U.S. patent application number 13/052504 was filed with the patent office on 2011-08-11 for optical system for cell imaging.
This patent application is currently assigned to Battelle Memorial Institute. Invention is credited to John S. Laudo.
Application Number | 20110194174 13/052504 |
Document ID | / |
Family ID | 35638175 |
Filed Date | 2011-08-11 |
United States Patent
Application |
20110194174 |
Kind Code |
A1 |
Laudo; John S. |
August 11, 2011 |
OPTICAL SYSTEM FOR CELL IMAGING
Abstract
A microscope system (10, 10', 10'', 10''') includes a laser (18)
or light emitting diode (18''') that generates source light having
a non-uniform spatial distribution. An optical system includes an
objective (40) defining a field of view, and an optical train (22,
22', 22'', 22''') configured to convert the source light into an
enlarged-diameter collimated light, to spatially homogenize the
enlarged-diameter collimated light, and to couple the homogenized
enlarged-diameter collimated light into the objective to provide
substantially uniform static illumination of the field of view. A
camera system (56) is statically optically coupled by the objective
with at least most of the field of view.
Inventors: |
Laudo; John S.; (Hilliard,
OH) |
Assignee: |
Battelle Memorial Institute
Columbus
OH
|
Family ID: |
35638175 |
Appl. No.: |
13/052504 |
Filed: |
March 21, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12168373 |
Jul 7, 2008 |
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13052504 |
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11261105 |
Oct 27, 2005 |
7397601 |
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12168373 |
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60631025 |
Nov 24, 2004 |
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60631026 |
Nov 24, 2004 |
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60631027 |
Nov 24, 2004 |
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Current U.S.
Class: |
359/385 |
Current CPC
Class: |
B01L 9/06 20130101; G02B
21/248 20130101; B01L 2200/025 20130101; G02B 21/06 20130101; G02B
27/095 20130101; G02B 27/48 20130101; G01N 33/5094 20130101; G02B
21/365 20130101; G02B 21/002 20130101 |
Class at
Publication: |
359/385 |
International
Class: |
G02B 21/06 20060101
G02B021/06 |
Claims
1. An optical system for imaging a field of view, the optical
system comprising: an optical train configured to adjust a
non-uniform distribution of source light to generate light having
an adjusted non-uniform spatial distribution; and an objective
configured to focus the light having the adjusted non-uniform
spatial distribution, the focusing defining illumination light that
substantially uniformly illuminates the field of view; wherein the
optical train comprises: a stationary beam homogenizer that adjusts
the source light to generate the light having the adjusted
non-uniform spatial distribution; and one or more focusing elements
that reduce a diameter of the light having the adjusted non-uniform
spatial distribution to couple the light having the adjusted
non-uniform spatial distribution into the objective.
2. The optical system as set forth in claim 1, wherein the source
light has a nonuniform Gaussian distribution, and the optical train
generates the light having an adjusted non-uniform spatial
distribution with reduced or eliminated Gaussian nonuniformity.
3. The optical system as set forth in claim 1, wherein the source
light has a nonuniform Lambertian distribution, and the optical
train generates the light having an adjusted non-uniform spatial
distribution with reduced or eliminated Lambertian
nonuniformity.
4. The optical system as set forth in claim 1, wherein the optical
train comprises only stationary components that are not rotated,
relatively oscillated, or relatively moved.
5. The optical system as set forth in claim 4, wherein the optical
train and the objective are arranged to be moved as a whole
respective to a sample to scan the field of view respective to the
sample.
6. The optical system as set forth in claim 1, further comprising:
a generally tubular sample holder defining an annular sample region
coinciding with the field of view.
7. The optical system as set forth in claim 1, further comprising:
an imaging optical sub-system including said objective, the imaging
optical sub-system configured to use said objective to acquire an
image of the field of view illuminated by said illumination
light.
8. An optical system for imaging a field of view, the optical
system comprising: an objective focused on a field of view; an
optical train generating and inputting illumination light into the
objective, said illumination light cooperating with focusing
performed by the objective to substantially uniformly illuminate
the field of view wherein the optical train includes a beam
homogenizer configured to reduce nonuniformity of source light; and
an imaging optical sub-system including said objective, the imaging
optical sub-system configured to use the objective to acquire an
image of the field of view substantially uniformly illuminated by
the optical train.
9. The optical system as set forth in claim 8, further comprising:
a sample holder configured to (i) define an annular sample region
coinciding with the field of view and (ii) rotate the annular
sample region respective to the objective.
10. An optical system for imaging a microscope field of view, the
optical system comprising: an objective focused on the microscope
field of view; and an optical train including: a stationary
collimator collimating source light into a collimated light beam
having a non-uniform distribution with a highest intensity central
beam region, a beam homogenizer disposed after the stationary
collimator in the optical train and configured to reduce
non-uniformity of the non-uniform distribution of the collimated
light beam by one of (i) a non-uniform absorption profile having
highest absorption in a central region corresponding to the highest
intensity central beam region and (ii) a refractive configuration
that refracts light from the highest intensity central beam region
into a lower intensity periphery of the light beam, and a
stationary beam reducer disposed after the beam homogenizer in the
optical train and coupling the collimated light into the
objective.
11. The optical system of claim 10, wherein the optical train
further comprises: a beam expander preceding the stationary
collimator in the optical train, the beam expander receiving and
expanding a laser beam to form the source light that is collimated
by the stationary collimator.
12. The optical system of claim 11, wherein the laser beam and the
source light have a Gaussian distribution.
13. The optical system of claim 10, wherein the beam homogenizer is
configured to reduce non-uniformity of the non-uniform distribution
of the collimated light beam by a non-uniform absorption profile
having highest absorption is in a central region corresponding to
the highest intensity central beam region.
14. The optical system of claim 10, wherein the beam homogenizer is
configured to reduce non-uniformity of the non-uniform distribution
of the collimated light beam by a refractive configuration that
refracts light from the highest intensity central beam region into
a lower intensity periphery of the light beam.
15. The optical system of claim 14, wherein the beam homogenizer
comprises a lens pair configured to reduce non-uniformity of the
non-uniform distribution of the collimated light beam by refracting
light from the highest intensity central beam region into a lower
intensity periphery of the light beam.
Description
[0001] This is a continuation application of application Ser. No.
12/168,373 filed Jul. 7, 2008 which is a continuation application
of application Ser. No. 11/261,105, filed Oct. 27, 2005 and since
issued as U.S. Pat. No. 7,397,601, which claims the benefit of U.S.
Provisional Application No. 60/631,025, filed Nov. 24, 2004, U.S.
Provisional Application No. 60/631,026, filed Nov. 24, 2004, and
U.S. Provisional Application No. 60/631,027, filed Nov. 24, 2004,
which is incorporated by reference herein in its entirety.
application Ser. No. 12/168,373, filed Jul. 7, 2008 is incorporated
by reference herein in its entirety. application Ser. No.
11/261,105, filed Oct. 27, 2005 is incorporated by reference herein
in its entirety. Provisional Application No. 60/631,025, filed Nov.
