table that is registered in advance.
[0464] The amount of culture fluid to be replaced may be calculated
and determined automatically using the weight and so on.
[0465] In this embodiment, the level of autofluorescence in the
background is detected using the acquired image; however, it may be
detected from an acquired image of a location where cells CE do not
exist. Alternatively, it may be detected by providing an optical
detector in the vicinity of the culture-fluid vessel 472.
[0466] According to the measurement procedure described above, as
shown in FIG. 43, a cell-tracking image showing the change in
position of individual cells over time can be obtained.
[0467] Next, the culturing and measurement procedures when using
the microplate 520 will be described with reference to FIGS. 44 and
45.
[0468] FIGS. 44 and 45 are flowcharts showing the culturing and
measurement when using the microplate 520.
[0469] First, sterilized water is supplied to the reservoir 521 in
the incubator box 500a (step S131).
[0470] Next, the PC is started up (step S132), and the main power
supplies for the detection unit 420 and the culture unit 470 are
turned on (step S133).
[0471] After that, the internal fan 522 inside the incubator box
500a is operated (step S134) to circulate the air inside the
incubator box 500a. Then, the CO.sub.2-concentration controller 495
is operated (step S135) to regulate the carbon dioxide
concentration in the culture gas supplied to the incubator box 500a
at 5%. Thereafter, the temperature controllers are operated (step
S13.6) to regulate the culture-fluid temperature, the culture-gas
temperature, and the temperature inside the insulated compartment
421 at about 37.degree. C.
[0472] After that, the door 427 of the detection unit 420 is opened
(step S137), the incubator box 500a is placed on the stage 422
(step S138), and the door 427 is closed (step S139).
[0473] Next, the transmission light source 423 is turned on (step
S140) to irradiate the cells CE with transmission light, and the
measurement conditions are set (step S141).
[0474] Then, by pressing the start-measurement button (step S142),
measurement of the cells CE commences.
[0475] First, scanning is carried out beforehand on predetermined
cells CE to perform autofocusing (step S143), and once the focus
position of each part has been determined, the shutter 435 is
opened (step S144).
[0476] Next, an image of the cells CE is acquired and output (step
S145). Here, the acquired image data is output to the memory of the
computer PC.
[0477] Then, if the required image acquisition has not yet been
completed, the operations from the autofocusing (step S143) to the
image acquisition and outputting (step S145) are repeated until the
required image acquisition has been completed (step S146). Here,
the required images are, for example, the images acquired using the
selected wavelength, the images acquired using the selected
magnification, and so forth.
[0478] Once the required image acquisition has been completed, the
X-axis motion stage 422X or the Y-axis motion stage 422Y is driven
by one step (step S147). Then, if the position to which the X-axis
motion stage 422X or the Y-axis motion stage 422Y has moved is
within the measurement region, the operations from the autofocusing
(step S143) to driving of the motion stage by a single step (step
S147) are repeated. These operations are repeated until the
position to which the X-axis motion stage 422X or the Y-axis motion
stage 422Y has moved is outside of the measurement region.
[0479] When the position to which the X-axis motion stage 422X or
the Y-axis motion stage 422Y has moved is outside the measurement
region, the shutter 435 is closed (step S149), and the X-axis
motion stage 422X and the Y-axis motion stage 422Y are moved to
their home positions (step S150).
[0480] Thereafter, after a predetermined measurement interval, the
operations from the autofocusing (step S143) to moving the stages
to their home positions (step S150) are repeated until the end of
the measurement time (step S151).
[0481] Once the measurement time ends (step S152), the door 427 is
opened (step S153), and the microplate 520 is removed from the
incubator box 500a (step S154). Then, the sterilized water is
removed from the reservoir 521 (step S155), and the door 427 is
closed (S156).
[0482] After that, the UV lamp 425 inside the insulated compartment
421 is illuminated (step S157) to sterilize the interior of the
insulated compartment 421, and the measurement is thus
completed.
[0483] As described above, the sterilization may be carried out at
the end of the measurement procedure, or alternatively, it may be
carried out at the beginning of the measurement procedure to
sterilize the insulated compartment 421 before measurement actually
takes place.
