U.S. patent application number 12/386091 was filed with the patent office on 2009-08-13 for methods for implement microscopy and microscopic measurement as well as microscope and apparatus for implementing them.
Invention is credited to Hiroshi Ishiwata.
Application Number | 20090201580 12/386091 |
Document ID | / |
Family ID | 34810102 |
Filed Date | 2009-08-13 |
United States Patent
Application |
20090201580 |
Kind Code |
A1 |
Ishiwata; Hiroshi |
August 13, 2009 |
Methods for implement microscopy and microscopic measurement as
well as microscope and apparatus for implementing them
Abstract
The invention relates to a microscope that enables a phase
object or surface pits and projections to be observed at a
relatively low image-formation magnification of 4 or lower over a
wide viewing range yet in a relatively narrow spatial frequency
distribution range. The microscope comprises a light source 2, an
illumination optical system 3 for guiding light from the light
source 2 to an object 4 under observation, a partial aperture
located substantially at the pupil position 28 of the illumination
optical system 3 and an image-formation optical system 15, 16, 18
for forming on the image-formation plane 19 an image 4 of the
object under observation illuminated by light passing through the
partial aperture, and further comprises an eyepiece optical system
6 or an image pickup optical system for viewing the image formed on
the image-formation plane 19. The diameter of the image of said
partial aperture at the pupil position 30 of the image-formation
optical system is set smaller than the pupil diameter of the
image-formation optical system, and at the pupil position 30 of the
image-formation optical system there is located wavefront
introduction means for introducing in the pupil position 30 of the
image-formation optical system a wavefront varying in size with the
pupil diameter.
Inventors: |
Ishiwata; Hiroshi; (Tokyo,
JP) |
Correspondence
Address: |
KENYON & KENYON LLP
1500 K STREET N.W., SUITE 700
WASHINGTON
DC
20005
US
|
Family ID: |
34810102 |
Appl. No.: |
12/386091 |
Filed: |
April 13, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11003728 |
Dec 6, 2004 |
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12386091 |
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Current U.S.
Class: |
359/368 |
Current CPC
Class: |
G02B 21/14 20130101;
G02B 21/361 20130101 |
Class at
Publication: |
359/368 |
International
Class: |
G02B 21/00 20060101
G02B021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 12, 2003 |
JP |
2003-414158 |
Feb 6, 2004 |
JP |
2004-030431 |
Claims
1-34. (canceled)
35. A microscope observation method, comprising: (a) illuminating a
sample with illumination light of an NA (numerical aperture)
smaller than a maximum NA of a condenser lens or an objective lens;
(b) entering light that transmits through or reflected from the
sample into the objective lens, wherein the objective lens is
provided with an absorption film having a similar shape as the
illumination light, the absorption film provided at a part slightly
larger than a part corresponding to the NA of the illumination
light on an exit pupil plane or a plane conjugate thereto; (c)
sequentially introducing wavefronts in a state inverting to each
other in the vicinity of a part where the wavefronts become flat
into the exit pupil plane of the objective lens; (d) recording each
observed image in a state where states of wavefronts invert to each
other in the vicinity of a part where the introduced wavefronts
become flat; and (e) carrying out image processing for each
recorded image and emphasizing contrast to observe the sample.
36. The microscope observation method according to claim 35,
wherein the wavefront introduction is carried out by shifting a
sight of the objective lens.
37. The microscope observation method according to claim 35,
wherein the wavefront introduction is carried out by control of a
variable shape mirror arranged at a position conjugate with the
exit pupil plane of the objective lens.
38. The microscope observation method according to claim 35,
wherein the illumination light is formed by providing an aperture
of a circle or orbicular zone shape smaller than a maximum NA on an
entrance pupil plane of the condenser lens, and being transmitted
through the aperture.
39. The microscope observation method according to claim 35,
wherein the illumination light is formed by using a substantial
point light source that illuminates a portion smaller than the
entrance pupil plane of the condenser lens.
Description
[0001] This application claims benefit of Japanese Application No.
2003-414158 filed in Japan on Dec. 12, 2003 and No. 2004-30431
filed in Japan on Feb. 6, 2004, the contents of which are
incorporated by this reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to methods for
implementing microscopy and microscopic measurements as well as a
microscope and apparatus for implementing them. More particularly,
the present invention relates to a method and microscope for
observing phase objects such as living cells and minute pits and
projections on semiconductor substrates, metal surfaces and glass
substrates and a method and apparatus for measuring the number,
morphology, distribution and so on of cells cultured in a culture
vessel.
[0003] Living cells, being colorless and transparent, cannot be
viewed through ordinary bright-field observations and, hence, are
viewed by means of phase contrast microscopes or differential
interference microscopes.
[0004] Like living cells, minute pits and projections occurring on
semiconductor substrates or metal surfaces, too, are observed under
differential interference microscopes, because of being incapable
of bright-field observations.
[0005] As set forth typically in patent publication 1, a phase
contrast microscope has a zonal aperture located at the pupil
position of a condenser lens therein and a phase film that is
similar in shape to the zonal aperture and is located at the pupil
position of an objective lens conjugate to the zonal aperture via
an object under observation, so that an image contrast proportional
to the phase distribution of the phase object is obtainable.
[0006] As set forth typically in patent publication 2, a
differential interference microscope has two doubly refracting
prisms one located at the pupil position of a condenser lens and
another at the pupil position of an objective lens with
interference of orthogonal two polarized light components slightly
displaced sideways, so that an image contrast proportional to the
differentiated value of the phase distribution of a phase object is
obtainable.
[0007] By the way, studies of growing and proliferating cells
sampled from within a living body and applying grown cells to
medical fields and pharmacometrics are now in the making.
[0008] Grown cells must be provided in a specific number and
amount. So far, growing cells in a culture vessel have been
observed under a microscope to visually measure how many cells are
there and to what degree cells are growing. In another effort,
images under observation are captured from a microscope in a CCD
camera or the like and the images are processed to determine the
number of cells (non-patent publication 3).
[0009] Patent Publication 1
[0010] JP(A) 7-225341
[0011] Patent Publication 2
[0012] JP(A) 8-122648
[0013] Patent Publication 3
[0014] JP(A) 2002-214228
[0015] Non-Patent Publication 1 [0016] "Optics", Vol. 30, No. 9
(2001), pp. 605 (33)-612 (40)
[0017] Non-Patent Publication 2 [0018] "Optics" by Hiroshi Kubota,
pp. 300-304 (1964, Iwanami Shoten, Publishers)
[0019] In both the phase contrast microscope and the differential
interference microscope, at the pupil position of each of an
illumination optical system and an image-formation optical system
there is located a device for the visualization of phase
distributions. To this end, the illumination optical system must
stay conjugate to the image-formation optical system. However, as
the magnification of a viewing optical system goes down to make a
viewing range wider, pupil aberration between the illumination
optical system and the image-formation optical system becomes too
large to keep the pupils in conjugate relations. This renders it
difficult to use both the phase contrast microscope and the
differential interference microscope for observations at
magnifications lower than 4.
[0020] For instance, when living cells are observed during growing
or minute pits and projections remaining on a semiconductor
substrate surface, faster observations are necessary over a wide
viewing range. However, both the phase contrast microscope and the
differential interference microscope offer a problem in this
regard.
[0021] Further, with living cells under observation by the phase
contrast microscope, the coexistence of an intensity component that
is not proportional to the phase difference of living cells and
called a halo renders observations difficult. Furthermore, the
coexistence of external perturbations such as illumination
variations renders observations much harder.
[0022] With visual observations and measurements of growing cells
in a culture vessel, there are problems such as longer time taken
for measurements all over the culture vessel, differences from
person to person, and lower repeatability.
[0023] In image processing, a cell portion is extracted depending
on an image contrast difference between the cell portion to be
detected and other cell-free portion. However, this is susceptible
of influences by S/N changes of the detected image due to changes
in the quantity distribution of illumination light or changes in
viewing environments. There is another requirement that the viewing
magnification be increased, ending up with another problem that the
time for making measurements all over the culture vessel surface
becomes long.
SUMMARY OF THE INVENTION
[0024] In view of such problems with the prior art as described
above, one object of the invention is to provide a method for
implementing microscopy which enables a phase object or surface
pits and projections to be observed at a relatively low
image-formation magnification of 4 or lower over a wide viewing
range yet in a relatively narrow spatial frequency distribution
range, and a microscope for carrying out that method.
[0025] Another object of the invention is to provide a method for
implementing microscopy and microscopic measurements which enables
the number, morphology, distribution, etc. of growing cells to be
measured without affected by external perturbations as well as a
microscope for carrying out that method and an apparatus for
implementing microscopic measurements.
[0026] According to the invention, the above objects are
accomplishable by the provision of a method for implementing
microscopy, characterized in that a partial aperture is located at
a pupil position of an illumination optical system in a microscope,
an object under observation is illuminated by light passing through
said partial aperture, and in a pupil position of an
image-formation optical system there is introduced a wavefront
varying in size with a pupil diameter to view an image formed on an
image-formation plane of said image-formation optical system.
[0027] The invention also provides a method for implementing
microscopy, characterized in that a substantial point light source
that gives out divergent light is used as an illumination light
source to form an image of said substantial point light source at a
pupil position of an illumination optical system in a microscope,
an object under observation is illuminated by the divergent light
from the image of said substantial point light source, and in a
pupil position of an image-formation optical system there is
introduced a wavefront varying in size with a pupil diameter to
view an image formed on an image-formation plane of said
image-formation optical system.
[0028] Preferably in each method, the image-formation optical
system should have an image-formation magnification of 4 or
lower.
[0029] The wavefront to be introduced in the pupil position of the
image-formation optical system may be either based on a
displacement from the focus position of the image-formation optical
system, or created by a variable-shape mirror that is located at
the pupil of the image-formation optical system or at or near a
position conjugate thereto.
[0030] When the partial aperture is located at the pupil position
of the illumination optical system the partial aperture comprises a
circular aperture or a zonal aperture, with satisfaction of
condition (10):
D.sub.0/10.ltoreq.D.sub.1.ltoreq.D.sub.0/3 (10)
where D.sub.1 is the aperture width of an image at the pupil
position of the image-formation optical system, and D.sub.0 is the
diameter of the pupil of the image-formation optical system.
[0031] It is preferable that an absorption film that conforms in
shape to the image of the partial aperture or the substantial point
light source at the pupil position of said image-formation optical
system and is slightly larger than that shape is located at the
pupil of said image-formation optical system or at or near a
position conjugate thereto, with satisfaction of condition (11)
.DELTA..ltoreq.D.sub.0/10 (11)
where .DELTA. is a margin width on the inner or outer peripheral
side of the absorption film with respect to the image of the
partial aperture or the substantial point light source at the pupil
position of the image-formation optical system, and D.sub.0 is the
diameter of the pupil of the image-formation optical system.
[0032] While the wavefront introduced in the pupil position of the
image-formation optical system is substantially symmetrically
changed, two images with inverted contrasts are taken of the same
object under observation, and a subtract operation is applied
between the two images thereby providing an enhanced contrast.
[0033] The invention provides a microscope, comprising a light
source, an illumination optical system for guiding light from the
light source to an object under observation, a partial aperture
located substantially at a pupil position of the illumination
optical system and an image-formation optical system for forming on
an image-formation plane an image of the object under observation
illuminated by light passing through the partial aperture, and
further comprising an eyepiece optical system or an image pickup
optical system for viewing the image formed on the image-formation
plane, characterized in that:
[0034] the diameter of an image of the partial aperture at the
pupil position of the image-formation optical system is set smaller
than the pupil diameter of the image-formation optical system,
wherein at the pupil position of the image-formation optical system
there is located wavefront introduction means for introducing in
the pupil position of the image-formation optical system a
wavefront varying in size with the pupil diameter.