24, 2004 is incorporated by reference herein in its entirety.
Provisional Application No. 60/631,026, filed Nov. 24, 2004 is
incorporated by reference herein in its entirety. Provisional
Application No. 60/631,027, filed Nov. 24, 2004 is incorporated by
reference herein in its entirety.
BACKGROUND
[0002] The following relates to the imaging arts. It is described
with particular reference to example embodiments that relate to
imaging of rare cells, such as epithelial cells, in the buffy coat
of a centrifuged blood sample. However, the following relates more
generally to illumination systems for generating a substantially
uniform static illumination across a large field of view and to
microscopes employing same.
[0003] In the technique of quantitative buffy coat analysis, a
whole blood sample is drawn and processed using anti-coagulant
additives, centrifuging, and so forth to separate the blood into
components including a buffy coat component comprised principally
of white blood cells. Rare cells of interest which are present in
the buffy coat, such as certain epithelial cells associated with
certain cancers, are tagged using a suitable fluorescent dye, and
fluorescence microscopic imaging is then used to count the
fluorescent dye-tagged cells of interest. Quantitative buffy coat
analysis is a promising non-invasive technique for screening for
certain cancers, for monitoring cancer treatment, and so forth.
[0004] The concentration of fluorescent dye-tagged rare cells in
the buffy coat is low. Optical scanning fluorescence microscopy
enables assessment of a large area of buffy coat sample by scanning
a field of view of a microscope relative to the buffy coat sample.
Scanning can be achieved by moving the microscope relative to the
buffy coat sample, by moving the buffy coat sample relative to the
microscope, or by some combination thereof. A large field of view
illuminated with high intensity uniform light is advantageous for
rapidly and accurately assaying the fluorescent dye-tagged rare
cells in the buffy coat sample. The illumination may also
advantageously employ monochromatic or narrow-bandwidth light so as
to facilitate spectral differentiation between rare cell
fluorescence and scattered illumination.
[0005] However, providing illumination at high intensity that is
uniform over a large field of view is difficult.
[0006] In the case of white light sources, filtering is typically
required to provide monochromatic or at least spectrally restricted
illumination. Spectral filtering blocks a large portion of the
optical output that lies outside the selected spectral range. Thus,
illuminating with a white light source is optically inefficient.
High intensity incandescent white light sources such as Xenon lamps
also produce substantial heat, which can adversely affect the
quantitative buffy coat analysis.
[0007] A laser light source is more optically efficient at
producing spectrally narrow light. For example, an argon laser
outputs high intensity narrow spectral lines at 488 nm and 514 nm,
and weaker lines at other wavelengths. These wavelengths are
suitable for exciting luminescence in certain tagging dyes that
luminesce at about 550 nm.
[0008] However, lasers typically output a tightly collimated beam
having a highly non-uniform Gaussian intensity profile or
distribution across a narrow beam cross-sectional area. Moreover,
the laser beam is coherent and typically exhibits a speckle pattern
due to interference amongst the wave fronts. The speckle pattern
can have spatial frequencies that overlap the typical size of rare
cells. The speckle pattern can also shift or change as the field of
view is scanned. These aspects of laser light substantially
complicate determination of whether a detected luminous feature is
a fluorescent dye-tagged rare cell, or an illumination
artifact.
[0009] Spatial uniformity can be improved using a beam homogenizer.
One type of beam homogenizer operates by providing an inverse
Gaussian absorption profile that substantially cancels the Gaussian
beam distribution. Another type of beam homogenizer employs two or
more lenses (or a compound lens) to refract the Gaussian beam in a
way that redistributes the light into a flattened spatial profile.
Using a beam homogenizer in microscopic fluorescence imaging is
problematic, however, because focusing of the homogenized beam by
the microscope objective can introduce additional beam
non-uniformities. Moreover, beam homogenizers generally do not
substantially reduce speckle non-uniformities.
[0010] In another approach, known as confocal microscopy, the laser
beam is rapidly rastered or scanned across the field of view. The
field of view is sampled rather than imaged as a whole. In this
dynamic approach, the portion is of the sample illuminated at any
given instant in time is much smaller than the field of view. By
rapidly rastering the focused laser beam over the field of view, an
image can be constructed from the acquired sample points. A uniform
illumination is, in effect, dynamically simulated through rapid
sampling of the field of view.
[0011] Confocal microscopy is an established technique. However,
the beam rastering adds substantial complexity and cost to the
microscope system. Confocal microscopy can also be highly sensitive
to small defects in a lenses or other optical component. Thus, very
high quality optics should be employed, which again increases
system cost.
INCORPORATION BY REFERENCE
[0012] U.S. application Ser. No. 10/263,974 filed Oct. 3, 2002 and
published as U.S. Publ. Appl. No. 2004/0067162 A1 on Apr. 8, 2004,
is incorporated by reference herein in its entirety. [0013] U.S.
application Ser. No. 10/263,975 filed Oct. 3, 2002 and published as
U.S. Publ. Appl. No. 2004/0067536 A1 on Apr. 8, 2004, is
incorporated by reference herein in its entirety. [0014] U.S.
patent application Ser. No. 11/261,306 filed Oct. 27, 2005,
entitled "Method and Apparatus for Detection of Rare Cells",
inventor Albert E. Weller, III, and corresponding to attorney
docket no. BATZ 2 00009, is incorporated by reference herein in its
entirety. [0015] U.S. patent application Ser. No. 11/261,104 filed
Oct. 27, 2005, entitled "Sample Tube Handling Apparatus", inventors
Steve Grimes, Thomas D. Haubert, and Eric R. Navin, and
corresponding to attorney docket no. BATZ 2 00010, is incorporated
by reference herein in its entirety.
BRIEF SUMMARY
[0016] According to one aspect, an optical system is disclosed for
imaging a microscope field of view. An objective is focused on the
microscope field of view. An optical train includes one or more
stationary optical components configured to receive source light
having a non-uniform spatial distribution and to output a corrected
spatial distribution to the objective that when focused by the
objective at the microscope field of view provides substantially
uniform static illumination over substantially the entire
microscope field of view.
[0017] According to another aspect, a microscope system is
disclosed. A laser, semiconductor laser diode, or light emitting
diode generates source light having a non-uniform spatial
distribution. An optical system includes (i) an objective defining
a field of view and (ii) an optical train configured to convert the
source light into an enlarged-diameter collimated light, spatially
homogenize the enlarged-diameter collimated light, and couple the
homogenized enlarged-diameter collimated light into the objective
to provide substantially uniform static illumination of the field
of view. A camera system is statically optically coupled by the
objective with at least most of the field of view.
[0018] According to another aspect, an optical system is disclosed
for imaging a microscope field of view. An objective is focused on
the microscope field of view. A stationary diffuser receives source
light having a non-uniform spatial distribution and diffuses the
source light to improve spatial uniformity. The diffused light is
used to provides substantially uniform static illumination over at
least most of the microscope field of view through the
objective.
