U.S. patent application number 09/795530 was filed with the patent office on 2001-08-30 for transformation of digital images.
Invention is credited to Hendrickson, Andrew, Herrera, Benn P., Kerschmann, Russell L..
Application Number | 20010017938 09/795530 |
Document ID | / |
Family ID | 23208478 |
Filed Date | 2001-08-30 |
United States Patent
Application |
20010017938 |
Kind Code |
A1 |
Kerschmann, Russell L. ; et
al. |
August 30, 2001 |
Transformation of digital images
Abstract
In general, the invention consists of a means for staining a
sample with a fluorescent dye, a means for producing a darkfield
image of the fluorescent stained sample, and a means of
transforming an image of a sample stained with one or more
darkfield dyes into an image stained with one or more brightfield
dyes for examination on a computer monitor. The process includes
applying a digital lookup table or other computational means in
order to convert the darkfield data to brightfield forms, and a
means of displaying said transformed information. Preferably, the
imaging means is a block face microscope, and the means for
transforming the images is a digital computer.
Inventors: |
Kerschmann, Russell L.;
(Mill Valley, CA) ; Hendrickson, Andrew; (San
Francisco, CA) ; Herrera, Benn P.; (San Francisco,
CA) |
Correspondence
Address: |
CLARK & ELBING LLP
176 FEDERAL STREET
BOSTON
MA
02110-2214
US
|
Family ID: |
23208478 |
Appl. No.: |
09/795530 |
Filed: |
February 27, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09795530 |
Feb 27, 2001 |
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09311789 |
May 13, 1999 |
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6195451 |
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Current U.S.
Class: |
382/133 |
Current CPC
Class: |
G06T 5/50 20130101 |
Class at
Publication: |
382/133 |
International
Class: |
G06K 009/00 |
Claims
What is claimed is:
1. An image production method comprising: a) producing a first
image of a sample; and c) using a digital processor to convert said
first image to an image that mimics a brightfield image of said
sample.
2. The method of claim 1, wherein said first image is produced
using a block-face microscope.
3. The method of claim 1, wherein said converting of said first
image to said second image comprises applying a lookup table to
said first image.
4. The method of claim 1, wherein said sample comprises two
fluorescent components.
5. The method of claim 1, wherein said sample is a biological
sample.
6. An image production method comprising: a) producing a first
image of a sample and a second image of said sample; and c) using a
digital processor to convert said first and second images to a
third image that mimics a brightfield image of said sample.
7. The method of claim 6, wherein said first and second images are
produced using a block-face microscope.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of and claims priority
from U.S. Utility Application No. 09/311,789, filed May 13, 1999
(now pending), hereby incorporated by reference.
COMPUTER PROGRAM LISTING APPENDIX
[0002] The attached computer program listing appendix, which
includes 42 pages of computer code (in the computer language C) for
the program described herein, is hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
[0003] The invention relates to the field of histology.
[0004] In present day practice, the preparation of organic tissue
samples and other material for transmission microscopy, both
visible light and electron microscopy, is normally carried out by
subjecting the sample to a series of chemical treatments
culminating in the production of a solid block in which the sample
is embedded. After the block is produced, thin sections of the
sample (with the surrounding embedding material) are cut from the
block and transferred to glass slides or other support. The
embedding material may then be chemically removed and the tissue
section stained with a variety of colored or fluorescent dyes,
immunohistochemical stains, or subjected to in situ hybridization
prior to examination.
[0005] In conventional histopathology, the most common brightfield
stain applied to clinically important tissue sections is the
hematoxylin and eosin (H&E) formulation. This method results in
staining of nucleic acids and other so-called "basophilic"
substances in the tissue section with a blue-purple coloration, and
proteins and other "acidophilic" or "eosinophilic" tissue
components with a pink coloration. This stain is used world-wide as
a general screening method for the examination of all tissue
components, to be followed in certain cases by special stains that
have affinities for specific tissue elements such as microorganisms
or nerve processes, and therefore enhance their appearance on
stained tissue sections.