[0035] The invention also provides a microscope, comprising a
substantial point light source for giving out divergent light, an
illumination light source for guiding light from the substantial
light source to an object under observation while the image of the
substantial point light source is once formed at a pupil position,
and an image-formation optical system for forming on an
image-formation plane an image of the object under observation
illuminated by light from the substantial point light source, and
further comprising an eyepiece optical system or an image pickup
optical system for viewing the image formed on the image-formation
plane, characterized in that:
[0036] the diameter of the image of the substantial point light
source at the pupil position of the image-formation optical system
is set smaller than the pupil diameter of the image-formation
optical system, wherein at the pupil position of the
image-formation optical system there is located wavefront
introduction means for introducing in the pupil position of the
image-formation optical system a wavefront varying in size with the
pupil diameter.
[0037] Preferably in each microscope, the image-formation optical
system should have an image-formation magnification of 4 or
lower.
[0038] The wavefront introduction means may comprise either means
for displacement and adjustment of the image-formation optical
system in the optical axis direction or a variable-shape mirror
that is located at the pupil of the image-formation optical system
or at or near a position conjugate thereto, and has a variable or
adjustable reflecting surface shape.
[0039] When the partial aperture is located at the pupil position
of the illumination optical system the partial aperture comprises a
circular aperture or a zonal aperture, with satisfaction of
expression (10):
D.sub.0/10.ltoreq.D.sub.1.ltoreq.D.sub.0/3 (10)
where D.sub.1 is the aperture width of an image at the pupil
position of the image-formation optical system, and D.sub.0 is the
diameter of the pupil of the image-formation optical system.
[0040] It is preferable that an absorption film that conforms in
shape to the image of the partial aperture or the substantial point
light source at the pupil position of said image-formation optical
system and is slightly larger than that shape is located at the
pupil of said image-formation optical system or at or near a
position conjugate thereto, with satisfaction of condition (11)
.DELTA..ltoreq.D.sub.0/10 (11)
where .DELTA. is a margin width on an inner or outer peripheral
side of the absorption film with respect to the image of the
partial aperture or the substantial point light source at the pupil
position of the image-formation optical system, and D.sub.0 is the
diameter of the pupil of the image-formation optical system.
[0041] Preferable in one specific embodiment of the invention, the
microscope should further comprise an image pickup optical system
for picking up the image formed on the image-formation plane, an
image pickup device located on the image pickup plane thereof, and
operation and storage means for storing and operating the image
picked up by the image pickup device.
[0042] With the methods for implementing microscopy and the
microscopes as recited above, living cells or minute pits and
projections on metal surfaces can be observed without recourse to
phase contrast microscopes or differential interference
microscopes. In particular, it is possible to observe phase
distributions or pit-and-projection distributions at a relatively
low image-formation magnification of 4 or lower at which
conventional phase contrast microscopes or differential
interference microscopes would run into difficulty. It is also
possible to cancel out illumination variations or halo components,
thereby obtaining an image intensity distribution of good contrast
commensurate with the phase distribution or pit-and-projection
distribution of an object under observation.
[0043] The invention provides a method for implementing microscopic
measurements, characterized in that by means of a microscope that
comprises an illumination optical system, a viewing optical system
including a unit for converting an object under observation into a
contrast image by interference and an image pickup device for
capturing therein a contrast image having a phase difference of
opposite sign upon interference of the image under observation, two
images comprising contrast images having a phase difference of
opposite sign are captured therein to apply a subtract operation to
the captured two images to form an image subjected to the subtract
operation, and at least one of the number of cells, the area taken
up by cells, the abundance distribution of cells and the abundance
density of cells in a viewing range is measured from the obtained
image subjected to the subtract operation.
[0044] Preferably in this case, a portion having an intensity value
of zero or nearly zero is extracted out of the intensity
distribution of the image subjected to the subtract operation to
take the portion as being a cell-free portion and other portion as
being a cell-containing portion, thereby making a separation
between the portions containing cells and no cells.
[0045] Alternatively, a portion having an intensity value of zero
or nearly zero is extracted out of the intensity distribution of
the image subjected to the subtract operation to allocate an
intensity value to one value and other portion to another one
intensity value for binarization.
[0046] The invention also provides a method of implementing
microscopic measurements, characterized in that by means of a
microscope that comprises an illumination optical system, a viewing
optical system including a unit for converting an object under
observation into a contrast image by interference and an image
pickup device for capturing therein a contrast image having a phase
difference of opposite sign upon interference of the image under
observation, images comprising contrast images having a phase
difference of opposite sign are captured therein to apply a
subtract operation and an add operation to the captured two images
to form two images wherein the image subjected to the subtract
operation is divided by the image subjected to the add operation to
measure information about the thickness of cells in a viewing
range.
[0047] The invention provides an apparatus for implementing
microscopic measurements, characterized by comprising a microscope
that comprises an illumination optical system, a viewing optical
system including a unit for converting an object under observation
into a contrast image by interference and an image pickup device
for capturing therein a contrast image having a phase difference of
opposite sign upon interference of the image under observation, and
a processing unit for capturing therein two images comprising
contrast images having a phase difference of opposite sign to apply
a subtract operation to the captured two images to form an image
subjected to the subtract operation, so that at least one of the
number of cells, the area taken up by cells, the abundance
distribution of cells and the abundance density of cells in a
viewing range is measured from the obtained image with the subtract
operation applied thereto.
[0048] Preferably in this case, the apparatus should further
comprise a separation unit for extracting a portion having an
intensity value of zero or nearly zero out of an intensity
distribution of the image subjected to the subtract operation to
take the portion as being a cell-free portion and other portion as
being a cell-containing portion, thereby making a separation
between the portions containing cells and no cells.
[0049] Alternatively, the apparatus may further comprise a
binarization unit for extracting a portion having an intensity of
zero or nearly zero out of the intensity distribution of the image
subjected to the subtract operation to allocate an intensity value
to one value and other portion to another one intensity value for
binarization.
[0050] The invention also provides an apparatus for implementing
microscope measurements, characterized by comprising a microscope
that comprises an illumination optical system, a viewing optical
system including a unit for converting an object under observation
into a contrast image by interference, and an image pickup device
for capturing therein a contrast image having a phase difference of
opposite sign upon interference of the image under observation, an
operation unit for capturing therein two images comprising contrast
images having a phase difference of opposite sign to apply a
subtract operation and an add operation to the captured two images
to form two images, and a thickness measurement unit for dividing
the image subjected to the subtract operation divided by the image
subjected to the add operation to measure information about the
thickness of cells in the viewing range.
[0051] In one specific embodiment of the apparatus, an aperture is
located substantially at the pupil position of the illumination
optical system, and the viewing optical system including a unit for
converting the object under observation into a contrast image by
interference is constructed such that the image of the object under
observation illuminated by the light passing through the aperture
in the illumination optical system is capture by the image pickup
device at positions displaced from the focus position toward the
near point side and the far point side.
[0052] With the method and apparatus for implementing microscopic
measurements as recited above, it is possible to form an image
subjected to the subtract operation thereby obtaining image
information of growing cells, which are less affected by external
perturbations such as illumination variations. In particular, a
portion with no cells distributed is little affected by external
perturbations.
[0053] By carrying out the extraction of a cell-free portion at
first, it is possible to make an easy separation between a
cell-containing portion and the cell-free portion.
[0054] Further, if the cell-containing portion is separated from
the cell-free portion for binarization, it is then possible to
calculate the area taken up by cells. Furthermore, if the average
area of cells is previously found, it is then possible to calculate
the number of cells.
[0055] Also, if the quotient of the images subjected to the
subtract operation and the add operation is found, it is then
possible to calculate the height distribution of cells. Further, if
a specific extraction range is set for binarization, it is then
possible to extract cell nuclei and, hence, obtain information
depending on the cell nuclei.
[0056] Still other objects and advantages of the invention will in
part be obvious and will in part be apparent from the
specification.
[0057] The invention accordingly comprises the features of
construction, combinations of elements, and arrangement of parts,
which will be exemplified in the construction hereinafter set
forth, and the scope of the invention will be indicated in the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] FIG. 1 is illustrative of the principles of the
invention.
[0059] FIGS. 2(a) and 2(b) are illustrative of the special
frequency characteristics of partially coherent image
formation.
[0060] FIG. 3 is illustrative of an image (a) of the pupil of an
illumination optical system formed by light that is not diffracted
by an object at the pupil of an image-formation optical system and
an image (b) of the pupil of the illumination optical system formed
by light that is subjected to first-order diffraction by the object
at the pupil of the image-formation optical system in the case
where the pupil of the illumination optical system is of zonal
shape.
[0061] FIG. 4 is illustrative in schematic of one example of the
inverted microscope according to the invention.
[0062] FIGS. 5(a) and 5(b) are illustrative of exemplary aperture
shapes of the aperture unit attached to or detached from the pupil
position of a condenser lens.
[0063] FIGS. 6(a) and 6(b) are illustrative of an image of the
aperture of FIG. 5 as projected onto the pupil plane of an
objective lens.
[0064] FIGS. 7(a) and 7(b) are illustrative of the configuration of
an absorption film in an absorption film unit attached to or
detached from the pupil plane of the objective lens with respect to
the aperture shape of the aperture unit of FIG. 5.
[0065] FIG. 8 is illustrative in schematic of one example of the
inverted microscope using a variable-shape mirror, similar to FIG.
4.
[0066] FIG. 9 is illustrative in schematic of one example of the
inverted microscope using an LED as an illumination light source,
similar to FIG. 4.
[0067] FIG. 10 is a schematic optical path diagram for the
arrangement of FIG. 4 applied to a reflecting microscope.
[0068] FIG. 11 is generally illustrative in schematic of one
example of the microscope capable of viewing and picking up the
contrast image of growing cells, making use of defocusing.
[0069] FIGS. 12(a), 12(b), 12(c) and 12(d) are illustrative of how
to measure states of growing cells according to the invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0070] First of all, the principles of creating image contrasts
proportional to the pit-and-projection phase distribution of a
phase object or pits and projections on a substrate surface
according to the invention are explained.
[0071] FIG. 1 is illustrative of the principles of the invention,
and provides a schematic illustration of an arrangement comprising
an object point and the entrance pupil, exit pupil and
image-formation plane of an image-formation optical system. As an
object under observation is located at a focus position as shown in
FIG. 1, light coming out of an object point under observation
spreads out in spherical waveform, entering the entrance pupil of
the image-formation optical system, whence the incident light takes
a spherical waveform of converging light, leaving the exit pupil of
the image-formation optical system and converging on the
image-formation plane to form an image thereon. The formed image is
free from any blurring, because there is no optical path (phase)
difference between light rays passing through the image-formation
optical system.
[0072] As the object point is displaced to a position indicated by
a dotted line, however, light from the object point enters the
entrance pupil in spherical waveform. The light incident on the
entrance pupil leaves the exit pupil, turning to a spherical
waveform of light going toward the image plane after displacement.
If observation is made on the image plane after displacement, there
is no optical path (phase) difference in the light; however, if
observation is made on the initial image-formation plane before
displacement, there is an optical path (phase) difference in each
light ray.
[0073] In the invention, this is utilized to locate the object
under observation at a position off the focus position of the
image-formation optical system to create phase differences in the
respective light rays passing through the image-formation optical
system.
[0074] Referring again to FIG. 1, there is the largest phase
difference between an axial chief ray through the image-formation
optical system and the maximum NA (numerical aperture) light
ray.
[0075] Accordingly, if the object under observation is viewed at a
position off the focus position of the image-formation optical
system, it is then possible to create the amount of phase
difference commensurate with the amount of displacement from the
focus position (the amount of defocusing) between the light
transmitted and the light diffracted through the object under
observation.
[0076] Therefore, that amount of phase difference functions
equivalently to a phase film used in phase contrast observations,
so that colorless, transparent growing cells, etc. can be observed
with an image contrast given in proportion to the phase
distribution of growing cells.
[0077] It is then preferable to form an image on the
image-formation plane of the image-formation optical system at a
lower image-formation magnification, because it is easier to limit
the angle of the diffracted light passing through the
image-formation optical system, with a much improved image
contrast.
[0078] When a phase object is observed, an image contrast
proportional to its phase difference is proportional to the phase
amount of the object under observation and the amount of a phase
difference given between transmitted light and diffracted light.