[0019] Numerous advantages and benefits of the present invention
will become apparent to those of ordinary skill in the art upon
reading the following detailed description of the preferred
embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The invention may take form in various components and
arrangements of components, and in various process operations and
arrangements of process operations. The drawings are only for the
purpose of illustrating preferred embodiments and are not to be
construed as limiting the invention.
[0021] FIG. 1 diagrammatically shows a microscope system including
an optical system for providing substantially uniform static
illumination over substantially the entire microscope field of
view.
[0022] FIG. 2 diagrammatically shows the microscope system of FIG.
1 with a modified optical system.
[0023] FIG. 3 diagrammatically shows the microscope system of FIG.
1 with another modified optical system.
[0024] FIG. 4 diagrammatically shows the microscope system of FIG.
1 with yet another modified optical system.
[0025] FIGS. 5-10 show various views of a test tube holder:
[0026] FIG. 5 shows a perspective view of the holder, with the
housing shown in phantom to reveal internal components.
[0027] FIG. 6 shows a side view of the holder, with the housing
shown in phantom.
[0028] FIG. 7 shows a perspective view of the test tube and
alignment and biasing bearings.
[0029] FIG. 8 shows a top view of the test tube holder including an
indication of bias force.
[0030] FIG. 9 shows a side view of a second end of the test tube
including a contoured base.
[0031] FIG. 10 shows a top view of the rotational coupler including
a contour configured to mate with the contoured base of the test
tube shown in FIG. 9.
[0032] FIGS. 11A and 11B show top views of another embodiment test
tube holder, with a test tube having an eccentric cross-section
loaded.
[0033] FIG. 12 shows a side view of a portion of a test tube holder
employing tilted roller bearings.
[0034] FIG. 13 shows a side view of a portion of a test tube holder
employing tilted roller bearings staggered along a test tube axis,
along with a float having helical ridges enables spiral scanning of
the test tube.
[0035] FIG. 14 shows a perspective view of a test tube holder that
holds the test tube horizontally and uses the test tube as a bias
force.
[0036] FIG. 15 shows a top view of a test tube holder employing
bushing surfaces as alignment bearings and a set of ball bearings
as bias bearings.
[0037] FIG. 16 diagrammatically depicts certain to measurement
parameters relevant in performing quantitative buffy coat analysis
using a buffy coat sample trapped in an annular gap between an
inside test tube wall and an outer surface of a float.
[0038] FIG. 17 diagrammatically shows a suitable quantitative buffy
coat measurement/analysis approach.
[0039] FIG. 18 diagrammatically shows another suitable quantitative
buffy coat measurement/analysis approach.
[0040] FIG. 19 diagrammatically shows a suitable image processing
approach for tagging candidate cells.
[0041] FIG. 20 shows a pixel layout for a square filter kernel
suitable for use in the matched filtering.
[0042] FIG. 21 shows a pixel intensity section A-A of the square
filter kernel of FIG. 20.
[0043] FIG. 22 diagrammatically shows a suitable user verification
process for enabling a human analyst to confirm or reject candidate
cells.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0044] With reference to FIG. 1, a microscope system 10 images a
microscope field of view coinciding with a buffy coat sample
disposed in a generally planar portion of an annular gap 12 between
a light-transmissive test tube wall 14 and a float wall 16 of a
float disposed in the test tube. Suitable methods and apparatuses
for acquiring and preparing such buffy coat samples are disclosed,
for example, in U.S. Publ. Appl. No. 2004/0067162 A1 and U.S. Publ.
Appl. No. 2004/0067536 A1.
[0045] The microscope field of view is generally planar in spite of
the curvatures of the test tube and the float, because the
microscope field of view is typically much smaller in size than the
radii of curvature of the test tube wall 14 and the float wall 16.
Although the field of view is substantially planar, the buffy coat
sample disposed between the light-transmissive test tube wall 14
and the float wall 16 may have a thickness that is substantially
greater than the depth of view of the microscope system 10.
[0046] The test tube is mounted in fixed position respective to the
microscope system 10 in a manner conducive to scanning the
microscope field of view across the annular gap. As will be
discussed, suitable mechanisms are preferably provided for
effectuating relative rotational and/or translational scanning of
the field of view over the annular gap containing the buffy coat
sample.
[0047] The microscope system 10 includes a laser 18, such as a gas
laser, a solid state laser, a semiconductor laser diode, or so
forth, that generates source light 20 (diagrammatically indicated
in FIG. 1 by dashed lines) in the form of a laser beam having an
illumination wavelength and a non-uniform spatial distribution that
is typically Gaussian or approximately Gaussian in shape with a
highest intensity in a central region of the beam and reduced
intensity with increasing distance from the beam center. An optical
train 22 is configured to receive the spatially non-uniform source
light 20 and to output a corrected spatial distribution.
[0048] A beam spreader includes a concave lens 24 that generally
diverges the laser beam, and a collimating lens 26 that collimates
the spread beam at a larger diameter that substantially matches the
diameter of a Gaussian spatial characteristic of a beam homogenizer
30. The beam homogenizer 30 flattens the expanded laser beam by
substantially homogenizing the Gaussian or other non-uniform
distribution of the source light to produce output light having
improved spatial uniformity.
[0049] In some embodiments, the beam homogenizer 30 operates by
having a spatially non-uniform absorption profile that corresponds
to an inverse-Gaussian. In such embodiments, the beam homogenizer
has highest absorption in a central region corresponding to the
highest intensity central region of the expanded laser beam, and
has a lower absorption, or no absorption, in the periphery
corresponding to the lower intensity outer regions of the expanded
laser beam.
[0050] In other embodiments, the beam homogenizer 30 refractively
redistributes the light to homogenize the light intensity across
the area of the expanded laser beam, for example using a suitable
lens pair. Refractive beam homogenizers refract light from the high
intensity central region of the expanded laser beam into the lower
intensity periphery regions.
[0051] A focusing lens 34 and cooperating lenses 36 reduce the
expanded and flattened or homogenized laser beam down to a desired
beam diameter for input to an objective 40 that is focused on the
microscope field of view. A dichroic mirror 44 is selected to
substantially reflect light at the wavelength or wavelength range
of the laser beam, and to substantially transmit light at the
fluorescence wavelength or wavelength range of the fluorescent dye
used to tag rare cells in the buffy coat sample.
[0052] The optical train 22 including the stationary optical
components 24, 26, 30, 34, 36 is configured to output a corrected
spatial distribution to the objective 40 that when focused by the
objective 40 at the microscope field of view provides substantially
uniform static illumination over substantially the entire
microscope field of view. The objective 40 focuses the corrected
illumination onto the microscope field of view. The objective 40
may include a single objective lens, or may include two or more
objective lenses. The focus depth of the microscope system 10 is
adjustable, for example by adjusting a distance between the
objective 40 and the light-transmissive test tube wall 14.
Additionally or alternatively, the focus depth may be adjusted by
relatively moving two or more lenses or lensing elements within the
objective 40.
