U.S. patent number 4,887,892 [Application Number 07/035,822] was granted by the patent office on 1989-12-19 for method and method and apparatus for control of light intensity for image analysis.
This patent grant is currently assigned to Cell Analysis Systems, Inc.. Invention is credited to James W. Bacus.
United States Patent |
4,887,892 |
Bacus |
December 19, 1989 |
Method and method and apparatus for control of light intensity for
image analysis
Abstract
An image apparatus for measuring true mass characteristics of
the specimen in real time is provided with means for controlling
the amount and intensity of background light so that the amount of
light is held substantially constant and at a level providing
reduced background and scattered light so that evaluations at
different times on the same or on different image analysis
apparatus of the same manufacture result in substantially identical
measurements of mass. The light control for the conventional light
microscope used with the image analysis apparatus includes four
variables which are the light intensity of the light bulb, the size
of the field iris, the size of the condenser iris, and the movement
the condenser lenses in a vertical or "Z" direction for focus.
Preferably, a fixed aperture means in the form of a cup attachment
is secured to the condenser iris optics to provide a fixed size
iris for the condenser. The controls for the variable condenser
iris are opened wide so that the less size aperature in the fixed
condenser iris is that which is controlling of the light passing
through the condenser optics. The preferred attachment device is a
cup-shaped member of one piece metal which is attached by a
threaded fastener in a non-invasive manner to the bottom of the
condenser iris and optics. A monitor displays the intensity of the
background light as a numerical value and the intensity of the
light bulb is adjusted to provide a substantially constant
background light transmitted to a CCD sensor or camera.
Inventors: |
Bacus; James W. (Hinsdale,
IL) |
Assignee: |
Cell Analysis Systems, Inc.
(Lombard, IL)
|
Family
ID: |
25164139 |
Appl.
No.: |
07/035,822 |
Filed: |
April 8, 1987 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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927285 |
Nov 4, 1986 |
|
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|
794937 |
Nov 4, 1985 |
4741043 |
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Current U.S.
Class: |
382/133; 382/274;
356/39; 348/79; 348/131 |
Current CPC
Class: |
G01N
15/1468 (20130101); G06K 9/00127 (20130101); G06K
9/00134 (20130101); G01N 15/1475 (20130101); G01N
21/6458 (20130101); G01N 2015/1018 (20130101); G01N
2201/127 (20130101) |
Current International
Class: |
G01N
15/14 (20060101); G06K 9/00 (20060101); G02B
021/08 (); G06K 009/28 (); G01N 033/48 (); H04N
007/18 () |
Field of
Search: |
;250/205 ;382/6
;350/523,526,528,508,502 ;128/633 ;356/39 ;358/107 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Primary Examiner: Henry; Jon W.
Attorney, Agent or Firm: Fitch, Even, Tabin &
Flannery
Parent Case Text
This application is a continuation-in-part of copending patent
application Ser. No. 927,285, filed Nov. 4, 1986 which in turn is a
continuation-in-part of Ser. No. 794,937, filed Nov. 4, 1985, now
U.S. Pat. No. 4,741,043.
Claims
What is claimed is:
1. An apparatus using a light microscope for image analysis
comprising:
a CCD sensor having pixels receiving transmitted light having
passed through the specimen and background light and for providing
an optical density value based on received light at each pixel,
a light source for illuminating a slide having a specimen
thereon,
means to adjust the light source to control the intensity of light
being applied to the specimen on the slide and to the CCD
sensor,
a field iris for receiving light from the light source and a
control means for adjusting the size of the field aperture in the
field iris,
a condenser optics means including a variable condenser iris which
is adjusted to control the amount of light passing therethrough
from the cone of light having passed through the field iris,
a means for adjusting the variable condenser iris between a wide
open and a substantially closed position,
said condenser optics means being movable in a 37 Z" direction to
adjust the focus of the light passing from the lenses and iris,
means for supporting a slide having a specimen for receiving
background light on one side of the slide and for providing
transmitted light and background light on the opposite side of the
slide for application to the face of the CCD sensor,
means to fix the condenser iris at a predetermined size to assist
in reducing the intensity and amount of light to a relatively
constant amount with a limited amount of light scatted at the face
of the CCD sensor,
a computer connected to the CCD sensor to provide measured values
of the background light, and
a monitor connected to the computer for displaying the actual value
of the background light so that the operator may adjust the means
for adjusting the intensity of the light source to provide a
numerical constant value for the background light.