The angle between the light diffracted and the light transmitted
through the object under observation varies with the shape of the
object under observation. A change in the angle between the
diffracted light and the transmitted light causes the amount of the
phase difference occurring between two light beams to differ even
at the same amount of defocusing. Therefore, if, for instance, the
amount of defocusing is changed for each cell under observation, it
is then possible to obtain much better image contrasts.
[0079] The phase difference of each light ray occurring by
defocusing is of varying sign between displacements of the object
under observation from the focus position of the image-formation
optical system toward a near point side and a far point side. In
images of growing cells or other object under observation, obtained
at positions displaced from the focus position of the
image-formation optical system toward the near point side and the
far point side, their image contrasts are inverted in
correspondence to the phase difference of the object under
observation.
[0080] A light-absorbing object such as dust deposited onto a
laboratory dish or the like can no longer behave as a phase object,
and there is no change in the image contrast even when a phase
difference is given thereto by defocusing. Thus, if an inter-image
operation is applied to an image picked up at a position displaced
toward the near point side and an image picked up at a position
displaced toward the far point side, it is then possible to isolate
an image component that is not affected by the phase difference
given by defocusing. Especially if a subtract operation is applied
to each pixel of two images, it is then possible to double the
image contrast of an image component commensurate with the phase
distribution of the object under observation, thereby eliminating
an image component bearing no phase information about dust, foreign
matters, illumination variations, etc.
[0081] It is noted that the above phase object-viewing method is
particularly useful in applications where the presence of phase
objects in the field must be recognized through an image-formation
optical system having an image-formation magnification of 4 or
lower.
[0082] The above principles are now explained with a little bit of
theory.
[0083] The formation I(x) of an image of a phase substance:
a(U)=exp{-i.phi.(U)} (1)
by partially coherent light is given by expression (6) referred to
in non-patent publication 1, specifically by the following
expression. It is herein noted that the expression numbers do not
correspond to those in non-patent publication 1.
I ( x ) = [ T ( 0 , 0 ) - 1 2 .intg. - .infin. .infin. { T ( f , 0
) + T ( 0 , - f ) } exp ( - 2 .pi. ifx ) .PHI. ( f ) .PHI. * ( f )
f ] + [ .intg. - .infin. .infin. { T ( f , 0 ) - T ( 0 , - f ) }
.PHI. ( f ) exp ( - 2 .pi. ifx ) f ] + [ .intg. - .infin. .infin. T
( f , f ) .PHI. ( f ) .PHI. * ( f ) f + .intg. - .infin. .infin. T
( f , - f ) .PHI. ( f ) .PHI. * ( - f ) exp ( - 4 .pi. ifx ) f ] (
2 ) ##EQU00001##
[0084] In expression (2), the encircled cross symbol (the first
term on the right side) is a convolution. It is noted that U,
f.sub.i (i=1, 2) and x are coordinates on an object plane, a pupil
plane and an image plane, respectively, and .phi.(U) is the phase
information of an object under observation.
[0085] Here .PHI.(f) is a Fourier transformation of .phi.(U), and
T(f.sub.1, f.sub.2) is given by expression (2) referred to in
non-patent publication 1, called the spatial frequency
characteristics (or TCC (transmission cross coefficient) given
by
T ( f 1 , f 2 ) = .intg. - .infin. .infin. S ( .xi. ) P ( f 1 +
.xi. ) P * ( f 2 + .xi. ) .xi. ( 3 ) ##EQU00002##
[0086] Here S(.xi.) is indicative of the intensity distribution of
the pupil of the illumination optical system, and P(.xi.) is
representative of the pupil function of the image-formation optical
system, where .xi. is position coordinates in the pupil.
[0087] Given an aberration-free optical system, P(.xi.) is a
function indicative of a pupil area, given by
P ( .xi. ) = p ( .xi. ) Here , p ( .xi. ) = { 1 ( .xi. .ltoreq. a ,
: pupil radius ) 0 ( Other ) ( 4 ) ##EQU00003##
[0088] When there is defocus or other aberration W(.xi.) (expressed
by a phase amount) in the image-formation optical system,
P(.xi.)=exp{-iW(.xi.)}p(.xi.) (5)
[0089] Of the above expression (2), the first and third terms on
the right side are higher-order terms with respect to the phase
.phi.(U), and the second term gives an image contrast proportional
to the phase distribution .phi.(U). Then, transformation of the
second term of expression (2) gives
T ( f , 0 ) - T ( 0 , - f ) = - 2 i .intg. - .infin. .infin. { W (
.xi. + f ) - W ( .xi. ) } S ( .xi. ) p ( .xi. ) p ( .xi. + f ) .xi.
( 6 ) ##EQU00004##
on condition that the amount of aberration W(.xi.) is small and
rotationally symmetric, and the function p(.xi.) indicative of the
pupil area of the image-formation optical system is rotationally
symmetric.
[0090] From the second term of expression (2) and expression (6),
it is understood that when the phase object is viewed, the phase
distribution can be visualized by offering defocus or other
aberration in such a way as to allow expression (6) to have a value
other than zero.
[0091] For a better understanding of the expansion of expressions,
the expansion is performed with one-dimensional variables in the
foregoing; however, the same also holds for expansion with
two-dimensional variables.
[0092] Schematic illustrations of expression (6) with the pupil of
the illumination optical system and the pupil of the
image-formation optical system are presented in FIGS. 2(a) and
2(b). Here FIGS. 2(a) and 2(b) illustrate that the intensity
distribution (aperture shape) S(.xi.) of the pupil of the
illumination optical system has a circular shape and a zonal shape,
respectively, and show displacements of the pupil of the
image-formation optical system by diffraction through the object.
Referring here to the case where the illumination optical system
has a zonal pupil, the pupil S(.xi.) of the illumination optical
system is displaced as illustrated in FIGS. 3(a) and 3(b) with an
interchanged coordinate system. FIG. 3(a) is the (hatched) portion
to be calculated with respect to W(.xi.) of expression (6), and
FIG. 3(b) is the (hatched) portion to be calculated with respect to
W(.xi.+f). More specifically, FIG. 3(a) corresponds to the image by
light not diffracted through the object of the pupil of the
illumination optical system at the pupil of the image-formation
optical system, and FIG. 3(b) corresponds to the image by light
subjected to first-order diffraction through the object of the
pupil of the illumination optical system at the pupil of the
image-formation optical system.
[0093] Therefore, if the aberration W(.xi.) at the pupil position
of the image-formation optical system is rotationally symmetric and
varies in size with radius, it is then possible to visualize the
phase distribution.
[0094] Consider here W(.xi.) upon defocusing. Wave aberration due
to defocusing is given by
.DELTA.L=-h.sup.2z/f.sup.2 (7)
where h is the height of a light ray in the pupil plane, f is the
focal length of a lens, z is the amount of defocusing, and .DELTA.L
is the optical path length at each point on the pupil, as set forth
in non-patent publication 2. The value of h/f is indicative of a
position on the pupil, equivalent to .xi. indicative of position
coordinates in the pupil. .DELTA.L, because of being an optical
path length difference at the position .xi., is given by
.DELTA.L=W(.xi.).times..lamda.=-.xi..sup.2z (8)
where .lamda. is a wavelength. Therefore,
W(.xi.)=-.xi..sup.2z/.lamda. (9)
That aberration W(.xi.) due to defocusing is rotationally symmetric
and increases in the minus direction in proportion to the square of
radius and, hence, can give an image contrast proportional to the
phase distribution.
[0095] Here in the coordinates for z, the right and left sides of
FIG. 1 are generally positive and negative with respect to the
focus position. Therefore, the sign of W(.xi.), too, varies between
defocusing from the focus toward the left (far point) side and
defocusing from the focus toward the right (near point) side.
[0096] From the above discussions, it is evident that the
aberration W(.xi.) is also achievable by means of optical means,
not by means of defocusing, which optical means gives wave
aberration that is likewise rotationally symmetric on the pupil
plane and has size varying with radius (such as a variable-shape
(deformable) mirror that will be specifically referred to
later).
[0097] Some examples of the microscope for reducing such principles
down to practice according to the invention are now explained.
[0098] FIG. 4 is illustrative in schematic of the inverted
microscope of one example according to the invention. This inverted
microscope shown generally at 1 is made up of a light source 2, an
illumination optical system 3 for guidance of light emanating from
light source 2, a viewing optical system 5 for forming the image of
an object 4 under observation, an eyepiece lens 6 for magnifying
the image of object 4 for visual observation, and a CCD camera 8
including an image pickup device 7 for picking up the image of
object 4. The CCD camera 8 is further connected with a personal
computer 21 for storage and operation of picked-up images and a TV
monitor 22.
[0099] The illumination optical system 3 is built up of, in order
from the side of light source 2, a collector lens 10, a deflecting
mirror 12 for deflection of the optical axis 11 of illumination
light, an interference filter 13 for turning light from the light
source 2 into quasi-monochromatic light, and a condenser lens
14.
[0100] The viewing optical system 5 is constructed of, in order
from the side of object 4 under observation, an objective lens 15
for forming the image of object 4, a first relay lens 16, a
deflecting mirror 17 for deflection of light from the objective
lens 15, and a second relay lens 18 for cooperating with the first
relay lens 17 to form the image of the objective lens 15 (the image
of object 4) on an image-formation plane.
[0101] Between the second relay lens 18 and the image-formation
plane 19 there is interposed a switchover mirror 20 that is a
half-silvered mirror for visually observing the image of object 4
by the eyepiece lens 6 and the CCD camera 8 or switching the visual
observation of the image of object 4 by the eyepiece lens 6 over to
that by the CCD camera 8 and vice versa.
[0102] At the pupil position 25 of the condenser lens 14 onto which
light from the light source 2 is projected, there is detachably
located an aperture unit 28 that includes a circular aperture 26
shown in FIG. 5(a) or a zonal aperture 27 shown in FIG. 5(b). The
aperture 26, 27 per se is located coaxially with respect to the
center of the pupil. The aperture unit 28 is formed of a shading
plate except the aperture 26, 27.
[0103] Here the aperture 26, 27 should preferably satisfy the
following condition (10) while projected onto the pupil plane 30 of
the objective lens 15 in the form of an aperture image 26', 27' as
shown in the form of an image on the pupil image 30 of the
objective lens 15 in FIGS. 6(a) and 6(b).
D.sub.0/10.ltoreq.D.sub.1.ltoreq.D.sub.0/3 (10)
Here d.sub.0 is the diameter of the pupil 30' of the objective lens
15, D.sub.1 is the aperture width (diameter) of the image 26' of
the circular aperture 26 on the pupil plane 30 of the objective
lens 15, and D.sub.1 is the aperture width of the image 27' of the
zonal aperture 27 on the pupil plane 30 of the objective lens 15.
What is here implied will be described later.
[0104] Further, an absorption film unit 31 is detachably located on
the pupil plane 30 of the objective lens 15. The absorption film
unit 31 includes an absorption film that conforms in shape to the
image of the aperture in the aperture unit 28 on the pupil plane
and is slightly larger in size than that shape. More specifically,
FIG. 7(a) shows the shape of an absorption film 32 on the
absorption film unit 31 commensurate with a circular aperture 26 in
the aperture unit 28, and FIG. 7(b) shows the shape of an
absorption film 33 on the absorption unit 31 commensurate with a
zonal aperture 27 in the aperture unit 28. As shown in FIGS. 7(a)
and 7(b), the shape of the absorption film 32, 33 provided on the
absorption unit 31 is circular in the case where the aperture 26 in
the aperture unit 28 is circular, and zonal in the case where the
aperture 27 in the aperture unit 28 is zonal. In addition, the
absorption film 32 is formed with a margin width .DELTA. on the
outer peripheral side of the image 26' of the aperture 26 on the
pupil plane 30 of the objective lens 15. Likewise, the absorption
films 33 are formed with margin widths .DELTA. on the outer and
inner peripheral sides of the image 27' of the aperture 27 on the
pupil plane 30 of the objective lens 15. Preferably, that margin
width should satisfy
.DELTA..ltoreq.D.sub.0/10 (11)
What is here implied will be described later.