[0053] The beam homogenizer 30 is designed to output a
substantially uniform homogenized beam for a Gaussian input beam of
the correct diameter. However, the objective 40 typically
introduces some spatial non-uniformity. Accordingly, one or more of
the stationary optical components, such as the spreading lens 24,
collimating lens 26, focusing lens 34, and/or focusing lenses 36
are optionally configured to introduce spatial non-uniformity into
the spatial distribution such that the beam when focused by the
objective 40 provides substantially uniform static illumination of
the microscope field of view. In some contemplated embodiments,
this corrective spatial non-uniformity is introduced by one or more
dedicated optical components (not shown) that are included in the
optical train 22 for that purpose.
[0054] The substantially uniform static illumination of the
microscope field of view causes fluorescence of any fluorescent
dye-tagged epithelial cells disposed within the microscope field of
view. Additionally, the fluorescent dye typically imparts a
lower-intensity background fluorescence to the buffy coat. The
fluorescence is captured by the objective 40, and the captured
fluorescence 50 (diagrammatically indicated in FIG. 1 by dotted
lines) passes through the dichroic mirror 44, and through an
optional filter 52 for removing any stray source light, to be
imaged by a camera system 56. The camera system 56 may, for
example, include a charge coupled device (CCD) camera for acquiring
electronic images that can be stored in a computer, memory card, or
other non-volatile memory for subsequent image processing.
[0055] With reference to FIGS. 2 and 3, other suitable microscope
systems are described.
[0056] FIG. 2 shows a microscope system 10' that is similar to the
microscope system 10 of FIG. 1, except that the optical train 22'
differs in that the stationary beam homogenizer 30 of FIG. 1 is
replaced by a stationary diffuser 30'. The diffuser 30' may, for
example, be a holographic diffuser available, for example, from
Physical Optics Corporation (Torrance, Calif.). Such holographic
diffusers employ a hologram providing randomizing non-periodic
optical structures that diffuse the light to impart improved
spatial uniformity. However, the diffusion of the light also
imparts some concomitant beam divergence. Typically, stronger
diffusion of the light tend to impart more spatial uniformity, but
also tends to produce greater beam divergence. Holographic
diffusers are suitably classified according to the
full-width-at-half-maximum (FWHM) of the divergence angle, with
larger divergence angles typically providing more diffusion and
greater light uniformity, but also leading to increased light loss
in the microscope system 10' due to increased beam divergence.
[0057] In some embodiments of the microscope system 10', the
diffuser 30' is a low-angle diffuser having a FWHM less than or
about 10.degree.. Lower angle diffusers are generally preferred to
provide less divergence and hence better illumination throughput
efficiency; however, if the divergence FWHM is too low, the
diffuser will not provide enough light diffusion to impart adequate
beam uniformity. Low diffusion reduces the ability of the diffuser
30' to homogenize the Gaussian distribution, and also reduces the
ability of the diffuser 30' to remove speckle.
[0058] With reference to FIG. 3, another embodiment microscope
system 10'' is similar to the microscope system 10', and includes
an optical train 22'' that employs a diffuser 30'' similar to the
diffuser 30' of the microscope system 10'. However, the diffuser
30'' is tilted at an angle .theta. respective to the optical path
of the optical train 22'' so as to substantially reduce a speckle
pattern of the source light 20. Without being limited to any
particular theory of operation, it is believed that the tilting
shifts the speckle pattern to higher spatial frequencies, in effect
making the speckle size smaller. The speckle size is spatially
shifted by the tilting such that the frequency-shifted speckle is
substantially smaller than an imaging pixel size.
[0059] In some embodiments, a tilt angle .theta. of at least about
30.degree. respective to the optical path of the optical train 22''
is employed, which has been found to substantially reduce speckle
for diffusers 30'' having a FWHM as low as about 5.degree.. On the
other hand, tilt angles .theta. of greater than about 45.degree.
have been found to reduce illumination throughput efficiency due to
increased scattering, even for a low-angle diffuser having a FWHM
of 5.degree..
[0060] With reference to FIG. 4, it is to be appreciated that the
microscope systems disclosed herein are suitable for other
microscopy applications besides imaging of samples contained in or
supported by test tubes. In FIG. 4, a microscope system 10'''
includes a light emitting diode (LED) 18''' as the light source,
rather than the laser 18 used in the previous microscope systems
10, 10', 10''. Because the LED 18''' outputs diverging source light
20''' rather than a collimated laser beam, an optical train 22'''
is modified in that the beam-expanding concave lens 24 is suitably
omitted, as shown in FIG. 4. Alternatively, a lens can be included
in the position of the lens 24, but selected to provide a suitable
divergence angle adjustment for collimation by the collimating lens
26. The optical train 10''' employs a diffuser 30''' similar to the
diffusers 30', 30''. The LED 18''' outputs incoherent light, and so
speckle is generally not present. However, the output of the LED
18''' typically does have a non-Gaussian distribution, for example
a Lambertian distribution. In view of these characteristics of the
source light 20''', the diffuser 30''' is not tilted, and in some
cases the diffuser 30''' can have a smaller divergence angle FWHM
than the untilted diffuser 30' used to impart spatial uniformity to
the laser beam source light 20 in the microscope system 10' of FIG.
2.
[0061] The microscope system 10''' of FIG. 4 further differs from
the microscope systems 10, 10', 10'' in that the microscope system
10''' images a sample disposed on a planar slide 60, which is
optionally covered by an optional cover glass 62. The slide 60 is
disposed on an x-y planar translation stage 64 to enable scanning
across the sample. It will be appreciated that the LED 18''' and
optical train 22''' are also suitable for imaging the buffy coat
sample disposed in the annular gap 12 between the
light-transmissive test tube wall 14 and float wall 16 shown in
FIGS. 1-3. Conversely, it will be appreciated that the laser 18 and
optical train 22, 22', 22'' are also suitable for imaging the
planar sample on the slide 60 shown in FIG. 4.
[0062] The optical trains 22, 22', 22'', 22''' have components
which are stationary in the sense that the components are not
rotated, relatively oscillated, or otherwise relatively moved. It
is, however, contemplated to move the optical train and the
objective 40 as a whole, and/or to include beam-steering elements,
or so forth, to enable relative scanning of the field of view
respective to the sample.
[0063] Suitable microscope systems for imaging an annular sample
contained in or supported by a test tube have been described. The
annular gap 12 typically has a thickness that is substantially
larger than a depth of view of the microscope objective 40. The
test tube wall 12 and float wall 16 are typically not uniform
across the entire surface of the test tube or float. While the
microscope objective 40 typically has an adjustable depth of focus
(adjusted by moving internal optical components and/or by moving
the objective 40 toward or away from the test tube wall 12), the
range of adjustment is limited. Accordingly, the test tube should
be held such that the surface proximate to the objective 40 is at a
well-defined distance away from the objective 40 as the test tube
is rotated and as the objective 40, or the test tube, is translated
along a tube axis.
[0064] Suitable test tube holders for achieving such aspects are
next described.