2. An apparatus using a light microscope for image analysis
comprising:
a CCD sensor having pixels receiving transmitted light having
passed through the specimen and background light and for providing
an optical density value based on received light at each pixel,
a light source for illuminating a slide having a specimen
thereon,
means to adjust the light source to control the intensity of light
being applied to the specimen on the slide and to the CCD
sensor,
a field iris for receiving light from the light source and a
control means for adjusting the size of the field aperture in the
field iris,
a condenser optics ;means including a variable condenser iris which
is adjusted to control the amount of light passing therethrough
from the cone of light having passed through the field iris,
a means for adjusting the variable condenser iris between a wide
open and a substantially closed position,
said condenser optics ;means being movable in a "Z" direction to
adjust the focus of the light passing from the lenses and iris,
means for supporting a slide having a specimen for receiving
background light on one side of the slide and for providing
transmitted light and background light on the opposite side of the
slide for application to the face of the CCD sensor,
means to fix the condenser iris at a predetermined size to assist
in reducing the intensity and amount of light to a relatively
constant amount with a limited amount of light scatter at the face
of the CCD sensor,
the means to fix the iris at said predetermined size comprising a
detachable member which has a fixed diameter opening, the size of
the fixed condenser iris opening being a predetermined fraction of
the size of the condenser variable iris when the condenser variable
iris is fully opened.
3. An apparatus in accordance with claim 2 in which the fixed iris
opening is about one-third less in size than the maximum wide open
size for the variable iris.
4. An apparatus in accordance with claim 3 which said detachable
member is a one-piece generally cup-shaped member having an opening
in a top wall and having an attachment flange which may be secured
to and detachably removed from the condenser optics.
5. An apparatus in accordance with claim 4 in which said cup-shaped
member has a bottom wall with a threaded screw hole therein, and in
which said cup shaped member has an opening whereby a threaded
screw may be passed through the opening into the threaded hole so
that the screw may readily attach and hold the cup-shaped member in
position.
6. An image display apparatus for a slide for a specimen including
in combination:
a light microscope having a light bulb and control for light
intensity,
said light microscope having a variable condenser iris and a
control for varying the size of the condenser iris,
said light microscope having a field iris and a control for
adjusting the size of the field iris to provide a cone of light
passing from the light bulb into and through the condenser
iris,
condenser optic means including condenser lenses for condensing the
light and for passing the light unto a transparent slide to provide
the background light for the slide and for a specimen on the
slide,
a CCD camera having pixels each receiving the transmitted light
having passed through the specimen and for receiving the background
light,
means for transforming the analog signals provided from the camera
into log transforms providing an optical density value for each of
the pixels,
a digital processing means for performing a true accurate
measurement in actual mass units from the log transforms
values,
means operable by the digital computer for converting the log
transforms numbers back into real time digital signals,
display monitor means connected to said means for conversion to
receive the digital signals and to provide a real time image of the
specimen for review, and
means in said digital computer for providing a background light
intensity value on said display monitor means so that the operator
may adjust the intensity of the light to a predetermined value for
the background light at the slide.
7. An apparatus in accordance with claim 6 in which a cup having a
fixed aperture is attached to the variable condenser lenses,
said variable condenser iris being capable of being opened by the
operator to a size larger than the fixed aperture size so that the
latter controls the amount of light being applied as background
light to the slide.
8. An apparatus in accordance with claim 7 in which a fastening
means fastens the fixed iris member for easy attachment and removal
from the condenser means.
Description
This invention relates to a method of and apparatus for measuring
cells with image analysis apparatus and more particularly to an
improved light control method and apparatus for use in such image
analysis apparatus.
The present invention is of particular use in a cell measuring
apparatus using image analysis as disclosed in co-pending patent
application Ser. No. 927,285, filed Nov. 4, 1986, which is hereby
incorporated by reference as if fully reproduced herein. The
present invention is not however limited to this specific apparatus
disclosed in the aforesaid application because it has utility with
other image analysis devices. The aforementioned patent application
discloses in considerable detail an apparatus which measures the
true optical density in mass units of a material in a cell such as
DNA or hemoglobin in real time using a light microscope and a CCD
camera which is receiving the image of the cell or cell nucleus for
measuring DNA content in the cell nucleus or measuring the amount
of hemoglobin present in red blood cells. The apparatus is also
capable of detecting and measuring other materials associated with
cells besides hemoglobin and DNA. The image analysis apparatus is
to be used by pathologists or others who may not be highly trained
in the use of such equipment so as to know how to control the light
and the image reaching the CCD camera to produce the desired
accurate, true mass unit measurements of DNA, hemoglobin, or other
materials.