[0105] With the instant example set up as described above,
illumination light from the light source 2 is projected onto the
pupil position of the condenser lens 14. Then, this illumination
light cooperates with the condenser lens 14 to illuminate the
object 4 under observation in the form of Kehler illumination. Upon
illumination of the object 4, the light passes through the object
4, entering the objective lens 15 so that the objective lens
cooperates with the first relay lens 16 and the second relay lens
18 to form the image of the object 4 under observation on an
image-formation plane 19. The image of the object 4 formed on the
image-formation plane 19 enters the eyepiece lens 6, forming an
image on the image pickup plane of the image pickup device 7 in the
CCD camera 8 by way of the switchover mirror 20. Thus, the object 4
can be observed at the focus position as usual.
[0106] On the other hand, when the aperture unit 28 is located at
the pupil position of the condenser lens 14, the image 26' of the
circular aperture 26 or the image 27' of the zonal aperture 27 in
the aperture unit 28 is projected onto the pupil plane 30 of the
objective lens 15 in such a way as to be coaxial with its pupil
30', as shown in FIG. 6.
[0107] Here the illumination light passing through the circular
aperture 26 or the zonal aperture 27 in the aperture unit 28 is
entered in the object 4 under observation, e.g., a growing living
cell having three-dimensional sizes, where it is broken down into
transmitted light, refracted light and diffracted light, leaving
the cell. When the contour of the cell is close in shape to a
sphere or ellipsoid, more light is subject to refraction, and when
the contour of the cell is flat, more light is subject to
transmission and diffraction. Some light refracted at a spherical
or ellipsoidal portion of the cell is larger than the NA of the
objective lens 15, and so is not captured in the objective lens
15.
[0108] In this example, defocusing is carried out by adjusting a
collimator mechanism (focus adjuster mechanism) suffixed to the
microscope to effect a minute amount of back or fore displacement
of the optical axis position of the objective lens 15 from a
position that is regarded as a focus position for ordinary
observations with respect to the object 4 under observation. With
the application of defocusing, the illumination light passing
through the circular aperture 26 or the zonal aperture 27 in the
aperture unit 28 behaves such that the light diffracted and the
light transmitted through the object 4 pass through different
portions in the pupil 30' of the objective lens 15 (see FIG. 3).
Between the transmitted light beam and the diffracted light beam
there is a phase difference that corresponds to the angle of
diffraction and the amount of a wavefront change based on
defocusing. That phase difference acts as is the case with a phase
film in a phase contrast microscope, forming on the image-formation
plane 19 an image intensity distribution proportional to the phase
distribution of the object 4 under observation.
[0109] The amount of the back or fore displacement of the objective
lens 15 must be such that the formed image is free of blurring. To
put it another way, that amount should preferably be smaller than
the value obtained by dividing the wavelength of the light source 2
used for observations by the square of the NA of the objective lens
15 used.
[0110] As already mentioned, the aperture width D.sub.1 of the
image 26' of the circular aperture 26 in the aperture unit 28 on
the image plane 30 or the image 27' of the zonal aperture 27 (the
diameter of the circular aperture 26) with respect to the diameter
D.sub.0 of the pupil 30' of the objective lens 15 should preferably
satisfy condition (10). In this connection, it can be appreciated
from the above expression (6) that at a site where the transmitted
light and the diffracted light overlap in the pupil 30', the phase
difference amount becomes zero at which there is no image intensity
distribution in the phase object. In other words, as the aperture
width of the image 26' or 27' of the aperture is too large with
respect to the diameter D.sub.0 of the pupil 30', the value of
expression (6) approximates to zero; the upper limit to that
aperture width is set at about 1/3 of the diameter D.sub.0 of the
pupil 30'.
[0111] As the aperture width of the image 26' or 27' of the
aperture is too small, conversely, the quantity of light available
for image observations becomes insufficient to observe bright
images; the lower limit thereto is set at about 1/10 of the
diameter D.sub.0 of the pupil 30'.
[0112] The principles of making a phase object visible according to
the invention are that in forming the image of the phase object by
interference of zero-order transmitted light with .+-. first- or
higher-order diffracted light through the phase object, a phase
difference is given to the diffracted light with respect to the
transmitted light to obtain an image intensity distribution. For
this reason, as the amplitude of the transmitted light is
relatively too large, there is a lowering of the contrast of the
image of the phase object to be visualized due to too bright a
background. However, if the amplitude of the transmitted light is
selectively decreased, it is then possible to make better the
contrast of the image intensity distribution formed by interference
with the diffracted light. It is thus desired to provide such an
absorption film unit 31 shown in FIG. 7(a) or 7(b) on the pupil
plane 30 of the objective lens 15, thereby absorb therein a part of
the light transmitted through the object 4 under observation
without undergoing diffraction. It is then desired that the
absorption film 32 or 33 on the absorption film unit 31 be formed
on the outer peripheral side of the aperture width of the image 26'
of the aperture 26 with a margin width .DELTA. or on the outer and
inner peripheral sides of the aperture width of the image 27' of
the aperture 27 with margin widths .DELTA. in such a way as to
satisfy condition (11). That is, at and near the image 26' or 27'
of the aperture 26 or 27 formed by the transmitted light on the
pupil plane 30 of the objective lens 15, there is an area that
mainly receives light due to the gradients of the refracting or
reflecting facets of the object 4 under observation with a phase
distribution (in the case of such a reflecting microscope as will
be described later). Thus, if the margin width .DELTA. of up to
about D.sub.0/10 is given to the outer peripheral side of the
aperture width of the image 26' of the aperture 26 or the outer and
inner peripheral sides of the aperture width of the image 27' of
the aperture 27, the components of light due to refraction or
reflection can then be effectively reduced with having little or no
influence on the image intensity distribution of the phase
distribution formed by the light diffracted through the object 4
under observation.
[0113] To obtain such effects, the absorption film 32, 33 should
preferably have a transmittance in the range of 10% to 30%.
[0114] As already described, the phase difference caused by
defocusing is of varying sign between displacements of the object 4
under observation from the focus position of the objective lens 15
toward a near point side and a far point side. In images of growing
cells or other object under observation, obtained at positions
displaced from the focus position of the objective lens 15 toward
the near point side and the far point side, their image contrasts
are inverted in correspondence to the phase difference of the
object 4 under observation. Therefore, if a subtract operation is
applied to each pixel of two images picked up by the CCD camera 8
at positions displaced toward the near and far point sides and
captured in the personal computer 21, it is then possible to double
the image contrast of an image component commensurate with the
phase distribution of the object 4 under observation. It is also
possible to obtain only the image intensity distribution
corresponding to the phase distribution of the object 4 under
observation, because the halo component of the image component
having no phase information about dust, foreign matters,
illumination variations, etc. that are not affected by wavefront
changes is canceled out down to zero.
[0115] In the above example, defocusing relying upon the collimator
mechanism of the microscope is utilized to create a phase
difference between the light diffracted and the light transmitted
through the object 4 under observation in the pupil 30' of the
objective lens 15. As already described, however, it is also
possible to introduce the phase difference in the position of the
pupil 30' of the objective lens 15 between the diffracted light and
the transmitted light by means of the variable-shape mirror
(deformable mirror), not by means of defocusing. This is
illustrated in FIG. 8 that is similar to FIG. 4.
[0116] The example of FIG. 8 is different from that of FIG. 1
primarily in that a variable-shape mirror 17' is located instead of
the deflecting mirror 17 of FIG. 4. The variable-shape mirror 17'
is located at a position conjugate to the pupil plane 30 of the
objective lens 15 via the first relay lens 16. If this
variable-shape mirror 17' is used to create rotationally symmetric,
variable wave aberration that is proportional to, for instance, the
square of radius with respect to the optical axis, as is the case
with defocusing, it is then possible to create a phase difference
between the diffracted light and the transmitted light, as is the
case with defocusing relying upon the collimator mechanism, so that
an image intensity distribution proportional to the phase
distribution of the object 4 under observation can be formed in the
image-formation plane 19. It is here noted that for the creation of
rotationally symmetric wave aberration at the pupil plane 30, it is
only needed to asymmetrically deform the decentered variable-shape
mirror 17' in such a way as to compensate for its decentration.
[0117] In this case, too, a change in the angle between the
diffracted light and the transmitted light causes the amount of
phase difference created between the two light beams to vary even
at the same amount of deformation of the variable-shape mirror 17'.
For instance, it is thus possible to obtain better image contrasts
by varying the amount of the deformation for each cell under
observation.
[0118] The phase difference of each light ray caused by the
deformation of the variable-shape mirror 17' is of varying sign
between when the deformation of the variable-shape mirror 17'
occurs at a position displaced toward the side of the objective
lens 15 and when it occurs at a position displaced toward the side
of the eyepiece lens 6. In images of growing cells or other object
4 under observation, obtained at positions displaced toward the
side of the objective lens 15 and the side of the eyepiece lens 6,
their image contrasts are inverted in correspondence to the phase
difference of the object 4 under observation.
[0119] In this example, too, the absorption film unit 31 is located
on the pupil plane 30 of the objective lens 15 to absorb a part of
the zero-order transmitted light through the object 4 under
observation as in the example of FIG. 4, thereby improving the
contrast of the image of the phase object to be visualized.
[0120] In this example, too, it is desired to satisfy conditions
(10) and (11).
[0121] It is here noted that even when, instead of locating the
absorption film unit 31 on the pupil plane 30 of the objective lens
15, an absorption film similar in shape to the absorption film 32
or 33 (when the image-formation magnification of the image of the
pupil 30' by the first relay lens 16 is not 1, the shape of the
absorption film 32 or 33 must be multiplied by that magnification)
is provided on the reflecting surface of the variable-shape mirror
17' located at a position conjugate to the pupil plane 30 of the
variable-shape mirror 17', similar effects are achievable. However,
it is desired that the transmittance of that absorption film be in
the range of 20% to 60% that is approximately twice as high as that
of the film 32 or 33, because light passes twice through the
absorption film provided on the front side of the reflecting
surface.
[0122] With the example of FIG. 8, control of the deformation of
the variable-shape mirror 17 is faster than collimation control of
the objective lens 15 as compared with the example of FIG. 4, so
that faster and more real-time visualization of the phase
distribution is achievable.
[0123] Both the examples of FIG. 4 and FIG. 8 take it for granted
that a light source having a certain area, like a halogen lamp, is
used as the light source 2 for illumination. The example shown in
FIG. 9, on the other hand, depends on the supposition that an LED
2' is used as the light source 2 for illumination. The LED 2' is
regarded substantially as a point light source that produces
substantially monochromatic, divergent light; in this example, an
illumination optical system 3 is built up of a collector lens 10, a
deflecting mirror 12 for deflecting the optical axis 11 of
illumination light and a condenser lens 14, so that light from the
LED 2' is focused on the pupil position of the condenser lens
14.
[0124] The instant example is different from that of FIG. 4 in that
the aperture unit 28, i.e., the circular aperture 26 or the zonal
aperture 27 is not located at the pupil position 25 of the
condenser lens 14, and is otherwise the same as that of FIG. 4.
[0125] This is because the LED 2' is thought of as a substantial
point source and so is the image of the LED 2' formed at the pupil
position 25 of the condenser lens 14; the contour of the image of
that LED 2' plays a role equivalent to the aperture 26 or 25. Since
the image of the LED 2' formed in the pupil 30' of the objective
lens 15 is also regarded substantially as a point, the light
transmitted and the light diffracted through the object 4 under
observation hardly overlap in the pupil 30', and so contribute to
the effective creation of the image intensity distribution of the
phase object. In addition, nearly all of the light diverged from
the LED 2' and captured in the collector lens 10 is utilized for
the illumination of the object 4 under observation, thereby
ensuring observation of bright images. It is here desired that the
image of the LED 2' be formed at the pupil position 25 of the
condenser lens 14 with a reduced magnification, because a diverging
light beam from the LED 2' can be converted into one having an
angle of divergence large enough to illuminate a wide surface of
the object under observation, and the image of the LED 2' formed in
the pupil 30' of the objective lens 15 can be much more reduced in
size.