[0065] With reference to FIGS. 5-10, a test tube holder 70 has
mounted therein a test tube 72 that is sealed by a test tube
stopper 73. The sealed test tube 72 contains a float 74 and blood
that has been suitably processed and centrifuged to separate out
components including red blood cells, plasma, and a buffy coat, for
example as described in U.S. Publ. Applications 2004/0067162 A1 and
2004/0067536 A1. The float 74 has a density which is less than that
of the packed red blood cells component (1.090 g/ml) and greater
than that of the plasma component (1.028 g/ml). Accordingly, after
centrifuging the float 74 is disposed along the test tube axis 75
(drawn and labeled in FIG. 6) between the packed red blood cell
layer and the plasma layer, that is, generally coincident with the
buffy coat. After centrifuging, the buffy coat is generally
disposed in the annular gap 12 between the test tube wall 14 and
the float wall 16. (See labeling in FIG. 6). Annular sealing ridges
76, 78 at ends of the float 74 engage an inside surface of the test
tube 72 when the test tube is at rest so as to seal the annular gap
12. During centrifuging, however, the test tube 72 expands to
provide fluid communication across the ridges 76, 78 so as to
enable the buffy coat to substantially collect in the annular gap
12.
[0066] At least one first alignment bearing, namely two radially
spaced apart first alignment bearings 80, 81 in the example test
tube holder 70, are disposed on a first side of the annular
sampling region 12. At least one second alignment bearing, namely
two second radially spaced apart alignment bearings 82, 83 in the
example test tube holder 70, are disposed on a second side of the
annular sampling region 12 opposite the first side of the annular
sampling region 12 along the test tube axis 75. The alignment
bearings 80, 81, 82, 83 are fixed roller bearings fixed to a
housing 84 by fastening members 85 (shown only in FIG. 8).
[0067] At least one biasing bearing, namely two biasing bearings
86, 87 in the example test tube holder 70, are radially spaced
apart from the alignment bearings 80, 81, 82, 83 and are spring
biased by springs 90 to press the test tube 72 against the
alignment bearings 80, 81, 82, 83 so as to align a side of the
annular sampling region 12 proximate to the objective 40 respective
to the alignment bearings 80, 81, 82, 83. In the example test tube
holder 70, the two first alignment bearings 80, 81 and the first
biasing bearing 86 are radially spaced apart by 120.degree.
intervals and lie in a first common plane 92 on the first side of
the annular sampling region 12. Similarly, the two second alignment
bearings 82, 83 and the second biasing bearing 87 are radially
spaced apart by 120.degree. intervals and lie in a second common
plane 94 on the second side of the annular sampling region 12. The
springs 90 are anchored to the housing 84 and connect with the
biasing bearings 86, 87 by members 98.
[0068] More generally, the bearings 80, 81, 86 and the bearings 82,
83, 87 may have radial spacings other than 120.degree.. For example
the biasing bearing 86 may be spaced an equal radial angle away
from each of the alignment bearings 80, 81. As a specific example,
the biasing bearing 86 may be spaced 135.degree. away from each of
the alignment bearings 80, 81, and the two alignment bearings 80,
81 are in this specific example spaced apart by 90.degree..
[0069] Optionally, the first common plane 92 also contains the
float ridge 76 so that the bearings 80, 81, 86 press against the
test tube 72 at the ridge 76, and similarly the second common plane
94 optionally also contains the float ridge 78 so that the bearings
82, 83, 87 press against the test tube 72 at the ridge 78. This
approach reduces a likelihood of distorting the annular sample
region 12. The biasing bearings 86, 87 provide a biasing force 96
that biases the test tube 72 against the alignment bearings 80, 81,
82, 83.
[0070] The housing includes a viewing window 100 that is elongated
along the tube axis 75. The objective 40 views the side of the
annular sample region 12 proximate to the objective 40 through the
viewing window 100. In some embodiments, the objective 40 is
linearly translatable along the test tube axis 75 as indicated by
translation range double-arrow indicator 104. This can be
accomplished, for example, by mounting the objective 40 and the
optical train 22, 22', 22'', or 22''' on a common board that is
translatable respective to the test tube holder 70. In another
approach, the microscope system 10, 10', 10'', 10''' is stationary,
and the tube holder 70 including the housing 84 is translated as a
unit to relatively translate the objective 40 across the window
100. In yet other embodiments, the objective 40 translates while
the optical train 22, 22', 22'', or 22''' remains stationary, and
suitable beam-steering components (not shown) are provided to input
the beam to the objective 40. The objective 40 is also focusable,
for example by moving the objective 40 toward or away from the test
tube 72 over a focusing range 106 (translation range 104 and
focusing range 106 indicated only in FIG. 6).
[0071] Scanning of the annular sampling region 12 calls for both
translation along the test tube axis, and rotation of the test tube
72 about the test tube axis 75. To achieve rotation, a rotational
coupling 110 is configured to drive rotation of the test tube 72
about the tube axis 75 responsive to a torque selectively applied
by a motor 112 connected with the rotational coupling 110 by a
shaft 114. The rotational coupling 110 of the example test tube
holder 70 connects with the test tube 72 at an end or base thereof.
At an opposite end of the test tube 72, a spring-loaded cap 116
presses against the stopper 73 of the test tube 72 to prevent the
rotation from causing concomitant translational slippage of the
test tube 72 along the test tube axis 75.
[0072] With particular reference to FIGS. 9 and 10, in some
embodiments the rotational coupling 110 is a contoured coupling
having a contour 120 configured to mate with a contoured base 122
of the test tube 72. In the illustrated example of FIGS. 9 and 10,
the contour 120 of the coupling 110 includes four depressions that
receive four nibs of the contoured base 122 of the test tube 72.
Other contour features can be employed.
[0073] In some embodiments, the contour 120 and contoured base 122
are keyed by suitable rotationally asymmetric features 124, 126
(shown in phantom in FIGS. 9 and 10) in the coupling 110 and test
tube base 122, respectively, to define an absolute rotational
position of the test tube 72 when the contoured base 122 of the
test tube 72 is mated with the contour 120 of the rotational
coupling 110. In this way, the absolute rotational position
(measured, for example as an absolute angle value in degrees) can
be maintained even if the test tube 72 is removed from and then
re-installed in the test tube holder 70.
[0074] In another approach for providing absolute angular position,
the test tube optionally includes fiducial markers, for example
optically readable reflective fiducial markers (not shown), to
indicate the absolute rotational position of the test tube.
[0075] In some embodiments, the second side alignment roller
bearings 82, 83 are omitted, and the rotational coupling 110
defines the at least one second alignment bearing disposed on the
second side of the annular sampling region 12 opposite the first
side of the annular sampling region 12 along the test tube axis 75.
In such embodiments, the rotational coupling acts as a mechanically
driven alignment bearing to provide both alignment and rotation of
the test tube 72. Optionally, in such embodiments the second side
bias bearing 87 is also omitted along with the corresponding roller
bearings 82, 83.