The present invention addresses the problem of how to mass produce
cell measuring apparatus using image analysis employing a
conventional, low cost, light microscope to illuminate the cells
and to provide an enlarged image to the face of a CCD camera and to
eliminate error in the mass measurements due to a high quantity of
glare or background light being associated with the image or due to
a non-uniform amount of light being used from one time to the next
time or from one apparatus to another apparatus. The problem
involves how to provide a uniform glare or background light at a
minimal level so that the scattered light hitting the image area on
the CCD camera is at a constant and a minimum or tolerable level.
If the scattered light reduces the optical density significantly,
then the true mass of the material in the cell may differ
substantially from the mass which will be measured from the reduced
density image.
Stated differently, the function of the light and optics in the
image analysis apparatus is to map accurately the cell or nucleus
image on the CCD camera and this is achieved by limiting the amount
of light and the amount of scattered light that hits the CCD face.
More specifically, due to the characteristics of the optics and the
tubes involved, some scattered and background light will reach the
pixels on the CCD camera face at which the pixels display an image
resulting from the optical absorption of the mass of the material
on the slide. The photons of scattered light which hit the pixels
showing the cell are thus weakened and produce an optical density
measurement less than is accurate. As will be explained, the
present invention is initially calibrated to correct for a minimal
amount of glare and it is desired that the apparatus be used at
this calibrated or correct glare setting.
With the present invention, the optical density measured for a cell
image is equal to the sum of the light measurements, that is the
light absorption from all the pixels from a cell or specimen
mounted on a transparent glass slide in the microscope. The amount
of transmitted light through the cell mass or object may be defined
as T = I.sub.t /I.sub.o where I.sub.o is the light about the cell
which has been produced by the light source and controlled by the
field diaphragm, the condenser diaphragm, and condenser optics
before hitting the microscope slide on which the cell is mounted in
the aforesaid apparatus. As the thickness of the cell object
increases, the transmitted light T falls off exponentially. To
avoid the complicated calculations for thickness based on
exponential calculations, the present invention, as more fully
disclosed in the co-pending application, uses the log transform in
which optical density (O.D.) equals -log [T]. The log transforms
are arranged in look-up tables in the digital computer and numbered
1 to 256 so that the output of the optical density from the CCD
camera for a given pixel receives a digital number of between 1 to
256 and this is applied to a look-up table which provides an output
log transformation and thereby an actual true optical density value
to the digital computer. The look-up tables incorporate therein a
glare correction to provide a calibrated optical density value
which allows a true optical density reading for the specimen when
the glare is at the calibrated setting. From the foregoing, it will
be seen that the log transformation assumes that the background
light I.sub.o is consistent because it is used as a constant
denominator in the expression T = I.sub.t /I.sub.o. Unless the
background light is carefully controlled to a substantially
constant value, the same image on a slide could have different mass
measurements from time to time on the same apparatus or two
different apparatus having different background light. On the other
hand, if the same slide is used in two different image apparatus of
this type, each having the background light control of this
invention, both apparatus should produce the same accurate and true
mass units of hemoglobin or DNA.
Another factor involved in the accuracy of the actual mass units of
DNA or hemoglobin being measured is that of light being passed
through narrow band pass filters at wave lengths other than the
peak wave, e.g. absorbed light for hemoglobin at a wave length 410.
By a band pass width of 10 nanometers. The narrow band way of
example, the light filters described herein have pass filter for
DNA measurement has a 630 nanometer peak and a 10 nanometer narrow
band pass. The width of the band pass filters are measured herein
as being 10 nanometers wide at about 50 percent of the transmission
peak when measured against the wave length in nanometers. Because
these filters are not perfect in screening out all but 410 or 630
nanometer light, light at other wavelengths hits the CCD camera and
there is response to this light wavelength because the CCD camera
responds to light wave lengths in the range of 400 to 800
nanometers. Thus, it is desirable to reduce the amount of light
being supplied because not all of the light has been filtered out
by the filters. Stated differently, the more light that is let in
by the bypass band filters, the more the light not at the selected
peak wavelength is present to reduce the accuracy of the true mass
units being measured.