[0126] In the instant example, it is desired that the absorption
film provided on the absorption film unit 31 be configured in
conformity with the image of the LED 2' in the pupil 30', and be
provided therearound with a margin width .DELTA. in such a way as
to satisfy condition (11).
[0127] In the instant example, too, it is acceptable to visualize
the phase distribution by deforming the variable-shape mirror 17',
not the deflecting mirror 17 of FIG. 9, located at a position
conjugate to the pupil plane 30 of the objective lens 15 as in the
example of FIG. 8 instead of effecting defocusing with the
collimator mechanism of the microscope.
[0128] By the way, the microscope according to the invention may
also be applied to a reflecting microscope like a metallurgical
microscope. A typical example of the phase distribution to be
visualized on the principles of the invention is minute pits and
projections found on a semiconductor or metal substrate.
[0129] FIG. 10 is a schematic optical path diagram for the
arrangement of FIG. 4 constructed in the form of a reflecting
microscope. This reflecting microscope shown generally at 1' is
made up of a light source 2, an illumination optical system 3 for
guidance of light emanating from light source 2, a viewing optical
system 5 for forming the image of an object 4 under observation, an
eyepiece lens 6 for magnifying the image of object 4 for visual
observation, and a CCD camera 8 including an image pickup device 7
for picking up the image of object 4. The CCD camera 8 is further
connected with a personal computer 21 for storage and operation of
picked-up images and a TV monitor 22.
[0130] The illumination optical system 3 is built up of, in order
from the side of light source 2, a collector lens 10, a deflecting
mirror 12 for deflection of an optical axis 11 of illumination
light, an interference filter 13 for turning light from the light
source 2 into quasi-monochromatic light, and a condenser lens
14.
[0131] The viewing optical system 5 is constructed of, in order
from the side of object 4 under observation, an objective lens 15
for forming the image of object 4, a half-silvered mirror 34 for
introduction of illumination light from the illumination optical
system 3, a first relay lens 16, a deflecting mirror 17 for
deflection of light from the objective lens 15, a second deflecting
mirror 38, and a second relay lens 18 for cooperating with the
first relay lens 17 to form the image of the objective lens 15 (the
image of object 4) on an image-formation plane.
[0132] Between the second relay lens 18 and the image-formation
plane 19 there is interposed a switchover mirror 20 that is a
half-silvered mirror for visually observing the image of object 4
by the eyepiece lens 6 and the CCD camera 8 or switching the visual
observation of the image of object 4 by the eyepiece lens 6 over to
that by the CCD camera 8 and vice versa.
[0133] At the pupil position 25 of the condenser lens 14 onto which
light from the light source 2 is projected, there is detachably
located an aperture unit 28 similar to that in the example of FIG.
4.
[0134] Further on the pupil plane 30 of the objective lens 15,
there is detachably located an absorption film unit 31 similar to
that in the example of FIG. 4.
[0135] In this example, the illumination light from the light
source 2 is projected onto the pupil position of the condenser lens
14. Then, that illumination light is reflected at the half-silvered
mirror 37 upon passing through the condenser lens 14, falling on
the object 4 under observation through the objective lens 15 for
Kehler illumination of the object 4. Upon the illumination of the
object 4, the light is reflected at the object 4 into the objective
lens 15. Upon incidence on the objective lens 15, the light passes
through the half-silvered mirror 37 to form an image of the object
4 on an image-formation plane 19 by means of the objective lens 15,
the first relay lens 16 and the second relay lens 18.
[0136] The image of the object 4 under observation formed on the
image-formation plane 19 is incident on the eyepiece lens 6, and
forms an image on the image pickup plane of the image pickup device
7 in the CCD camera 8 by means of the switchover mirror 20. Thus,
the object 4 under observation can be observed by the reflecting
microscope at the focus position as usual.
[0137] On the other hand, when the aperture unit 28 is located at
the pupil position of the condenser lens 14, illumination light
from the light source 2 passes successively through the collector
lens 10, the interference filter 13 and a front lens 35 forming a
part of the condenser lens 14, illuminating the circular aperture
26 or the zonal aperture 27 in the aperture unit 28. Passing
through the circular aperture 26 or the zonal aperture 27, the
light passes through a rear lens 36 forming another part of the
condenser lens 14 and the half-silvered mirror 37, forming an image
26' or 27' of the aperture on the pupil plane 30 of the objective
lens 15. Thereafter, the light passes through the objective lens
15, falling on the object 4 under observation in the form of
collimated light. The light reflected at and the light diffracted
through the object 4 reenter the objective lens 15, at which they
are reflected, again forming the image 26' or 27' of the aperture
26 or 27 on the pupil plane 30 of the objective lens 15.
[0138] Therefore in this example, too, defocusing is carried out by
adjusting a collimator mechanism (focus adjuster mechanism)
suffixed to the microscope to effect a minute amount of back or
fore displacement of the optical axis position of the objective
lens 15 from a position that is regarded as a focus position for
ordinary observations with respect to the object 4 under
observation. With the application of defocusing, the illumination
light passing through the circular aperture 26 or the zonal
aperture 27 in the aperture unit 28 behaves such that the light
diffracted through and the light transmitted at the object 4 pass
through different portions in the pupil 30' of the objective lens
15 (see FIG. 3). Between the reflected light beam and the
diffracted light beam there is a phase difference that corresponds
to the angle of diffraction and the amount of a wavefront change
based on defocusing. That phase difference forms on the
image-formation plane 19 an image intensity distribution
proportional to the amount of pits and projections of the object 4
under observation.
[0139] In this reflecting microscope, too, the absorption film unit
31 could be located on the pupil plane 30 of the objective lens 15,
as is the case with the example of FIG. 4, to allow a part of the
zero-order transmitted light through the object 4 under observation
so that the contrast of the image regarding the amount of pits and
projections to be visualized can be improved.
[0140] In the instant reflecting microscope, too, it is desired to
satisfy condition (10) and condition (11).
[0141] It is here noted that as the absorption film unit 31 is
located on the pupil plane 30 between the objective lens 15 and the
half-silvered mirror 37, the whole luminance of the illumination
light after passing through the aperture 26 or the aperture 27
becomes weak upon falling on the object 4 under observation. To
avoid this, it may be preferable to locate the absorption film unit
31 at a position conjugate to the pupil plane 30 between the
half-silvered mirror 37 and the image-formation plane 19.
[0142] In the instant reflecting microscope, too, it is acceptable
to visualize the phase distribution by deforming the variable-shape
mirror 17', not the deflecting mirror 17 of FIG. 10, located at a
position conjugate to the pupil plane 30 of the objective lens 15
as in the example of FIG. 8 instead of effecting defocusing with
the collimator mechanism of the microscope.
[0143] As is the case with the example of FIG. 9, it is acceptable
to use the LED 2' in place of the light source 2, thereby
dispensing with the aperture unit 28 located at the pupil position
25 of the condenser lens 14.
[0144] With the examples other than that of FIG. 4, too, the
contrast of the image intensity distribution is invertible by
inverting the wave aberration introduced onto the pupil plane 30 of
the objective lens 15 or a position conjugate thereto. Accordingly,
if, as is the case with the example of FIG. 4, the image before its
contrast is inverted and the image with its contrast inverted are
picked up by the CCD camera 8 and captured in the personal computer
21 to perform a subtract operation for each pixel of the two
images, it is then possible to double the image contrast of the
image component corresponding to the phase or pit-and-projection
distribution of the object 4 under observation. It is also possible
to obtain only the image intensity distribution corresponding to
the phase or pit-and-projection distribution of the object 4 under
observation, because the halo component of the image component
having no phase information on dust, foreign matters, illumination
variations, etc. that are not affected by wavefront changes is
canceled out down to zero.
[0145] As can be understood from the foregoing, the microscopy of
the invention, and the microscope used to implement it enables
living cells or minute pits and projections on metal surfaces, etc.
to be observed without recourse to phase contrast microscopes or
differential interference microscopes. In particular, it is
possible to observe phase distributions or pit-and-projection
distributions at a relatively low magnification such as an
image-formation magnification of up to 4, at which conventional
phase contrast microscopes or differential interference microscopes
encounter difficulty. It is also possible to cancel out
illumination variations or halo components, thereby obtaining an
image intensity distribution of good contrast corresponding to the
phase or pit-and-projection distribution of an object under
observation.
[0146] The invention is now explained specifically with reference
to the method and apparatus for determining or measuring the state
of growing cells using the principles of visualizing the phase
object according to the invention.
[0147] The basics of the method and apparatus for determining or
measuring the state of growing cells according to the invention are
that when the image to be observed of growing cells that are
generally a phase object is converted by interference into a
contrast image, the sign of a phase difference between interfering
light beams is inverted to obtain two images with inverted
contrasts to enhance the contrast of a contrast image of the
growing cells, so that the number, morphology, distribution or the
like of the growing cells can be reliably determined or measured.
Methods for converting phase objects by interference into contrast
images include, in addition to such defocusing, and wave
aberration-introducing methods as described above, phase difference
methods and differential inference methods (non-patent publication
1).
[0148] Specific examples of the microscopic measurement apparatus
of the invention are now explained with the microscope capable of
observing and picking up the contrast image of growing cells
inclusive of contrast inversion, utilizing such a defocusing method
as described above.
[0149] FIG. 11 is generally illustrative in schematic of the
microscope 1 capable of observing and picking up the contrast
images of growing cells utilizing such defocusing. The microscope
of the instant example is constructed in the form of an inverted
microscope 1 that is made up of a light source 2, an illumination
optical system 3 for guidance of light emanating from light source
2, a viewing optical system 5 for forming a contrast image of
growing cells at an internal bottom position of a laboratory dish
4' on an X-Y stage 40, an eyepiece lens 6 for magnifying the
contrast image of growing cells at the internal bottom position of
the laboratory dish 4' for visual observation, and a CCD camera 8
including an image pickup device 7 for picking up the image of
growing cells at the internal bottom position of the laboratory
dish 4'. The CCD camera 8 is further connected with a personal
computer 21 for storage and operation of picked-up images and a TV
monitor 22, with focus signals from the personal computer 21 sent
out to an objective lens displacement mechanism 43 via a driver
circuit.
[0150] The illumination optical system 3 is built up of, in order
from the side of the light source 2, a collector lens 10, a
deflecting mirror 12 for deflection of the optical axis 11 of
illumination light, an interference filter 13 for turning light
from the light source 2 into quasi-monochromatic light, and a
condenser lens 14.
[0151] The viewing optical system 5 is constructed of, in order
from the side of the laboratory dish 4' with growing cells placed
thereon, an objective lens 15 for forming the image of growing
cells at the internal bottom position of the laboratory dish 4', a
first relay lens 16, a deflecting mirror 17 for deflection of light
from the objective lens 15, and a second relay lens 18 for
cooperating with the first relay lens 17 to form the image by the
objective lens 15 (the image of object 4) on an image-formation
plane.
[0152] Between the second relay lens 18 and the image-formation
plane 19 there is interposed a switchover mirror 20 that is a
half-silvered mirror for visually observing the contrast image of
growing cells by the eyepiece lens 6 and the CCD camera 8 or
switching the visual observation of the image of object 4 by the
eyepiece lens 6 over to that by the CCD camera 8 and vice
versa.
[0153] At the pupil position 25 of the condenser lens 14 onto which
light from the light source 2 is projected, there is detachably
located an aperture unit 28 that includes a partial aperture
comprising a circular aperture or a zonal aperture. The aperture
unit 28 is formed of a shading plate except the partial
aperture.
[0154] With the instant example set up as described above,
illumination light from the light source 2 is projected onto the
pupil position of the condenser lens 14. Then, this illumination
light cooperates with the condenser lens 14 to illuminate the
growing cells at the internal bottom position of the laboratory
dish 4' in the form of Kehler illumination. Upon illumination of
the growing cells, the light passes through the growing cells,
entering the objective lens 15 so that the objective lens 15
cooperates with the first relay lens 16 and the second relay lens
18 to form an image of the growing cells on the image-formation
plane 19. The contrast image of the growing cells formed on the
image-formation plane 19 enters the eyepiece lens 6, forming an
image on the image pickup plane of the image pickup device 7 in the
CCD camera 8 by way of the switchover mirror 20. Thus, the growing
cells can be observed at the focus position as usual.