[0076] On the other hand, in some other contemplated embodiments
the rotational coupling 110 is omitted, and one or more of the
roller bearings 81, 82, 83, 84, 86, 87 are mechanically driven to
rotate the test tube 72. In such embodiments, the driven roller
bearings serve as the rotational coupling. The driven bearings can
be one or more of the alignment bearings 81, 82, 83, 84, or can be
one or more of the biasing bearings 86, 87.
[0077] In order to install the test tube 72 in the test tube holder
70, the housing 84 is provided with a hinged lid or door 130 (shown
open in FIG. 5 and closed in FIG. 6). When the hinged lid or door
130 is opened, the spring-loaded cap 116 is lifted off of the
stopper 73 of the test tube 72. Optionally, the support members 98
that support the biasing bearings 86, 87 include a manual handle or
lever (not shown) for manually drawing the biasing bearings 86, 87
away from the test tube 72 against the biasing force of the springs
90 so as to facilitate loading or unloading the test tube 72 from
the holder 70.
[0078] The test tube holder 70 advantageously can align the
illustrated test tube 72 which has straight sides. The test tube
holder 70 can also accommodate and align a slightly tapered test
tube. The held position of a tapered test tube is indicated in FIG.
6 by a dashed line 134 which indicates the tapered edge of a
tapered test tube. The illustrated tapering 134 causes the end of
the test tube closest to the rotational coupling 110 to be smaller
diameter than the end of the test tube closest to the spring-loaded
cap 116. As indicated in FIG. 6, the biasing of the biasing
bearings 86, 87 presses the test tube against the alignment
bearings 81, 82, 83, 84 to maintain alignment of the portion of the
annular sample region 12 proximate to the objective 40 in spite of
the tapering 134. It will be appreciated that the holder 70 can
similarly accommodate and align a test tube having an opposite
taper in which the end closes to the rotational coupling 110 is
larger in diameter than the end closest to the spring-loaded cap
116.
[0079] In the case of a substantial tapering, or in the case a test
tube that has a highly eccentric or non-circular cross-section, the
biasing against the alignment bearings 81, 82, 83, 84 will not
completely compensate for the tapering or cross-sectional
eccentricity or ellipticity. This is because the radial spacing
apart of the first alignment bearings 81, 82 and the radial spacing
apart of the second alignment bearings 83, 84 allows a narrower
tube to extend a further distance into the gap between the first
alignment bearings 81, 82 and into the gap between the second
alignment bearings 83, 84.
[0080] With reference to FIGS. 11A and 11B, a modified test tube
72' having an elliptical cross-section is more precisely aligned by
employing a set of three bearings per supported float ridge, in
which the three bearings include only one alignment bearing 81' and
two or more biasing bearings 86'. The alignment bearing 81' is at
the same radial position as the objective 40 (shown in phantom in
FIGS. 11A and 11B). As the elliptical test tube 12' rotates, the
imaged side that is biased against the alignment bearings 81'
remains precisely aligned with the radially coincident objective 40
whether the imaged side correspond to the short axis of the
elliptical test tube 72' (FIG. 11A), or whether the imaged side
correspond to the long axis of the elliptical test tube 72' (FIG.
11B).
[0081] With reference to FIG. 12, in another variation, bearings
140 are tilted respective to the tube axis 75 of the test tube 72
to impart force components parallel with the tube axis 75 to push
the test tube 72 into the rotational coupling 110. In this
arrangement, the spring-loaded cap 116 is optionally omitted,
because the tilting of the bearings 140 opposes translational
slippage of the test tube 72 during rotation.
[0082] With reference to FIG. 13, in another variation, a modified
float 74' includes spiral ridges 76', and tilted bearings 142 are
spaced along the tube axis 75 in accordance with the spiral pitch
to track the spiraling sealing ridges 76' responsive to rotation of
the test tube 72. In this approach, the tilted bearings 142 impart
a force that causes the test tube 72 to translate along the tube
axis 75, so that the objective 40 can be maintained at a fixed
position without translating while scanning annular gap 12'. In
this approach, the roller bearings 142 are suitably motorized to
generate rotation of the test tube 72. That is, the roller bearings
142 also serve as the rotational coupling.
[0083] With reference to FIG. 14, in another variation, the
mechanical bias can be provided by a mechanism other than biasing
bearings. In example FIG. 14, the test tube 72 is arranged
horizontally resting on alignment bearings 181, 182, 183, 184 with
the objective 40 mounted beneath the test tube 72. A weight 186 of
the test tube 72 including the float 74 (said weight
diagrammatically indicated in FIG. 14 by a downward arrow 186)
provides as the mechanical bias pressing the test tube 72 against
the alignment bearings 181, 182, 183, 184. In other contemplated
embodiments, a vacuum chuck, positive air pressure, magnetic
attraction, or other mechanical bias is employed to press the test
tube against the alignment bearings. The alignment bearings 181,
182, 183, 184 can be rotated mechanically so that the alignment
bearings 181, 182, 183, 184 serve as the rotational coupling, or a
separate rotational coupling can be provided.
[0084] With reference to FIG. 15, the bearings can be other than
roller bearings. For example, the bearings can be rollers, ball
bearings, or bushing surfaces. In the variant test tube holder
shown in FIG. 15, a housing 200 provides an anchor for a spring 202
that presses a set of biasing ball bearings 204 against the test
tube 72 to press the test tube 72 against alignment bearings 211,
212 defined by bushing surfaces of the housing 200. Other types of
bearings can be used for the biasing and/or alignment bearings that
support the test tube as it rotates.
[0085] In the illustrated embodiments other than the embodiment of
FIG. 13, the test tube is not translated within the tube holder,
and instead the translative component of the scanning is achieved
by translating the objective 40, or by translating the test tube
and tube holder as a unit. In other contemplated embodiments, it is
contemplated to keep the objective fixed and to translate the test
tube within the test tube housing, for example by including a
linear translation capability in the shaft 114 connecting the motor
112 with the rotational coupling 112 so as to translate the test
tube 72 along the test tube axis 75.
[0086] Suitable microscope systems and test tube holders have been
described for imaging an annular sample region contained in or
supported by a test tube. It is to be understood that the annular
sampling region can be other than the illustrated fluid sample
contained in the gap 12 between the test tube wall 14 and the float
wall 16. For example, the annular sample region can be a film or
coating adhered on an outside surface of the test tube, or the
annular sample region can be a film or coating adhered on an inside
surface of the test tube. Moreover, the term "test tube" is to be
broadly construed as encompassing other tubular sample holders
besides the illustrated conventional test tube 72. For example, the
test tube could be a cylindrical rod that has been inserted into a
contained volume, solid object, or other subject of interest so as
to coat an outside of the cylindrical rod with a sample of the
subject of interest, or the test tube can be a cylindrical
geological core sample, or so forth.
[0087] Having described suitable microscope systems and test tube
holders for acquiring data from an annular slide or annular
sampling region contained in or supported by a test tube, suitable
processing approaches for identifying or quantifying fluorescent
dye tagged cells in an annular biological fluid layer are now
described.