[0006] Methods have been introduced for en bloc staining, wherein
the entire sample is stained by immersion before being subjected to
infiltration and embedment. Sections are then cut from the block
for transmission microscopy, or the cut face of the block itself is
imaged in a process called block face microscopy or surface imaging
microscopy. In the latter method, including that implemented in
U.S. Pat. No. 4,960,330, a sample that has been stained en bloc
with either conjugated or unconjugated fluorescent dyes is
subsequently infiltrated by and embedded in a medium, commonly a
plastic polymer, that is heavily opacified or otherwise treated to
allow for the suppression of images of tissue originating from more
than a small number of microns deep within the block. This results
in the production of a thin, "virtual section" closely resembling a
conventional glass-slide mounted tissue section.
[0007] Block face microscopy is advantageous over standard
brightfield microscopy in that block face methods allow for the
generation of high-quality microscopy images of biological tissue
and other materials without the need to manufacture glass histology
slides. The elimination of this requirement permits full automation
of the histopathologic process, reducing incremental costs for each
additional section produced, and consequently allowing for much
greater amounts of information to be collected from each
sample.
[0008] In block face microscopy, the digital virtual section as
captured unmodified from the block face is a dark field image
resulting from the colored emissions from the fluorescence-stained
sample appearing against a black background representing the
opacified polymer in which the sample is infiltrated and embedded.
In contrast, conventional optical transmission microscopy,
including that practiced in most surgical pathology laboratories
and other medically-related microscopy-based diagnostic facilities,
produces a brightfield image because thin slices of tissue and
other material are stained with standard non-fluorescent dyes and
are then trans-illuminated with a white or near-white light source,
resulting in a background that is brighter, rather than darker than
the tissue image.
[0009] In order to optimize block face microscopy images for
clinical diagnosis and other purposes, it is preferable that the
raw darkfield images captured from the face of the block be
transformed and displayed as the more familiar images encountered
in brightfield microscopy.
SUMMARY OF THE INVENTION
[0010] In general, the invention consists of a means for staining a
sample with a fluorescent dye, a means for producing a darkfield
image of this fluorescent stained sample, and a means of
transforming the darkfield images into bright field images for
examination on a computer monitor. The process includes applying a
digital lookup table or other computational means in order to
convert the darkfield data to brightfield forms, and a means of
displaying the transformed information. Preferably, the imaging
means is a block face microscope, and the means for transforming
the images is a digital computer.
[0011] Accordingly, in a first aspect, the invention features an
image production method that includes: (a) staining a sample with a
fluorescent dye; (b) producing a first image of the resultant
fluorescent-dyed sample; and (c) using a digital processor to
convert the first image to a second image that mimics an image of
the sample stained with a non-fluorescent dye.
[0012] In a preferred embodiment, the converting of the first image
to the second image includes applying a lookup table to the first
image. In preferred embodiments, the modifying includes inverting
the lookup table, or adjusting the color ranges of either the first
image or the second image to mimic the color ranges of the sample
stained with a non-fluorescent dye.
[0013] The sample can be stained with one dye or with two or more
dyes. A preferred sample is a biological sample.
[0014] The invention also features an image production method that
includes: (a) staining a sample with a first and a second
fluorescent dye; (b) producing a first image of the sample stained
with the first dye and a second image of the sample stained with
the second dye; and (c) using a digital processor to convert the
first and second images to a third image that mimics an image of
the sample stained with hematoxylin and eosin.
[0015] The invention features an image production method that
includes: (a) staining a sample with a fluorescent dye; (b)
producing a first image of the sample stained with the dye and a
second image of the sample stained with the dye; and (c) using a
digital processor to convert the first and second images to a third
image that mimics an image of the sample stained with hematoxylin
and eosin.
[0016] The invention also features an image production method that
includes: (a) staining a sample with the plurality of fluorescent
dyes; (b) producing a first image of one of the dyes in the sample;
and (c) using a digital processor to convert the first image to a
second image that mimics an image of the sample stained with one
non-fluorescent dye.
[0017] In another aspect, the invention features an image
production method that includes: (a) staining the sample with the
plurality of fluorescent dyes; (b) producing a first image that
mimics the sample stained with a subset of fluorescent dyes; and
(c) using a digital processor to convert the first image to a
second image that mimics an image of the sample stained with one or
more non-fluorescent dyes.
[0018] Preferably, the first and second images of the invention are
produced using an apparatus that includes a block-face microscope.