[0155] In this example, defocusing is carried out by using a focus
signal from the personal computer 21 to effect a minute amount of
back or fore displacement of the optical axis position of the
objective lens 15 from a position that is regarded as a focus
position for ordinary observations via an objective lens
displacement mechanism 43. With the application of defocusing, the
illumination light passing through the partial aperture in the
aperture unit 28 behaves such that the light diffracted and the
light transmitted through the growing cells pass through different
portions in the pupil 30' of the objective lens 15 (see FIG. 3).
Between the transmitted light beam and the diffracted light beam
there is a phase difference that corresponds to the angle of
diffraction and the amount of a wavefront change based on
defocusing. That phase difference acts as is the case with a phase
film in a phase contrast microscope, forming on the image-formation
plane 19 an image intensity distribution proportional to the phase
distribution of the growing cells under observation.
[0156] The amount of the back or fore displacement of the objective
lens 15 has to be such that the formed image is free of blurring.
To put it another way, that amount should preferably be smaller
than the value obtained by dividing the wavelength of the light
source 2 used for observations by the square of the NA of the
objective lens 15 used.
[0157] As already described, the phase difference of each light ray
caused by defocusing is of opposite sign between displacements of
the object under observation from the focus position of the
objective lens 15 toward a near point side and a far point side. In
the images of growing cells under observation, captured by the
image pickup device 7 in the personal computer 21 at positions
displaced from the focus position of the objective lens 15 toward
the near point side and the far point side, their image contrasts
are inverted in correspondence to the phase difference of the
object under observation, as shown in FIGS. 12(a) and 12(b).
Specifically, FIG. 12(a) shows on the left side a contrast image of
growing cells obtained by defocusing the objective lens 15 toward
the far point side, and on the right side a schematic gradation
indicative of the image intensity at a scanning line position
indicated by a broken line on the left side of FIG. 12(a).
Likewise, FIG. 12(b) shows on the left side a contrast image of
growing cells obtained by defocusing the objective lens 15 toward
the near point side, and on the right side a schematic gradation
indicative of the image intensity at a scanning line position
indicated by a broken line on the left side of FIG. 12(b). It is
here noted that defocusing is carried out in much the same amount.
A background area free of any cells shows a substantially constant
gradation n. Upon defocusing toward the near point side (far point
side) as shown in FIG. 12(a), cells and cell nuclei have some
bright contrast against the background. Conversely, upon defocusing
toward the far point side (near point side) as shown in FIG. 12(b),
cells and cell nuclei have some dark contrast against the
background.
[0158] Thus, the sign of the phase difference goes into reverse
whenever the direction of defocusing changes. With a subtract
operation performed in the personal computer 21 thereby taking a
difference between the values of associated individual pixels of
the two contrast images (FIGS. 12(a) and 12(b)) taken of the
growing cells with inverted contrasts and creating a new pixel,
there is obtained such an image as shown on the left side of FIG.
12(c), wherein the intensity change of illumination light within
the viewing range or the light intensity change due to foreign
matters or the like is canceled out down to zero. In the case of a
phase distribution such as one of growing cells, on the other hand,
the contrast of an image is inverted by the inversion of the sign
of a phase difference. Therefore, if a subtract operation is
applied to an image comprising two contrast images of opposite
phase-difference sign, then the contrast of the image doubles as
shown on the right side of FIG. 12(c) (on condition that the amount
of defocusing on the near point side must be the same as that on
the far point side).
[0159] Thus, the gradation (intensity value) of a cell-free portion
of the image obtained by the subtract operation becomes zero.
Accordingly, it is preferable that a portion having a gradation of
zero or nearly zero is extracted in the personal computer 21 from
the image to which the subtract operation is applied (the left side
of FIG. 12(c)) as the cell-free portion, and that cell-free portion
is eliminated in the personal computer 21 from the viewing range to
take the rest as a cell-containing portion. In other words, a
portion of the image subjected to the subtract operation and having
an intensity of substantially zero is indicative of a portion
having a phase of substantially zero and containing no cell. On the
other hand, although another portion of the image which is
subjected to the subtract operation and whose intensity is not
substantially zero is indicative of a portion corresponding to the
phase difference, yet it entails intensity variations of
illumination light and intensity variations due to the influences
of dewdrops deposited on the lid of a culture vessel, etc., failing
to precisely reflect the phase difference. Therefore, extracting
and excluding the cell-free portion out of the viewing range to
determine the cell-containing area is preferable to direct
extraction of the cell-containing portion, because influences of
external perturbations are held back.
[0160] As shown by a one-dotted line on the right side of FIG.
12(c), a threshold value for binarization may be given to a portion
at or near the intensity of zero of the image to which the subtract
operation is applied (the left side of FIG. 12(c)) to allocate the
intensity of zero to pixels in an area below the threshold value
and the intensity of 1 to pixels greater than the threshold value,
so that such a binarized image as shown in FIG. 12(d) is formable.
This binarized image is then indicative of the abundance
distribution of cells.
[0161] If the data about that binarized image is added up for each
pixel and the sum is divided by the total number of pixels in the
viewing range, the abundance density of cells in the viewing range
is then calculated. If the area of one pixel on an actual object
surface (the internal bottom of the laboratory dish 4') is
multiplied by the sum, it is then possible to calculate the total
area of cells present in the viewing range, and if that total area
is divided by the average area of one cell, it is then possible to
calculate the number of cells present in the viewing range.
[0162] Alternatively, if the number of pixels in which cells are
present is divided by the total number of pixels in the image, it
is then possible to find out the average density of cells and the
rate taken up by cells in the viewing range. Also, if the average
number of pixels taken up by one cell is previously determined, it
is then possible to divide the number of pixels in which cells are
present by the average number of pixels for one cell, thereby
finding out the number of cells present in the viewing range.
[0163] Apart from binarization, not only the subtract operation but
also an add operation is applied to two images captured in the
personal computer 21 (the left images in FIG. 12(a) and FIG.
12(b)).
[0164] As described above, image information equivalent to the
phase distribution of the object under observation (growing cells)
is obtainable from the image obtained by means of the subtract
operation. Remaining affected by illumination variations, etc.,
however, that image information still fails to provide any precise
indication of the phase distribution. Therefore, if the add
operation is performed in the personal computer 21 to obtain the
sum of associated individual pixels of two images (FIGS. 12(a) and
12(b)) with inverted contrasts and create a new pixel, information
about the intensity distribution such as illumination variations in
the viewing range is then obtainable, because the phase
distribution is canceled out by the inverted distribution of a
portion with cells present therein. By dividing the image obtained
by the subtract operation by the image obtained by that add
operation, the influences of illumination variations, etc. can be
so held back that image information proportional to the phase
distribution is obtainable.
[0165] The same cells, cultured in the laboratory dish 4', could be
thought of as having substantially identical refractive indices,
and the obtained phase distribution could be equivalent to
information about the height of the cells. Accordingly, the height
distribution of the growing cells could be determined or measured
from the intensity (gradation) distribution of the thus obtained
image (the image obtained by dividing the image obtained by the
subtract operation by the image obtained by the add operation).
[0166] Some growing cells vary in thickness between their nucleus
portions and other portions, with the nucleus portion swelling up
as shown at N on the right side of FIG. 12(c). In the above image
obtained by dividing the image obtained by the subtract operation
by the image obtained by the add operation, an image with cell
nucleus portions extracted off is obtained by binarization image
processing wherein zero is assigned to a portion less than a
specific threshold value (that is larger than a value equivalent to
the height of a nucleus-free portion and smaller than a value
equivalent to the height of a nucleus-containing portion) and one
is allocated to other portions. The area of the nucleus of each
cell may be determined from the binarized image.
[0167] By multiplying the above binarized image indicative of a
nucleus distribution by the image indicative of the previous phase
distribution (the image obtained by dividing the image obtained by
the subtract operation by the image obtained by the add operation),
the height distribution of the nucleus portion can be calculated.
The flatness of the nucleus may also be determined from the
information about the nucleus area and height of each cell.
[0168] Further, simultaneously with the capture in the personal
computer of the two images (the left image in FIG. 12(a) and the
right image in FIG. 12(b)) having a phase difference of opposite
sign upon interference, another aperture unit having a zonal
aperture for the illumination of a dark field, instead of the
aperture unit 28 having a partial aperture for defocusing, is
inserted in the pupil position 25 of the condenser lens 14 to take
in a dark field image of the same growing cells using dark field
illumination. If that dark field image is multiplied by the above
binarized image indicative of a nucleus distribution, it is then
possible to obtain a dark field image of the nucleus portion and,
hence, calculate the scattering distribution of the nucleus.
[0169] In the foregoing explanation of the invention, defocusing is
used as the means for obtaining two contrast images that are the
images to be observed of the growing cells that are generally a
phase object and have a phase difference of opposite sign upon
interference for image formation; however, it is acceptable to make
use of wave aberration introduction. Referring again to FIG. 11,
the aperture unit 28 having a partial aperture is located at the
pupil position 25 of the condenser lens 14, and in the pupil plane
30 of the objective lens 15 there is introduced a wavefront that
varies in size with the diameter of the pupil. To this end, for
instance, the surface of the deflecting mirror 15 at a position
conjugate to the pupil plane 30 of the objective lens 15 is
deformed. If, in this case, that deformation is carried out in the
same shape but with opposite sign, then two contrast images with a
phase difference of opposite sign are then obtainable, as described
above.
[0170] Alternatively, it is acceptable to rely on the principles of
the differential interference microscope.
[0171] This is explained with reference to FIG. 11. Instead of the
aperture unit 28, a diffusion screen-providing first prism is
inserted between orthogonal polarized light components by a
Nomarski or Wollaston prism at the pupil position 25 of the
condenser lens 11, and the second prism comprising a Nomarski or
Woolaston prism to return that diffusion screen back is positioned
at the pupil plane 30 of the objective lens 15 conjugate to the
first prism, a polarizer is located on the entrance side of the
first prism, and an analyzer is located at the exit side of the
second prism. By taking two images while the amount of retardation
between the two polarized light components is inverted, it is
possible to obtain two contrast images having a phase difference of
opposite sign, as described above.
[0172] While the method for implement microscopy and microscopic
measurements according to the invention, the microscope for
implementing them, and the apparatus for implementing microscopic
measurements according to the invention have been described with
reference to the principles and examples of the invention, it is
understood that the invention is in no sense limited thereto, and
could be modified in various forms and ways.
[0173] The method for implement microscopy and microscopic
measurements according to the invention, the microscope for
implementing them, and the apparatus for implementing microscopic
measurements according to the invention, for instance, could be
embodied as follows.
[0174] (1) A method for implementing microscopy, characterized in
that a partial aperture is located at the pupil position of an
illumination optical system in a microscope, an object under
observation is illuminated by light passing through said partial
aperture, and in the pupil position of an image-formation optical
system there is introduced a wavefront varying in size with the
diameter of the pupil to view an image formed on an image-formation
plane of said image-formation optical system.
[0175] (2) The method for implementing microscopy according to (1)
above, characterized in that the image-formation magnification of
said image-formation optical system is 4 or lower.
[0176] (3) The method for implementing microscopy according to (2)
above, characterized in that the wavefront introduced in the pupil
position of said image-formation optical system is based on a
displacement from the focus position of said image-formation
optical system.
[0177] (4) The method for implementing microscopy according to (3)
above, characterized in that an absorption film that conforms in
shape to the image of said partial aperture or said substantial
point light source at the pupil position of said image-formation
optical system and is slightly larger than said shape is located at
the pupil of said image-formation optical system or at or near a
position conjugate thereto, with satisfaction of expression
(11)
.DELTA..ltoreq.D.sub.0/10 (11)
where .DELTA. is a margin width on an inner or outer peripheral
side of said absorption film with respect to the image of said
partial aperture or said substantial point light source at the
pupil position of said image-formation optical system, and D.sub.0
is the diameter of the pupil of said image-formation optical
system.