In laboratory conditions where experts are employed to control the
amount of light, they have a number of choices, including the
changing of the lights to control its intensity or the use of
different objective condenser lenses so as to use more accurate
light sources and lenses. However, when it is desired to use a
commercial and conventional light microscope in order to reduce the
cost of the system, it is desired not to have a large number of
different types of objective lenses or to have changes in light
sources or other variables with which the user may not be familiar
and may be unable to manipulate properly. With conventional light
microscopes, there are four sources of light variation which are
(1) the field diaphragm which changes the size of the cone of light
in the typical light microscope; (2) a condenser optic or optics
assembly which is movable in the Z or vertical direction to change
the focus; (3) a condenser iris or diaphragm which is controlled in
size by an adjustable knob, and (4) a light intensity control for
changing the intensity of light from the light source. The
condenser iris opening is generally reduced by about one-third from
the maximum variable opening for the usual sizes of condenser
lenses provided with the light microscope. However, the operator
has nothing but manual vision as a guide in determining the size of
the iris opening; and this is the most difficult variable to
control of the four variables. The area of the opening of the
condenser iris varies with the square of the radius of the opening
so that minor differences in estimating the size of the iris
opening diameter can result in significantly varying amounts of
light passing through the condenser iris. Thus, the varying amounts
of glare could, if not properly adjusted, close to the glare
correction already in the look-up tables, result in error in the
optical density readings for the specimen cells.
In accordance with an object of the present invention, there has
been provided a new and improved, simplified control for the light
used in image analysis apparatus for making true measurements of a
cell constituent such as hemoglobin, DNA, or other materials.
Another object of the invention is to provide a low cost attachment
to the apparatus disclosed in the aforesaid application which
allows the control of the condenser iris and the amount of
background light being transmitted.
A general object of the invention is to provide a new and improved
light control method and apparatus in an image analysis cell
measuring apparatus .
These and other objects and advantages of the invention will become
apparent from the following detailed description, taken in
conjunction with the accompanying drawings in which:
FIG. 1 is a view of the preferred apparatus having a light
microscope and being operated in accordance with the novel features
of the invention.
FIG. 2 is an enlarged view of the condenser optics and the slide on
a stage of the light microscope shown in FIG. 1.
FIG. 2A is a functional block diagram of the image analysis system
of FIG. 1.
FIG. 3 is a diagrammatic illustration, of the CCD camera connected
to the look-up tables and the digital computer and to an image
display.
FIG. 4 is an enlarged fragmentary view of the microscope including
operating controls for making adjustments.
FIG. 5 is a diagrammatic illustration of the light controls for
providing an image on the face of the CCD sensor.
FIG. 6 is a graph showing absorption versus hemoglobin for a narrow
pass band filter used with the apparatus shown in FIG. 5.
FIG. 7 shows that the CCD sensor will be affected by light from 400
through 800 nanometers.
FIGS. 8, 9, 10 and 11 depict a condenser lens optics with a fixed
iris attachment.
As shown in the drawings for purposes of illustration, the
invention is embodied in a method and apparatus which is fully
disclosed in Patent Application 927,285, filed Nov. 4, 1986,
entitled "Analysis Method and Apparatus Method for Biological
Specimens," the application and is incorporated by reference, and
may be referred for further detailed description of the apparatus
shown and described hereinafter.
As shown in the drawings for purposes of the illustration, the
invention is embodied in an image display apparatus, such as shown
in FIG. 1, which includes a conventional light microscope 15 of a
generally commercially available type such as sold by the Reichert
Company. As best seen in the FIGS. 1, 2 and 2A the microscope
includes a stage or platform 11 on which is mounted the glass slide
or support 14 which is preferably of a transparent glass material
and is a common, commercially available specimen slide used for
medical analysis. The slide 14 will typically contain a specimen 20
in the form of a cell which is being analyzed for a specific mass
of materials such as DNA for a ploidy analysis for cancer or the
amount of hemoglobin in a red blood the cell.
As best seen in FIGS. 2A and 5, the light imaging system for the
specimen 20 on the slide 14 includes an adjustable intensity or
variable light source 17 which supplies light through field optics
19 which includes a variable field iris 22A which may be adjusted
in size by operation of a control such as a control knob 21 shown
in FIG. 4 which has a conventional wire leading back to the iris
22A and its control nut (not shown) which adjusts the size of the
opening 22 (FIG. 5) for the field diaphragm or iris 18. Light
having passed through the field optics 19 is then directed to the
condenser optics 24 which includes an adjustable diaphragm or iris
device 25 and condenser lenses 26 which are moveable vertically or
in the Z direction by turning an outer adjustment ring 27 (FIG.
9).
The condenser iris or diaphragm device 25 is shown in considerable
detail in FIGS. 8-11 and includes a central variable iris 28
through which the light passes between a series of leaves 29 which
may be opened or closed to change the shape or the dimension of the
variable iris opening 28 as will be described in greater detail
hereinafter. At the front of the microscope is a suitable control
32 (FIG. 4) such as a knob labeled condenser the turning of which
adjusts the position of the leaves 29 and the size of the variable
aperture 28 through which the light passes into the lens 26 and
then passes to the slide 14 on which is located the specimen 20.