The dye used in the invention can include a metachromatic dye
(e.g., acridine orange).
[0019] By "conjugated" dye is meant a dye that is bound to a
molecule having aspecificity for tissue elements. Exemplary
conjugated dyes include, without limitation, fluorescently-coupled
antibodies and fluoresc dna probes.
[0020] By "unconjugated" dye is meant a dye that is inherently
fluorescent.
[0021] By "infiltration" is meant treating the tissue with a liquid
or series of liquids which penetrate throughout the tissue to the
molecular level and are then transformed into a solid in order to
render the sample rigid.
[0022] By "embedding" or "embedment" is meant positioning the
infiltrated tissue in a mold and surrounding it with a substance
(usually the same as the infiltrating substance) which is then
hardened to form an encasing block. The embedding substance thus
serves to provide rigid support and to facilitate the cutting
process.
[0023] By "sectioning" is meant cutting from the block thin slices
which may then be mounted on glass slides or other support.
[0024] By "staining" is meant treating a material with a colored or
fluorescent substance that associates with the material on the
molecular level.
[0025] "Fluorescence" or "darkfield" staining is accomplished using
unconjugated dyes (i.e., dyes that are naturally fluorescent when
excited with light of the proper wavelength) or conjugated dyes
(i.e., molecules that bind to the sample and that are attached,
either directly or indirectly, to a fluorochrome). Examples of
conjugated dyes include without limitation: (i) a primary antibody
that binds to an antigen and a secondary antibody, containing a
fluorochrome, that binds to the primary antibody; and (ii) a
molecule that has, covalently bound to it, a fluorochrome.
Fluorescence staining results in images that have a black
background, while "standard" or "brightfield" staining is
accomplished using dyes that are usually non-fluorescent and
results in images with a bright, or white background. It is
understood that the same dye could be useful for both fluorescence
and standard microscopy.
[0026] "Metachromatic" dyes constitute a subset of histochemical
stains, many of which are fluorescent. Like most fluorochromes,
these compounds absorb light of a specific wavelength and re-emit
it at a longer wavelength or wavelengths. The spectral properties
of metachromatic dyes are strongly influenced by their proximity to
target molecules, such that their emission wavelength is altered.
Thus, these dyes change colors when they combine with certain types
of materials. In some cases, the metachromatic dye may emit light
only when bound to its target, and is otherwise not visible. An
exemplary metachromatic dye is acridine orange. Other metachromatic
dyes are known to those skilled in the art and include, without
limitation, those listed in Conn's Biological Stains, Williams
& Wilkens 9th Ed. 1977 (hereby incorporated by reference), and
those available from Molecular Probes, Inc. (Eugene, Oreg.). Most
of the latter dyes are the subject of issued U.S. patents, all of
which are hereby incorporated by reference. The Molecular Probes
metachromatic dyes, which tend to label proteins, are catalogued as
falling into the following three categories: bezoxadiazole
derivatives, napthalene derivatives, and pyrene derivatives.
[0027] By this method, image data on biological tissue and other
materials may be generated efficiently by means of block face
microscopy, and displayed in the most useful and familiar form as
bright field images.
[0028] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWING
[0029] The FIGURE is a schematic illustration of the method of the
invention, including applying a lookup table to a first image to
yield a second image, yielding modified color.
DETAILED DESCRIPTION OF THE INVENTION
[0030] We have discovered a method for imaging a sample, such as a
tissue sample, with one or more darkfield dyes and transforming the
image to a second image that mimics an image of a sample stained
with one or more brightfield dyes. The method of transforming a
first image to a second image can involve applying a lookup table
to the first image.
[0031] Lookup tables
[0032] The transformation of a first image to a desired second
image generally uses a collection of conversion values referred to
as a lookup table. A lookup table consists of a list of color value
relationships describing a one-to-one transform and may be
generally computational according to certain criteria set by the
user of the imaging system. Means for transforming digital images
include general methods such as transfer functions that apply a
mathematical formula to each data element. Examples include image
color inversion transformations that may act by subtracting all
data elements from some constant value.