[0178] (5) The method for implementing microscopy according to (3)
above, characterized in that while the wavefront introduced in the
pupil position of said image-formation optical system is
substantially symmetrically changed, two images with inverted
contrasts are taken of the same object under observation, and a
subtract operation is applied between the two images thereby
providing an enhanced contrast.
[0179] (6) The method for implementing microscopy according to (3)
above, characterized in that while the wavefront introduced in the
pupil position of said image-formation optical system is
substantially symmetrically changed, two images with inverted
contrasts are taken of the same object under observation, a
subtract operation is applied between the two images to form an
image with the subtract operation applied thereto, and at least one
of the number of cells, the area taken up by cells, the abundance
distribution of cells and the abundance density of cells in a
viewing range is measured from the obtained image with the subtract
operation applied thereto.
[0180] (7) The method for implementing microscopy according to (3)
above, characterized in that while the wavefront introduced in the
pupil position of said image-formation optical system is
substantially symmetrically changed, two images with inverted
contrasts are taken of the same object under observation, and a
subtract operation and an add operation are applied between the two
images to form two images wherein the image subjected to the
subtract operation is divided by the image subjected to the add
operation to measure information about the thickness of cells in a
viewing range.
[0181] (8) The method for implementing microscopy according to (2)
above, characterized in that said partial aperture comprises a
circular aperture or a zonal aperture, with satisfaction of
condition (10):
D.sub.0/10.ltoreq.D.sub.1.ltoreq.D.sub.0/3 (10)
where D.sub.1 is the aperture width of the image at the pupil
position of said image-formation optical system, and D.sub.0 is the
diameter of the pupil of said image-formation optical system.
[0182] (9) The method for implementing microscopy according to (8)
above, characterized in that an absorption film that conforms in
shape to the image of said partial aperture or said substantial
point light source at the pupil position of said image-formation
optical system and is slightly larger than said shape is located at
the pupil of said image-formation optical system or at or near a
position conjugate thereto, with satisfaction of condition (11)
.DELTA..ltoreq.D.sub.0/10 (11)
where .DELTA. is a margin width on the inner or outer peripheral
side of said absorption film with respect to the image of said
partial aperture or said substantial point light source at the
pupil position of said image-formation optical system, and D.sub.0
is the diameter of the pupil of said image-formation optical
system.
[0183] (10) The for implementing microscopy according to (2) above,
characterized in that while the wavefront introduced in the pupil
position of said image-formation optical system is substantially
symmetrically changed, two images with inverted contrasts are taken
of the same object under observation, and a subtract operation is
applied between the two images thereby providing an enhanced
contrast.
[0184] (11) The method for implementing microscopy according to (2)
above, characterized in that while the wavefront introduced in the
pupil position of said image-formation optical system is
substantially symmetrically changed, two images with inverted
contrasts are taken of the same object under observation, a
subtract operation is applied between the two images to form an
image with the subtract operation applied thereto, and at least one
of the number of cells, the area taken up by cells, the abundance
distribution of cells and the abundance density of cells in a
viewing range is measured from the obtained image with the subtract
operation applied thereto.
[0185] (12) The method for implementing microscopy according to (2)
above, characterized in that while the wavefront introduced in the
pupil position of said image-formation optical system is
substantially symmetrically changed, two images with inverted
contrasts are taken of the same object under observation, and a
subtract operation and an add operation are applied between the two
images to form two images wherein the image subjected to the
subtract operation is divided by the image subjected to the add
operation to measure information about the thickness of cells in a
viewing range.
[0186] (13) The method for implementing microscopy according to (1)
above, characterized in that the wavefront introduced in the pupil
position of said image-formation optical system is based on a
displacement from the focus position of said image-formation
optical system.
[0187] (14) The method for implementing microscopy according to
(13) above, characterized in that said partial aperture comprises a
circular aperture or a zonal aperture, with satisfaction of
condition (10):
D.sub.0/10.ltoreq.D.sub.1.ltoreq.D.sub.0/3 (10)
where D.sub.1 is the aperture width of the image at the pupil
position of said image-formation optical system, and D.sub.0 is the
diameter of the pupil of said image-formation optical system.
[0188] (15) The method for implementing microscopy according to
(13) above, characterized in that an absorption film that conforms
in shape to the image of said partial aperture or said substantial
point light source at the pupil position of said image-formation
optical system and is slightly larger than said shape is located at
the pupil of said image-formation optical system or at or near a
position conjugate thereto, with satisfaction of condition (11)
.DELTA..ltoreq.D.sub.0/10 (11)
where .DELTA. is a margin width on the inner or outer peripheral
side of said absorption film with respect to the image of said
partial aperture or said substantial point light source at the
pupil position of said image-formation optical system, and D.sub.0
is the diameter of the pupil of said image-formation optical
system.
[0189] (16) The method for implement microscopy according to (13)
above, characterized in that while the wavefront introduced in the
pupil position of said image-formation optical system is
substantially symmetrically changed, two images with inverted
contrasts are taken of the same object under observation, and a
subtract operation is applied between the two images thereby
providing an enhanced contrast.
[0190] (17) The method for implementing microscopy according to
(13) above, characterized in that while the wavefront introduced in
the pupil position of said image-formation optical system is
substantially symmetrically changed, two images with inverted
contrasts are taken of the same object under observation, a
subtract operation is applied between the two images to form an
image with the subtract operation applied thereto, and at least one
of the number of cells, the area taken up by cells, the abundance
distribution of cells and the abundance density of cells in a
viewing range is measured from the obtained image with the subtract
operation applied thereto.
[0191] (18) The method for implementing microscopy according to
(17) above, characterized in that a portion having an intensity
value of zero or nearly zero is extracted out of the intensity
distribution of the image subjected to the subtract operation to
take said portion as being a cell-free portion and other portion as
being a cell-containing portion, thereby making a separation
between the portions containing cells and no cells.
[0192] (19) The method for implementing microscopy according to
(18) above, characterized in that a portion having an intensity
value of zero or nearly zero is extracted out of the intensity
distribution of the image subjected to the subtract operation to
allocate the intensity value to one value and other portion to
another one intensity value for binarization.
[0193] (20) The method for implementing microscopy according to
(13) above, characterized in that while the wavefront introduced in
the pupil position of said image-formation optical system is
substantially symmetrically changed, two images with inverted
contrasts are taken of the same object under observation, and a
subtract operation and an add operation are applied between the two
images to form two images wherein the image subjected to the
subtract operation is divided by the image subjected to the add
operation to measure information about the thickness of cells in a
viewing range.
[0194] (21) The method for implementing microscopy according to (1)
above, characterized in that while the wavefront introduced in the
pupil position of said image-formation optical system is
substantially symmetrically changed, two images with inverted
contrasts are taken of the same object under observation, and a
subtract operation is applied between the two images thereby
providing an enhanced contrast.
[0195] (22) The method for implementing microscopy according to (1)
above, characterized in that while the wavefront introduced in the
pupil position of said image-formation optical system is
substantially symmetrically changed, two images with inverted
contrasts are taken of the same object under observation, a
subtract operation is applied between the two images to form an
image with the subtract operation applied thereto, and at least one
of the number of cells, the area taken up by cells, the abundance
distribution of cells and the abundance density of cells in a
viewing range is measured from the obtained image with the subtract
operation applied thereto.
[0196] (23) The method for implementing microscopy according to
(22) above, characterized in that a portion having an intensity
value of zero or nearly zero is extracted out of the intensity
distribution of the image subjected to the subtract operation to
take said portion as being a cell-free portion and other portion as
being a cell-containing portion, thereby making a separation
between the portions containing cells and no cells.
[0197] (24) The method for implementing microscopy according to
(22) above, characterized in that a portion having an intensity
value of zero or nearly zero is extracted out of the intensity
distribution of the image subjected to the subtract operation to
allocate the intensity value to one value and other portion to
another one intensity value for binarization.
[0198] (25) The method for implement microscopy according to (1)
above, characterized in that while the wavefront introduced in the
pupil position of said image-formation optical system is
substantially symmetrically changed, two images with inverted
contrasts are taken of the same object under observation, and a
subtract operation and an add operation are applied between the two
images to form two images wherein the image subjected to the
subtract operation is divided by the image subjected to the add
operation to measure information about the thickness of cells in a
viewing range.
[0199] (26) A method for implementing microscopy, characterized in
that a substantial point light source that gives out divergent
light is used as an illumination light source to form the image of
said substantial point light source at the pupil position of an
illumination optical system in the microscope, an object under
observation is illuminated by divergent light from the image of
said substantial point light source, and in the pupil position of
an image-formation optical system there is introduced a wavefront
varying in size with the diameter of the pupil to view an image
formed on an image-formation plane of said image-formation optical
system.
[0200] (27) The method for implementing microscopy according to
(26) above, characterized in that the image-formation magnification
of said image-formation optical system is 4 or lower.
[0201] (28) The method for implementing microscopy according to
(27) above, characterized in that the wavefront introduced in the
pupil position of said image-formation optical system is based on a
displacement from the focus position of said image-formation
optical system.
[0202] (29) The method for implementing microscopy according to
(26) above, characterized in that the wavefront introduced in the
pupil position of said image-formation optical system is based on a
displacement from the focus position of said image-formation
optical system.
[0203] (30) The method for implementing microscopy according to
(27) above, characterized in that the wavefront introduced to be in
the pupil position of said image-formation optical system is
created by a variable-shape mirror that is located at the pupil of
said image-formation optical system or at or near a position
conjugate thereto and has a variable or adjustable reflecting
surface shape.
[0204] (31) A method for implementing microscopy, characterized in
that by means of a microscope that comprises an illumination
optical system, a viewing optical system including a unit for
converting an object under observation into a contrast image by
interference and an image pickup device for capturing therein a
contrast image having a phase difference of opposite sign upon
interference of the image under observation, two images comprising
contrast images having a phase difference of opposite sign are
captured therein to apply a subtract operation to the thus captured
two images, and at least one of the number of cells, the area taken
up by cells, the abundance distribution of cells and the abundance
density of cells in a viewing range is measured from the obtained
image with the subtract operation applied thereto.
[0205] (32) The method for implementing microscopy according to
(31) above, characterized in that a portion having an intensity
value of zero or nearly zero is extracted out of the intensity
distribution of the image subjected to the subtract operation to
take said portion as being a cell-free portion and other portion as
being a cell-containing portion, thereby making a separation
between the portions containing cells and no cells.
[0206] (33) The method for implementing microscopy according to
(31) above, characterized in that a portion having an intensity
value of zero or nearly zero is extracted out of the intensity
distribution of the image subjected to the subtract operation to
allocate the intensity value to one value and other portion to
another one intensity value for binarization.
[0207] (34) A method for implementing microscopy, characterized in
that by means of a microscope that comprises an illumination
optical system, a viewing optical system including a unit for
converting an object under observation into a contrast image by
interference and an image pickup device for capturing therein a
contrast image having a phase difference of opposite sign upon
interference of the image under observation, two images comprising
contrast images having a phase difference of opposite sign captured
therein to apply a subtract operation and an add operation to the
thus captured two images to form two images wherein the image
subjected to the subtract operation is divided by the image
subjected to the add operation to measure information about the
thickness of cells in a viewing range.
[0208] (35) A microscope, comprising a light source, an
illumination optical system for the guidance of light from the
light source to an object under observation, a partial aperture
located substantially at the pupil position of the illumination
optical system and an image-formation optical system for forming on
an image-formation plane an image of the object under observation
illuminated by light passing through the partial aperture, and
further comprising an eyepiece optical system or an image pickup
optical system for viewing the image formed on the image-formation
plane, characterized in that:
[0209] the diameter of the image of said partial aperture at the
pupil position of said image-formation optical system is set
smaller than the diameter of the pupil of said image-formation
optical system, wherein at the pupil position of said
image-formation optical system there is located wavefront
introduction means for introducing in the pupil position of said
image-formation optical system a wavefront varying in size with the
diameter of the pupil.