[0484] The autofocusing may be carried out for each measurement of
the cells CE, as described above, but it need not be carried out
for each measurement.
[0485] With the configuration described above, since the thermal
environment is maintained by the insulated compartment 421 and the
humidity environment and the culture-fluid environment are
maintained by the chamber 510 disposed inside the insulated
compartment 421, the humidity environment and the culture-fluid
environment are influenced by the thermal environment, and
therefore the thermal environment inside the chamber 510 is also
maintained.
[0486] Accordingly, sudden changes and non-uniformities in the
thermal environment, which might cause damage to the cells CE, are
moderated via the humidity environment and the culture-fluid
environment, and therefore, it is possible to reduce damage caused
to the cells CE.
[0487] Also, since the dimensions of the chamber 510 are small
compared to the insulated compartment 421, it is easier to maintain
and control the humidity environment and the culture-fluid
environment, thus making it relatively difficult to cause damage to
the cells CE.
[0488] Since the cells CE can be examined through the insulated
compartment 421, the incubator box 500, and the chamber 510, it is
possible to carry out examination while culturing the cells CE,
without causing any damage to the cells CE. Accordingly, behavior
that the cells exhibit during culturing can be accurately measured
over time.
[0489] It is possible to measure in real time the reaction of the
cells CE in the object under examination while changing the culture
conditions. For example, the existence and level of expression of
proteins can be measured, and changes in the level of expression
with time can be accurately measured.
[0490] Furthermore, deterioration of the activity of the cells CE
by handling them in a first examination can be prevented, which
enables multiple examinations of the same cells CE. Also, since
multiple examinations of the same cells CE can be carried out at
intervals, it is not necessary to control the experimental
protocol.
[0491] Since the detection section 440 examines the cells CE in the
chamber 510 via the insulated compartment 421 and the incubator
500, it is not necessary to insert and remove the cells CE from the
chamber 510 during examination, and therefore, the cells CE can
remain inside the chamber 510 during examination. Accordingly, it
is possible to accurately examine the same position in each
examination. Also, contamination can be prevented during
examination, which can prevent the cells from being stressed.
[0492] Furthermore, by controlling the environmental conditions
inside the chamber 510 (for example, the combination of carbon
dioxide concentration and humidity), it is possible to prevent the
performance of the detection section 440 from deteriorating.
[0493] Moreover, since the cells CE are contained inside the
chamber 510, which is disposed inside the insulated compartment 421
and the incubator box 500, a certain distance can be maintained
between the cells CE and the environment outside the incubator box
500 compared to a case where chamber 510 is not provided.
Therefore, it is possible to reduce the effects of electric and
magnetic fields from outside the incubator box 500, such as those
from the driving motors of the X-axis motion stage 422X and the
Y-axis motions stage 422Y and magnets provided in the door 427.
Eleventh Embodiment
[0494] Next, an eleventh embodiment will be described with
reference to FIGS. 46A to 48.
[0495] The basic construction of the biological-specimen
examination system of this embodiment is the same as the tenth
embodiment, but the constructions of the detection unit and the
culture unit are different from those in the tenth embodiment.
Therefore, in this embodiment, only the detection unit and the
culture unit will be described using FIGS. 46A to 48, and a
description of the chamber and so on will be omitted.
[0496] FIG. 46A is an elevational view of a biological-specimen
examination system 600 of this embodiment, and FIG. 46B is a side
view thereof.
[0497] As shown in FIGS. 46A and 46B, the biological-specimen
examination system 600 includes an inverted microscope (microscope
imaging apparatus) 610 and a culture stage 620. The inverted
microscope 610 and the culture stage 620 may be integrated or they
may be constructed so as to be detachable from each other.
[0498] If the culture system 620 can be attached to and detached
from the inverted microscope 610, an existing inverted microscope
can also be used. In such a case, even though a stage driving motor
is disposed in the vicinity of the cells CE due to constraints on
the shape and the construction involved with attaching the culture
stage 620, for example, the effect of electric and magnetic fields
on the cells CE can be suppressed.