[0033] Alternatively, each data element in the image may be altered
according to some higher-order computed result based upon analyses
of patterns or other features in the image. For example, the value
of every data element in some region of interest within an image
may be summated, and this total value used to determine the general
color of the region as it is displayed. Methods involving such
higher level computations to determine a stain simulation are
referred to in the invention as "computed stains."
[0034] In general, a lookup table, such as an RGB (red, green, and
blue) lookup table, is applied to each channel (e.g., the red
channel, the green channel, etc.) of the darkfield image. In the
course of converting the image from a darkfield image to a
brightfield image, each source darkfield sample value (e.g., each
pixel) is used as an index to reference the lookup table in order
to ascertain the corresponding value for the brightfield image. The
colors obtained for each channel are then combined to create the
RGB brightfield image. Methods for color mapping and described, for
example, in J. C. Russ (1995) The image processing handbook. 2nd
edition CRC Press, Ann Arbor, Mich., hereby incorporated by
reference.
[0035] Additive colorspace conversion
[0036] In one example, a table of RGB colors is created with one
entry for each possible darkfield value. For example, if the source
(i.e., the darkfield image) has a range from 0 to 255 (wherein 0 is
black and 255 is white), a table of 256 entries is created. Each
entry in the table is filled with a color that can be obtained as
follows:
[0037] H=target color hue
[0038] S=target color saturation
[0039] B=target color brightness*I/max_sample_value (e.g.,
8-bit=255)
[0040] where I is the index of the current lookup table value being
calculated, and ranges from 0 to max_sample value (e.g., 255). The
resulting HSB color is converted to RGB colorspace and is inserted
into the lookup table at position I.
[0041] The result is that each lookup table entry is a
brightness-scaled version of the target brightfield image color.
The colors in the table span a range from black, ascending in
brightness up to the last entry, which is the unmodified target
image color.
[0042] For example, if the target color were primary red, then the
lookup table would start at black, gradually increase from darker
reds to brighter reds, to the last entry, which would be primary
red.
[0043] For each set of source channels, the RGB colors obtained
from the lookup table are added (e.g., red to red, green to green,
blue to blue) to obtain one RGB color. The values are limited so
that the max_sample_value (e.g., 255 for 8-bit) is not exceeded. A
brightening value is then added to each RGB value. This value is
obtained by taking the "darkness" of the original, darkfield image
and converting it as follows:
[0044] max_sample_value--maximum(source channel 1, source channel
2, . . . )
[0045] The largest of the source sample values is subtracted from
the max_sample_value to obtain a darkness level. The darkness level
is added to each component of the new RGB color to brighten it by
that amount. The result is that source sample values that were very
dark become very bright.
[0046] Subtractive colorspace conversion
[0047] In a second method for converting a darkfield image to one
resembling a brightfield image, a lookup table of RGB colors is
created as described above, except that each entry in the table is
filled with a color that can be obtained as follows:
[0048] H=target color hue
[0049] S=target color saturation*I/max_sample_value (e.g.,
8-bit=255)
[0050] B=target color brightness
[0051] The resulting HSB color is converted to RGB colorspace and
is inserted into the lookup table at position I.
[0052] The result is that each lookup table entry is a
saturation-scaled version of the target brightfield image color.
The colors in the table span a range from white, ascending in
saturation up to the last entry, which is the unmodified target
image color.
[0053] For example, if the target color were primary red, the
lookup table would start at white, increasing from lighter reds
(pinks) to more saturated reds until the last entry, primary
red.
[0054] Thus, using the foregoing method, each source channel is
converted to an RGB color. These colors are blended as follows.
[0055] The RGB colors are converted to CMY (cyan, magenta, and
yellow) colors. CMY is subtractive color space. The CMY colors are
added (e.g., the cyan values from each source channel are added to
produce a single cyan value, the magenta values from each source
channel are added to produce a single magenta value, etc.). As
described herein, it is desirable to restrict the values to the
maximum allowed (i.e., the max_sample_value). Following summation,
the resulting CMY color is reconverted to RGB colorspace.
[0056] In the case of the conversion of a darkfield image stained
with acridine orange to a brightfield image stained with H&E,
the increase is from black to the target color (in the case of
additive colorspace) or white to the target color (in the case of
subtractive colorspace) is linear. Those skilled in the art will
recognize that other lookup tables, including non-linear or even
discontinuous lookup tables, are also useful in the invention.