[0210] (36) The microscope according to (35) above, characterized
in that the image-formation magnification of said image-formation
optical system is 4 or lower.
[0211] (37) The microscope according to (36) above, characterized
in that said wavefront introduction means comprises means for
displacement and adjustment of said image-formation optical system
in the optical axis direction.
[0212] (38) The microscopy according to (37) above, characterized
in that an absorption film that conforms in shape to the image of
said partial aperture or said substantial point light source at the
pupil position of said image-formation optical system and is
slightly larger than said shape is located at the pupil of said
image-formation optical system or at or near a position conjugate
thereto, with satisfaction of condition (11)
.DELTA..ltoreq.D.sub.0/10 (11)
where .DELTA. is a margin width on an inner or outer peripheral
side of said absorption film with respect to the image of said
partial aperture or said substantial point light source at the
pupil position of said image-formation optical system, and D.sub.0
is the diameter of the pupil of said image-formation optical
system.
[0213] (39) The microscope according to (37) above, characterized
by further comprising an image pickup optical system for picking up
the image to be formed on said image-formation plane, an image
pickup device located on the image pickup plane thereof, and
operation and storage means for operation and storage of an image
picked up by said image pickup device.
[0214] (40) The microscope according to (37) above, characterized
by further comprising a processing unit for capturing two images
comprising contrast images having a phase difference of opposite
sign by means of said microscope and applying a subtract operation
to the thus captured two images to form an image subjected to the
subtract operation, so that at least one of the number of cells,
the area taken up by cells, the abundance distribution of cells and
the abundance density of cells in a viewing range is measured from
the obtained image with the subtract operation applied thereto.
[0215] (41) The microscope according to (37) above, characterized
by further comprising an operation unit for capturing two images
comprising contrast images having a phase difference of opposite
sign and applying a subtract operation and an add operation to the
thus captured two images, and a thickness measurement unit for
dividing the image subjected to the subtract operation by the image
subjected to the add operation to measure information about the
thickness of cells in a viewing range.
[0216] (42) The microscope according to (36) above, characterized
in that said partial aperture comprises a circular aperture or a
zonal aperture, with satisfaction of condition (10):
D.sub.0/10.ltoreq.D.sub.1.ltoreq.D.sub.0/3 (10)
where D.sub.1 is the aperture width of the image at the pupil
position of said image-formation optical system, and D.sub.0 is the
diameter of the pupil of said image-formation optical system.
[0217] (43) The microscope according to (36) above, characterized
in that an absorption film that conforms in shape to the image of
said partial aperture or said substantial point light source at the
pupil position of said image-formation optical system and is
slightly larger than said shape is located at the pupil of said
image-formation optical system or at or near a position conjugate
thereto, with satisfaction of condition (11)
.DELTA.=D.sub.0/10 (11)
where .DELTA. is a margin width on the inner or outer peripheral
side of said absorption film with respect to the image of said
partial aperture or said substantial point light source at the
pupil position of said image-formation optical system, and D.sub.0
is the diameter of the pupil of said image-formation optical
system.
[0218] (44) The microscope according to (36) above, characterized
by further comprising an image pickup optical system for picking up
the image to be formed on said image-formation plane, an image
pickup device located on the image pickup plane thereof, and
operation and storage means for operation and storage of an image
picked up by said image pickup device.
[0219] (45) The microscope according to (36) above, characterized
by further comprising a processing unit for capturing two images
comprising contrast images having a phase difference of opposite
sign by means of said microscope and applying a subtract operation
to the thus captured two images to form an image subjected to the
subtract operation, so that at least one of the number of cells,
the area taken up by cells, the abundance distribution of cells and
the abundance density of cells in a viewing range is measured from
the obtained image with the subtract operation applied thereto.
[0220] (46) The microscope according to (36) above, characterized
by further comprising an operation unit for capturing two images
comprising contrast images having a phase difference of opposite
sign by means of said microscope and applying a subtract operation
and an add operation to the thus captured two images, and a
thickness measurement unit for dividing the image subjected to the
subtract operation by the image subjected to the add operation to
measure information about the thickness of cells in a viewing
range.
[0221] (47) The microscope according to (35) above, characterized
in that said wavefront introduction means comprises means for
displacement and adjustment of said image-formation optical system
in the optical axis direction.
[0222] (48) The microscope according to (47) above, characterized
in that said partial aperture comprises a circular aperture or a
zonal aperture, with satisfaction of condition (10):
D.sub.0/10.ltoreq.D.sub.1.ltoreq.D.sub.0/3 (10)
where D.sub.1 is the aperture width of the image at the pupil
position of said image-formation optical system, and D.sub.0 is the
diameter of the pupil of said image-formation optical system.
[0223] (49) The microscope according to (36) above, characterized
in that an absorption film that conforms in shape to the image of
said partial aperture or said substantial point light source at the
pupil position of said image-formation optical system and is
slightly larger than said shape is located at the pupil of said
image-formation optical system or at or near a position conjugate
thereto, with satisfaction of condition (11)
.DELTA..ltoreq.D.sub.0/10 (11)
where .DELTA. is a margin width on the inner or outer peripheral
side of said absorption film with respect to the image of said
partial aperture or said substantial point light source at the
pupil position of said image-formation optical system, and D.sub.0
is the diameter of the pupil of said image-formation optical
system.
[0224] (50) The microscope according to (47) above, characterized
by further comprising an image pickup optical system for picking up
the image to be formed on said image-formation plane, an image
pickup device located on the image pickup plane thereof, and
operation and storage means for operation and storage of an image
picked up by said image pickup device.
[0225] (51) The microscope according to (47) above, characterized
by further comprising a processing unit for capturing two images
comprising contrast images having a phase difference of opposite
sign by means of said microscope and applying a subtract operation
to the thus captured two images to form an image subjected to the
subtract operation, so that at least one of the number of cells,
the area taken up by cells, the abundance distribution of cells and
the abundance density of cells in a viewing range is measured from
the obtained image with the subtract operation applied thereto.
[0226] (52) The microscope according to (51) above, characterized
by further comprising a separation unit for extracting a portion
having an intensity value of zero or nearly zero out of the
intensity distribution of the image subjected to the subtract
operation to define said portion as being a cell-free portion and
other portion as being a cell-containing portion, thereby making a
separation between the portions containing cells and no cells.
[0227] (53) The microscope according to (51) above, characterized
by further comprising a binarization unit for extracting a portion
having an intensity of zero or nearly zero out of the intensity
distribution of the image subjected to the subtract operation to
allocate the intensity value to one value and other portion to
another one intensity value for binarization.
[0228] (54) The microscope according to (47) above, characterized
by further comprising an operation unit for capturing two images
comprising contrast images having a phase difference of opposite
sign by means of said microscope and applying a subtract operation
and an add operation to the thus captured two images, and a
thickness measurement unit for dividing the image subjected to the
subtract operation by the image subjected to the add operation to
measure information about the thickness of cells in the viewing
range.
[0229] (55) The microscope according to (35) above, characterized
by further comprising an image pickup optical system for picking up
the image to be formed on said image-formation plane, an image
pickup device located on the image pickup plane thereof, and
operation and storage means for operation and storage of an image
picked up by said image pickup device.
[0230] (56) The microscope according to (35) above, characterized
by further comprising a processing unit for capturing two images
comprising contrast images having a phase difference of opposite
sign by means of said microscope and applying a subtract operation
to the thus captured two images to form an image subjected to the
subtract operation, so that at least one of the number of cells,
the area taken up by cells, the abundance distribution of cells and
the abundance density of cells in a viewing range is measured from
the obtained image with the subtract operation applied thereto.
[0231] (57) The microscope according to (56) above, characterized
by further comprising a separation unit for extracting a portion
having an intensity value of zero or nearly zero out of the
intensity distribution of the image subjected to the subtract
operation to define said portion as being a cell-free portion and
other portion as being a cell-containing portion, thereby making a
separation between the portions containing cells and no cells.
[0232] (58) The microscope according to (56) above, characterized
by further comprising a binarization unit for extracting a portion
having an intensity of zero or nearly zero out of the intensity
distribution of the image subjected to the subtract operation to
allocate the intensity value to one value and other portion to
another one intensity value for binarization.
[0233] (59) The microscope according to (35) above, characterized
by further comprising an operation unit for capturing two images
comprising contrast images having a phase difference of opposite
sign by means of said microscope and applying a subtract operation
and an add operation to the thus captured two images, and a
thickness measurement unit for dividing the image subjected to the
subtract operation by the image subjected to the add operation to
measure information about the thickness of cells in the viewing
range.
[0234] (60) A microscope, comprising a substantial point light
source for giving out divergent light, an illumination light source
for guiding light from the substantial light source to an object
under observation while the image of the substantial point light
source is once formed at a pupil position, and an image-formation
optical system for forming on an image-formation plane the image of
the object under observation illuminated by light from the
substantial point light source, and further comprising an eyepiece
optical system or an image pickup optical system for viewing the
image formed on the image-formation plane, characterized in
that:
[0235] the diameter of the image of said substantial point light
source at the pupil position of said image-formation optical system
is set smaller than the diameter of the pupil of said
image-formation optical system, wherein at the pupil position of
said image-formation optical system there is located wavefront
introduction means for introducing in the pupil position of said
image-formation optical system a wavefront varying in size with the
diameter of the pupil.
[0236] (61) The microscope according to (60) above, characterized
in that the image-formation magnification of said image-formation
optical system is 4 or lower.
[0237] (62) The microscope according to (61) above, characterized
in that said wavefront introduction means comprises means for
displacement and adjustment of said image-formation optical system
in the optical axis direction.
[0238] (63) The microscope according to (60) above, characterized
in that said wavefront introduction means comprises means for
displacement and adjustment of said image-formation optical system
in the optical axis direction.
[0239] (64) The microscope according to (60) above, characterized
in that said wavefront introduction means comprises a
variable-shape mirror that is located at the pupil of said
image-formation optical system or at or near a position conjugate
thereto and has a variable or adjustable reflecting surface
shape.
[0240] (65) An apparatus for implementing microscopic measurements,
characterized by comprising a microscope that comprises an
illumination optical system, a viewing optical system including a
unit for converting an object under observation into a contrast
image by interference and an image pickup device for capturing
therein a contrast image having a phase difference of opposite sign
upon interference of the image under observation, and a processing
unit for capturing therein two images comprising contrast images
having a phase difference of opposite sign to apply a subtract
operation to the thus captured two images to form an image
subjected to the subtract operation, so that at least one of the
number of cells, the area taken up by cells, the abundance
distribution of cells and the abundance density of cells in a
viewing range is measured from the obtained image with the subtract
operation applied thereto.
[0241] (66) The apparatus for implementing microscopic measurements
according to (65) above, characterized by further comprising a
separation unit for extracting a portion having an intensity value
of zero or nearly zero out of the intensity distribution of the
image subjected to the subtract operation to define said portion as
being a cell-free portion and other portion as being a
cell-containing portion, thereby making a separation between the
portions containing cells and no cells.
[0242] (67) The apparatus for implementing microscopic measurements
according to (65) above, characterized by further comprising a
binarization unit for extracting a portion having an intensity of
zero or nearly zero out of the intensity distribution of the image
subjected to the subtract operation to allocate the intensity value
to one value and other portion to another one intensity value for
binarization.
[0243] (68) An apparatus for implementing microscopic measurements,
characterized by comprising a microscope that comprises an
illumination optical system, a viewing optical system including a
unit for converting an object under observation into a contrast
image by interference, and an image pickup device for capturing
therein a contrast image having a phase difference of opposite sign
upon interference of the image under observation, an operation unit
for capturing in said microscope two images comprising contrast
images having a phase difference of opposite sign to apply a
subtract operation and an add operation to the thus captured two
images to form two images, and a thickness measurement unit for
dividing the image subjected to the subtract operation divided by
the image subjected to the add operation to measure information
about the thickness of cells in the viewing range.
* * * * *