[0088] With reference to FIG. 16, certain measurement parameters
are diagrammatically illustrated. The objective 40 images over a
field of view (FOV) and over a depth of view located at a focus
depth. In FIG. 16, the focus depth is indicated respective to the
objective 40; however, the focus depth can be denoted respective to
another reference. In some embodiments, the depth of view of the
objective 40 is about 20 microns, while the annular gap 12 between
the test tube wall 14 and the float wall 16 is about 50 microns.
However, the depth of focus corresponding to the annular gap 12 can
vary substantially due to non-uniformities in the test tube and/or
the float or other factors. It is expected that the annular gap 12
is located somewhere within an encompassing depth range. In some
embodiments, an encompassing depth range of 300 microns has been
found to be suitable. These dimensions are examples, and may be
substantially different for specific embodiments depending upon the
specific objective 40, light-transmissive test tube, float, the
type of centrifuging or other sample processing applied, and so
forth.
[0089] With reference to FIG. 17, one suitable data acquisition
approach 300 is diagrammatically shown. In process operation 302,
analysis images are acquired at a plurality of focus depths
spanning the encompassing depth range. To avoid gaps in the depth
direction, the number of analysis images acquired in the operation
302 should correspond to at least the encompassing depth range
divided by the depth of view of the objective 40.
[0090] In some embodiments, the analysis images are processed in
optional operation 304 to identify one or more analysis images at
about the depth of the biological fluid layer (such as the buffy
layer) based on image brightness. This optional selection takes
advantage of the observation that typically the fluorescent dye
produces a background fluorescence that is detected in the acquired
analysis images as an increased overall image brightness. Image
brightness can be estimated in various ways, such as an average
pixel intensity, a root-mean-square pixel intensity, or so
forth.
[0091] In an image processing operation 306, the analysis images,
or those one or more analysis images selected in the optional
selection operation 304, are processed using suitable techniques
such as filtering, thresholding, or so forth, to identify observed
features as candidate cells. The density of dye-tagged cells in the
biological fluid layer is typically less than about one dye-tagged
cell per field of view. Accordingly, the rate of identified
candidate cells is typically low. When a candidate cell is
identified by the image processing 306, a suitable candidate cell
tag is added to a set of candidate cell tags 310. For example, a
candidate cell tag may identify the image based on a suitable
indexing system and x- and y-coordinates of the candidate cell
feature. Although the density of rare cells is typically low, it is
contemplated that the image processing 306 may nonetheless on
occasion identify two or more candidate cells in a single analysis
image. On the other hand, in some analysis images, no candidate
cells may be identified.
[0092] At a decision point 312, it is determined whether the sample
scan is complete. If not, then the field of view is is moved in
operation 314. For example, the field of view can be relatively
scanned across the biological fluid sample in the annular gap 12 by
a combination of rotation of the test tube 72 and translation of
the objective 40 along the test tube axis 75. Alternatively, using
the tube holder of FIG. 13, scanning is performed by moving the
test tube 72 spirally. For each new field of view, the process
operations 302, 304, 306 are repeated.
[0093] Once the decision point 312 indicates that the sample scan
is complete, a user verification process 320 is optionally employed
to enable a human analyst to confirm or reject each cell candidacy.
If the image processing 306 is sufficiently accurate, the user
verification process 320 is optionally omitted.
[0094] A statistical analysis 322 is performed to calculate
suitable statistics of the cells confirmed by the human analyst.
For example, if the volume or mass of the biological fluid sample
is known, then a density of rare cells per unit volume or per unit
weight (e.g., cells/milliliter or cells/gram) can be computed. In
another statistical analysis approach, the number of confirmed
cells is totaled. This is a suitable metric when a standard buffy
sample configuration is employed, such as a standard test tube,
standard float, standard whole blood sample quantity, and
standardized centrifuging processing. The statistical analysis 322
may also include threshold alarming. For example, if the cell
number or density metric is greater than a first threshold, this
may indicate a heightened possibility of cancer calling for further
clinical investigation, while if the cell number or density exceeds
a second, higher threshold this may indicate a high probability of
the cancer calling for immediate remedial medical attention.
[0095] With reference to FIG. 18, a modified acquisition approach
300' is diagrammatically shown. In modified process operation 304',
the focus depth for maximum background fluorescence intensity is
first determined using input other than analysis images, followed
by acquisition 302' of one or a few analysis images at about the
focus depth for maximum background fluorescence. For example, the
search process 304' can be performed by acquiring low resolution
images at various depths. To avoid gaps in the depth direction, the
number of low resolution images acquired in the operation 304'
should correspond to at least the encompassing depth range divided
by the depth of view of the objective 40. In another approach, a
large-area brightness sensor (not shown) may be coupled to the
captured fluorescence 50 (for example, using a partial mirror in
the camera 56, or using an intensity meter built into the camera
56) and the focus of the objective 40 swept across the encompassing
depth range. The peak signal of the sensor or meter during the
sweep indicates the focus providing highest brightness.
[0096] With the depth of the biological fluid sample determined by
the process operation 304', the acquisition process 302' acquires
only one or a few analysis images at about the identified focus
depth of highest brightness. To ensure full coverage of the
biological fluid layer, the number of acquired analysis images
should be at least the thickness of the annular gap 12 divided by
the depth of view of the objective 40. For example, if the annular
gap 12 has a thickness of about 50 microns and the depth of view is
about 20 microns, then three analysis images are suitably
acquired--one at the focus depth of highest brightness, one at a
focus depth that is larger by about 15-25 microns, and one at a
focus depth that is smaller by about 15-25 microns.
[0097] An advantage of the modified acquisition approach 300' is
that the number of acquired high resolution analysis images is
reduced, since the focus depth is determined prior to acquiring the
analysis images. It is advantageous to bracket the determined focus
depth by acquiring analysis images at the determined focus depth
and at slightly larger and slightly smaller focus depths. This
approach accounts for the possibility that the rare cell may be
best imaged at a depth that deviates from the depth at which the
luminescence background is largest.
[0098] With reference to FIG. 19, a suitable embodiment of the
image processing 306 is described, which takes advantage of a
priori knowledge of the expected rare cell size to identify any
cell candidates in an analysis image 330. In a matched filtering
process 332, a suitable filter kernel is convolved with the image.
The matched filtering 332 employs a filter kernel having a size
comparable with the expected size of an image of a rare cell in the
analysis image 330.
[0099] With continuing reference to FIG. 19 and with brief further
reference to FIGS. 20 and 21, in some embodiments a square filter
kernel 334 is employed. The kernel 334 includes a central positive
region of pixels each having a value of +1, and an outer negative
region of pixels each having a value of -1. The area of the
positive region should be about the same size as the area of the
negative region. Points outside of either the inner or outer region
have pixel values of zero. Optionally, other pixel values besides
+1 and -1 can be used for the inner and outer regions,
respectively, so as to give the filter a slightly positive or
slightly negative response.