[0057] Staining with darkfield dyes
[0058] One method for computationally generating an image of a
sample stained with a brightfield dye or combination of dyes (e.g.,
H&E) is to stain the sample with a mixture of fluorochrome
stains that differentially bind to various tissue structures or,
alternatively, with a single fluorescent stain that produces more
than one color when applied to a sample (i.e., a metachromatic
dye). The image conversion methods described herein allow for
conversion of the darkfield image of a sample stained with one or
more darkfield dyes to mimic an image of the sample stained with
one or more brightfield dyes.
[0059] In one example, a sample containing fungal organisms is
stained with a combination of fluorescent dyes, one of which
displays higher affinity for the fungal organisms and another of
which displays higher affinity for components of the tissues. A
darkfield image of the sample stained with these dyes would reveal
fungal organisms labeled with a distinctive color relative to the
tissue. Upon conversion to a brightfield image (using standard
methods such as those described herein), the differential color
properties of the original image are transformed to a brightfield
image of tissue containing fungal organisms.
[0060] In a second example, a darkfield image of a sample
containing fungal organisms, which has been stained with a single,
monochromatic dye, is transformed to yield a polychromatic
brightfield image by assigning different brightfield colors to
various grayscale intensity values in the original darkfield image.
for example, if the fungal organisms display a greater affinity for
the monochromatic dye than does the tissue, the difference in
intensity can be used to yield one set of brightfield colors for
the brightly stained sample (i.e., the fungal organisms) and a
second set for the dimly stained sample (i.e., the tissue). Other
distinctive properties, such as size, can also be used to
computationally assign brightfield colors.
[0061] Encoding multiple stains in a single sample
[0062] A sample can be stained with multiple dyes, while only one
or a subset of these dyes is imaged. At another time, the other
dyes from the same sample can be imaged using different filter
cubes or excitation methods. Thus, one sample can contain many more
dyes than are imaged at one time.
[0063] Dyes that are imaged at the same time can be distinguished
from each other using filter cubes that restrict the wavelengths of
emitted light that reaches the detector. Alternatively, individual
dyes can be distinguished using spectral analysis or other higher
order analysis. Such methods for imaging tissue are described in
Levenson, R. M. and Farkas, D. L. (1997) Proc. SPIE, 2983: 123-135;
Levenson, R. M. and Young, D. A. (1991) In: M. J. Dunn (Editor),
Proc. International Meeting on Two-Dimensional Electrophoresis.
Dept. of Cardiothoracic Surgery, National Heart and Lung Inst.
(UK), London, UK; and Levenson, R. M., Brinckmann, U. G., Androphy,
E., Schiller, J., Turek, L., Chin, M., Broker, T. R., Chow, L. T.
and Young, D. A. (1987) In: B. M. Steinberg, J. L. Brandsma and L.
B. Taichman (Editors), Cancer Cells V. Papillomaviruses. Cold
Spring Harbor Press, New York, pp. 137-144, all of which are hereby
incorporated by reference.
[0064] The following examples are to illustrate the invention. They
are not meant to limit the invention in any way.
EXAMPLES
Example I
Single Dye Method for H&E
[0065] The metachromatic dye acridine orange is a fluorescent dye
in which the color of the fluorescence produced upon staining
tissue and other material is dependent on the chemical composition
of the tissue element to which it is bound. For example, nucleic
acids emit a yellow color upon staining with acridine orange, while
cytoplasmic proteins simultaneously emit a green color when so
stained. Thus, the color differentiation of tissue elements
resulting from staining of tissue and other materials with acridine
orange is similar to that seen with H&E, except that different
specific colors are produced, and in addition, acridine orange,
being a fluorescent stain, produces a darkfield, rather than a
brightfield, image.
[0066] A specific algorithm for making display data that appears as
a brightfield H&E stain from a fluorescent sample using
initially stained using acridine orange and captured with a digital
camera is as follows. The input image is digitized by the camera,
yielding pixels which, are black or only have red and green values.
The output pixels represent an RGB image that will appear as a
brightfield image stained with Hematoxylin and Eosin.