[0499] Also, when examining the cells CE using the inverted
microscope 610, for example, the culture stage 620 can be attached
to the inverted microscope 610; at other times (for example, when
culturing the cells CE), the inverted microscope 610 can be removed
from the microscope.
[0500] FIG. 47 is a plane view of the culture stage 620, and FIG.
48 is a perspective view of the culture stage 620.
[0501] As shown in FIGS. 46A, 46B, and 47, the culture stage 620
includes a frame 621, openable/closable lid 622 provided on the
upper surface of the frame 621, an X-axis motion stage 422X, a
Y-axis motion stage 422Y, a small or strip-shaped heater 620H, a
heatsink 623, a fan 624, and culture-gas supply connector 625.
[0502] The frame 621 is preferably formed of a highly opaque,
corrosion-resistant material, such as anodized aluminum or
stainless steel, like SUS 316. More preferably, from the viewpoint
of thermal insulation, a material having a low thermal conductivity
is selected.
[0503] The interior of the frame 621 is divided into a measurement
area 626 for performing examination of cultured cells and a
non-measurement area 627 only for culturing cells. Thus, cell
culturing is performed in the culture stage 620. A microplate 520
that holds the cells is accommodated inside the culture stage 620,
and the culture stage 620 is configured so as to allow examination
of the cells in the microplate 520 from outside the culture stage
620. In the description given here, the microplate 520 is used as a
culture vessel, as shown in FIGS. 47 and 48, but a dish or flask
may also be used.
[0504] The fan 624 and the culture-gas supply connector 625 are
disposed in a side wall in the non-measurement area 627 in the
frame 621. Also, the heater 620H and the heatsink 623 for
dissipating heat from the heater 620H are disposed in an area where
the fan 624 and the culture-gas supply connector 625 are not
disposed (including the measurement area 626).
[0505] The fan 624 causes convection of the air inside the culture
stage 620 and is arranged so as not to blow directly onto the
incubator box 500a.
[0506] The temperature inside the culture stage 620 is raised by
means of the heater 620H and is regulated at 36.5.degree.
C.+0.5.degree. C.
[0507] As shown in FIGS. 47 and 48, the X-axis motion stage
422.times. and the Y-axis motion stage 422Y are provided on the
bottom surface of the frame 621. The X-axis motion stage 422.times.
and the Y-axis motion stage 422Y are driven by, for example, motors
and ball screws.
[0508] A small or strip-shaped heater (not shown) is attached to
the Y-axis motion stage 422Y. The heater is disposed at a position
such that the microplate 520 is uniformly heated.
[0509] The measurement area 626 and the non-measurement area 627
are formed by dividing the interior of the culture stage 620 in the
X-direction by means of a top plate 628 and a pair of partition
seats 629, which are fixed to the frame 621. The region to the left
of the partition seats 629 in FIG. 47 constitutes the measurement
area 626, and the region to the right constitutes the
non-measurement area 627.
[0510] A partition plate 629a that is formed in substantially the
same shape as the cross-sectional shape of the frame 621 is
attached to the X-axis motion stage 422X.
[0511] By moving the X-axis motion stage 422X towards the
non-measurement area 627, side faces at both ends (the ends in the
Y-direction) of the partition plate 629a come into contact with the
partition seats 629, and the partition plate 629a is thus
positioned so as to divide the space inside the frame 621 into two
portions in the left-to-right (X-axis) direction.
[0512] Furthermore, since the edge of the partition plate 629a at
the top plate 628 is positioned in contact with the lower surface
of the top plate 628, the measurement area 626 and the
non-measurement area 627 can define two spaces that are separated
from each other.
[0513] At the upper opening of the measurement area 626, a glass
lid 630 is removably attached to the frame 621 and is disposed so
as to cover the upper opening of the measurement area 626. The
glass lid 630 can be attached by, for example, screwing the glass
lid 630 to the frame 621, or by means of a lock mechanism, a hook,
a magnet, and so forth.
[0514] The glass lid 630 may be constructed such that the entire
surface or substantially the entire surface except for the
peripheral frame portion is formed of the glass plate 631, or it
may be formed with a minimum possible area so long as it does not
obstruct measurement. In order to suppress the reflection of light
during transmission examination and incidence examination, it is
preferable to use an optical glass material having an
anti-reflection film (AR coat) coated on both sides thereof as the
glass plate 631.