[0100] With continuing reference to FIG. 19, the matched filtering
removes or reduces offsets caused by background illumination, and
also improves the signal-to-noise ratio (SNR) for rare cells. The
signal is increased by the number points in the positive match
area, while the noise is increased by the number of points in both
the positive and negative match areas. The gain in SNR comes from
the fact that the signal directly adds, while the noise adds as the
root-mean-square (RMS) value or as the square root of the number of
samples combined. For a filter with N positive points and N
negative points, a gain of N/ (2N) or (N/2) is obtained.
[0101] The square filter kernel 334 is computationally advantageous
since its edges align with the x- and y-coordinate directions of
the analysis image 330. A round filter kernel 334' or
otherwise-shaped kernel is optionally used in place of the square
filter kernel 334. However, the round filter kernel 334' is more
computationally expensive than the square filter kernel 334.
Another advantage of the square filter kernel 334 compared with the
round filter kernel 334' is that the total filter edge length of
the square filter 334 is reduced from twice the detection size to
1.414 times the detection size. This reduces edge effects, allowing
use of data that is closer to the edge of the analysis image
330.
[0102] The size of the filter kernel should be selected to
substantially match the expected image size of a dye-tagged cell in
the analysis image 330 to provide the best SNR improvement. For
example, the square filter is kernel 334 with a positive (+1)
region that is ten pixels across is expected to provide the best
SNR improvement for a cell image also having a diameter of about
ten pixels. For that matched case, the signal is expected to
increase by about a factor of 78 while the noise is expected to
increase by about a factor of 14, providing a SNR improvement of
about 5.57:1. On the other hand, the SNR improvement for a smaller
eight pixel diameter cell using the same square filter is expected
to be about 3.59:1. The SNR improvement for a larger fourteen pixel
diameter cell using the same square filter is expected to be about
3.29:1.
[0103] The matched filter processing 332 can be implemented in
various ways. In one approach, each point in the input image is
summed into all points in the output image that are in the positive
inner region. Then all the points in the output image that are in
the outer negative region but not in the inner positive region are
subtracted off. Each point in the input image is touched once,
while each point in the output image is touched the outer-box pixel
area count number of times.
[0104] In another suitable approach, for each point in the output
image, all points from the input image that are within the positive
inner box are read and summed. All points outside the positive
inner box but within the negative outer box are then subtracted.
While each output image pixel is touched only once, each input
image pixel is touched by the outer-box pixel count.
[0105] In another suitable approach, two internal values are
developed for the current row of the input image: a sum of all
points in the row in the negative outer box distance, and a sum of
all points in the row in the inner positive box distance. All
output image column points at the current row have the input image
sum of all points in the outer-box subtracted from them. All the
output image column points within the inner positive box get the
sum of the input image row points in the inner positive box
distance added in twice. The row sums can be updated for the next
point in the row by one add and one subtract. This reduces the
execution cost to be on the order of the height of the filter
box.
[0106] In the matched filter processing 332, various edge
conditions can be employed. For example, in one approach, no output
is produced for any point whose filter overlaps an edge of the
analysis image 330. This approach avoids edge artifacts, but
produces an output image of reduced usable area. In another
suitable example edge condition, a default value (such as zero, or
a computed mean level) is used for all points off the edge.
[0107] With continuing reference to FIG. 19, binary thresholding
processing 338 is applied after the matched filtering 332. A
difficulty in performing the thresholding 338 is selection of a
suitable threshold value. Threshold selection is complicated by a
likelihood that some analysis images will contain no cells, or only
a single cell, or only a couple or few cells. In one approach, a
the threshold is selected as a value that is a selected percentage
below the peak pixel intensity seen in the filtered data. However,
this threshold will cause noise to be detected when no cells are
present, since in that case the peak pixel value will be in the
noise. Another approach is to use a fixed threshold. However, a
fixed threshold may be far from optimal if the background intensity
varies substantially between analysis images, or if the matched
filtering substantially changes the dynamic range of the pixel
intensities.
[0108] In the illustrated approach, the threshold is determined by
processing 340 based on the SNR of the unfiltered analysis image
330. By first determining the standard deviation of the input
image, the expected noise at the filter output can be computed. The
noise typically rises by the square root of the number of pixels
summed, which is the outer-box area in pixel counts. In some
embodiments, the threshold is set at approximately 7-sigma of this
noise level. As this filter does not have an exact zero DC
response, an appropriate mean level is also suitably summed to the
threshold.
[0109] The thresholding 338 produces a binary image in which pixels
that are part of a cell image generally have a first binary value
(e.g., "1") while pixels that are not part of a cell image
generally have second binary value (e.g., "0"). Accordingly,
connectivity processing 344 is performed to identify a connected
group of pixels of the first binary value corresponding to a cell.
The connectivity analysis 344 aggregates or associates all first
binary value pixels of a connected group as a cell candidate to be
examined as a unit. The center of this connected group or unit can
be determined and used as the cell location coordinates in the
candidate cell tag.
[0110] With reference to FIG. 22, a suitable embodiment of the
optional user verification processing 320 is described. A tag is
selected for verification in a selection operation 350. In a
display operation 352, the area of the analysis image containing
the candidate cell tag is displayed, optionally along with the
corresponding area of analysis images adjacent in depth to the
analysis image containing the candidate cell. Displaying the
analysis images that are adjacent in depth provides the reviewing
human analyst with additional views which may fortuitously include
a more recognizable cell image than the analysis image in which the
automated processing 306 detected the cell candidate. The human
analyst either confirms or rejects the candidacy in operation 354.
A loop operation 356 works though all the candidate cell tags to
provide review by the human analyst of each candidate cell. The
statistical analysis 322 operates on those cell candidate tags that
were confirmed by the human analyst.
[0111] Example data acquisition and analysis processing has been
described with reference to FIGS. 16-22 in the context of
quantitative buffy coat analysis using the annular sample in the
annular gap 12 between the test tube wall 14 and the float wall 16.
However, it will be appreciated that the processing is readily
applied to other sample scanning approaches, such as the scanning
of the planar sample slide 60 depicted in FIG. 4.
[0112] The example embodiments principally relate to quantitative
buffy coat analysis. However, it will be appreciated that the
apparatuses and methods disclosed herein are applicable to other
types of bioassays. For example, the cells can be stained rather
than fluorescently tagged, or the cells may have an intrinsic
optical signature (fluorescence, contrast, or so forth) that
enables assessment by optical microscopy. The features assessed may
be other than rare cells. For example, the assessed features may be
cell fragments, bacteria, or multi-cellular structures. The sample
may be a biological sample other than a buffy coat sample.
[0113] The invention has been described with reference to the
preferred embodiments. Obviously, modifications and alterations
will occur to others upon reading and understanding the preceding
detailed description. It is intended that the invention be
construed as including all such modifications and alterations
insofar as they come within the scope of the appended claims or the
equivalents thereof.
[0114] Having thus described the preferred embodiments, the
invention is now claimed to be:
* * * * *