[0067] Let:
[0068] Sr, Sg=the source pixel values for red and green
[0069] Dr, Dg, Db=the destination pixel values for red green and
blue
[0070] Pmax=the maximum value of a pixel (i.e. 8-bit=28=256,
10-bit=210=1024, etc.)
[0071] Hr, Hg, Hb=the RGB indices of a Hematoxylin brightfield
color
[0072] Er, Eg, Eb=the RGB indices of an Eosin brightfield color
[0073] Dr=Pmax--max(Sr, Sg)+(Sr/Pmax)*Hr+(Sg/Pmax)*Er
[0074] Dg=Pmax--max(Sr, Sg)+(Sr/Pmax)/Hg+(Sg/Pmax)*Eg
[0075] Db=Pmax--max(Sr, Sg)+(Sr/Pmax)/Hb+(Sg/Pmax)*Eb
[0076] The invention thus makes possible the conversion of images
of acridine orange stained tissues into images resembling H&E
stained tissues by means of applying a color lookup table or other
means of color transformation to the images of acridine orange
stained tissue.
[0077] Those skilled in the art of H&E staining will recognize
that there is variability in the hue and saturation of tissue
stained with each component due to variations in the production or
storage of the staining solution. Thus, more than one look-up table
can be used to convert a darkfield image of a sample stained with
acridine orange to mimic a brightfield image stained with H&E.
Appendix A lists computer code (in the computer language C) for a
program that generates lookup tables for specified target-stained
colors suitable for transforming a darkfield image of a sample
stained with acridine orange to a brightfield image of the sample
stained with H&E.
Example II
Multiple Dye Method for H&E
[0078] Fluorescent stain mixtures that differentially color
basophilic and acidophilic substances in tissue may be substituted
for acridine orange, and suitable lookup tables or other means
created to transform the resulting darkfield images into those
which resemble standard brightfield H&E images. One alternative
formulation is a combination of propidium iodide (to stain nucleic
acids) and eosin Y (to stain proteins). In one example, a biopsy of
cancerous tissue is treated with propidium iodide, a yellow
fluorescent stain that binds in a quantitative manner to nucleic
acids, and is commonly used for the quantitation of DNA in flow
cytometric studies of tumor cell nucleic acid content. The tissue
is also stained with eosin Y to color green the non-nuclear
components of the tissue. According to the invention, a darkfield
image captured from this tissue sample is transformed into a
brightfield image in which the background color appears white, the
non-nuclear portion of the tissue appears pink, and the nuclei
appear blue. Alternatively, the fluorescence intensity of each
nucleus is quantitatively measured to approximate the DNA content
of the nucleus, which quantity relates by a color lookup table or
other means to the color to be applied to the brightfield image of
each nucleus, such that each nucleus will display a different color
according to the total amount of DNA contained therein, thus
resulting in a "computed stain" that relates to DNA content.
Example III
Method for producing a polychromatic image from a monochromatic
dye
[0079] In a third example, by use of the invention, an image of a
monochromatic stain is converted to a polychromatic image
resembling an image of a sample stained with a polychromatic stain
or multiple monochromatic stains. One example is the application of
the blue-white stain Fluorescence Brightener 28 to tissue samples
containing fungal organisms. Fluorescence Brightener 28 strongly
binds to fungal bodies and makes them appear much more prominent
than other structures in darkfield images, such as the cells of the
infected organism. Hence, in an image with pixels representing 256
shades of gray (in which a pixel value of 0 is black, and a pixel
value of 255 is white), the pixels representing the fungal bodies
have high pixel values.
[0080] In one example, the fungal bodies have a pixel value greater
than X, while the non-fungal structures each have a pixel value
less than X. A color lookup table is created to transform the more
brightly staining fungal bodies (i.e, all pixels greater than X)
into a first brightfield color such as dark purple-grey; the
remaining structures of the tissue (i.e., all pixels between X and
0) are transformed into a second brightfield color such as pale
green. Similarly, the black background (i.e., all pixels with
values of 0) is made to appear white, thus producing a brightfield
appearance. This coloration combination results in an image closely
resembling the Grocott methenamine-silver stain, which is commonly
employed in surgical pathology laboratories to stain fungus in
tissue sections.
OTHER EMBODIMENTS
[0081] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims. Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the claims.
* * * * *