[0515] The anti-reflection film may be coated on both sides of the
glass plate 631, as described above, or it may be coated on only
one side of the glass plate 631.
[0516] The glass lid 630 may be removed as required when carrying
out various tasks, for example, when changing the objective lens of
the inverted microscope 610, when cleaning the inside of the
measurement area 626, and so forth.
[0517] The glass lid 630 may be provided with an observation hole
into which the objective lens of the inverted microscope 610 is
inserted. Also, a rubber sheet may be disposed in the observation
hole to occupy a gap between the objective lens and the observation
hole. The sheet is preferably disposed so as to prevent relative
motion between the objective lens and the culture stage 620.
[0518] At the upper opening of the non-measurement area 627, the
openable/closable lid 622 is attached so that it can be opened and
closed by means of a hinge or the like. When closed, one edge of
the openable/closable lid 622 is in contact with the top plate 628
so as to be supported.
[0519] The openable/closable lid 622 is entirely formed of an
opaque material (for example, the same material as the frame 621)
and is provided with an observation-hole cover 632 that blocks the
observation hole or a UV-irradiation-hole cover 633 that blocks a
UV irradiation hole, as required.
[0520] The observation hole is an opening (window) formed in the
openable/closable lid 622. The observation-hole cover 632 is
formed, for example, of a glass plate, a resin plate, or the like
having low transmittance and is inserted in the observation hole.
Also, the observation-hole cover 632 is formed of the same opaque
material as the openable/closable lid 622 and may be attached in
such a manner that it can be either detached or opened and
closed.
[0521] The UV-irradiation-hole is an opening (window) formed in the
openable/closable lid 622. The UV-irradiation-hole cover 633 is
formed, for example, of the same opaque material as the
openable/closable lid 622 and is attached in such a manner that it
can be opened and closed, or detached from the UV irradiation
hole.
[0522] The UV-irradiation-hole cover 633 is removed when
irradiating the interior of the non-measurement area 627 with
ultraviolet (UV) light to sterilize it. A handheld UV lamp can be
used for the UV irradiation.
[0523] The incubator box 500a, which contains the microplate 520,
is held on the Y-axis motion stage 422Y. Since the incubator box
500a is the same as that described in the tenth embodiment, the
same components are assigned the same reference numerals, and a
description thereof shall be omitted.
[0524] Culture gas is supplied to the connector 523 of the
incubator box 500a via a culture-gas supply tube 634 from a
culture-gas supply connector 625 of the culture stage 620.
[0525] With the configuration described above, since the
biological-specimen examination system 600 according to the present
invention includes the integrated inverted microscope 610, a
biological specimen can be examined using the inverted microscope
610. Therefore, more detailed examination can be carried out
compared to a case where the inverted microscope 610 is not
provided.
[0526] When measuring and culturing the cells, in order that the
cells are not affected by ambient light, the entire
biological-specimen examination system 600 may be surrounded with a
blackout curtain.
[0527] Also, instead of cells, the biological specimen to be
measured may be various other types of biological specimen, such as
bacteria, microorganisms, ova, and so forth.
[0528] With the biological-specimen examination system of this
embodiment, since it is possible to reduce the image acquisition
time, special phenomena that occur in the biological specimen only
for a short time can be observed, and the accuracy of the
examination results of the biological specimen can be improved.
[0529] Furthermore, with the biological-specimen examination system
of this embodiment, an advantage is afforded in that, even when the
brightness of the biological specimen changes over time, it is
possible to effectively observe such changes in the biological
specimen over time. Also, it is possible to accurately examine a
wide range of biological specimens, from specimens having low
brightness to specimens having high brightness.
[0530] Moreover, with the biological-specimen examination system of
this embodiment, since the time required for measuring the
measurement region once can be shortened, the motion of the
biological specimen in part of the measurement region does not vary
substantially over time. Thus, the accuracy of the examination
results when examining the biological specimen over time can be
improved.
* * * * *