The image on the slide and the background light passes through an
objective lens 35 and then passes to a beam splitter 31 (FIG. 2A)
such as a prism which passes the light onto the television camera
or CCD sensor 18 and up to the microscope eyepieces 23A. An image
38 of the cell specimen 20 is shown diagrammatically on the face 39
of the CCD sensor in FIG. 5; and it is this image which will be
generating the analog signal which will be then converted to a
digital signal which is representative of the mass of the substance
being analyzed in the specimen such as DNA or hemoglobin or other
material as will be hereinafter described.
The apparatus shown in FIG. 1 and described in the aforesaid patent
application measures actual mass in actual true units such as
picograms and reports out the same and it is important that the
accuracy of the equipment be such that each of the various machines
in the field if given the same slide would report out the same mass
for the same cell being analyzed and that this mass be actual and a
true mass of the constituent such as DNA or hemoglobin rather than
a relative number. The problem with providing such consistent
results and accuracy from machine to machine and from location to
location using different operators at the various installations,
such as in different pathology laboratories throughout the country,
is that the light control used has a significant effect on the
optical density of the image 38 on the face of the CCD sensor
18.
More specifically, the operator using the equipment may adjust the
control 33 which is a knob labeled "intensity" in FIG. 4 to adjust
the voltage for the lamp 17 which thereby controls the intensity of
the light supplied to the optics. The operator at the station may
also adjust the size of the field diaphragm by turning the knob 21
labeled diaphragm which is connected by a wire to the leads for the
iris 22 of the field optics 19. The cone of light from the field
iris is reduced in size to that which is the smallest needed to
fully illuminate the specimen 20 so that there is a limited amount
of background light being passed through the optics to the face of
the CCD sensor 18. The cone of light, having been set at a
particular size, is then passed through the condenser optics 24 at
which is located the condenser iris which is controlled by the
condenser variable knob 32 which is adjusted down to approximately
one-third of the maximum opening, in this instance, by turning the
control knob to move the iris leaves 29 inwardly, as shown in FIG.
11. This adjusts the condenser iris to a size which limits the
amount of light which is passing through the transparent slide 20
at the area surrounding the specimen on the glass slide. The
condenser lenses 26 are also movable in a vertical or "Z" direction
by operating the control ring 27 shown in FIG. 9 for changing the
optics until the proper focus of the field diaphragm iris is
obtained and this therefore also controls the amount of light being
passed onto and through the slide and eventually to the microscope
eyepieces 23A and on to the face 39 of the CCD sensor 18. The above
manipulations are performed while the operator views the cell image
40 on the monitor 37 for focus and light control. Thus it will be
seen tat there are four different variables that need to be
adjusted to control the intensity and amount of light which is
hitting the microscope slide on which is mounted the specimen; and
these four variables are the intensity of the light source, the
size of the field iris or diaphragm, the size of the condenser iris
or diaphragm, and the "Z" position of the condenser lenses.
To obtain the accurate readings of actual mass units for the
particular material being measured such as DNA, hemoglobin or the
like, it is important that the amount of scattered light be limited
in the analysis and that the scattered or background glare be
fairly uniform from machine to machine and from operation to
operation so that the results are all fairly accurate as to the
particular mass actual being measured. Also, the amount of
scattered light or glare should closely match the amount of glare
or scattered light for which the optical density values in the
look-up tables have been adjusted. As best seen in FIG. 5A, the
light coming to the slide is designated I.sub.0 as shown in the
FIG. 5 and this is often termed hereinafter as the background light
I.sub.o which passes readily through the transparent slide at the
area about the specimen 20. At the specimen 20, some of the
background light I.sub.0 is absorbed and the light which is
transmitted through the object is called transmitted light I.sub.t.
The I.sub.t passes through the objects to provide the image 38 on
the face 39 of the CCD sensor. The transmitted light T = I.sub.t
/I.sub.o. As the specimen thickness increases, the I.sub.t which is
the light having passed through the cell decreases exponentially
with increased thickness of the material being measured, for
example DNA or hemoglobin. As will be explained in greater detail
hereinafter in connection with FIGS. 2A and 3, the CCD camera
provides a analog signal which is digitized by an analog to digital
convertor and this digitized number from 1 to 256 is sent to a
first look-up table 50 and the look-up table contains the optical
densities each of which is a log transform equal to the formula OD
= -log[T]where OD is the optical density and is the transmitted
light where T =I.sub.t /I.sub.o. Thus, a log transform may be used
directly by the digital computer for each of the pixels in the 512
.times. 512 pixel array without having to make exponential
calculations with respect to finding the actual optical density for
a given pixel. The CCD sensor 18 is preferably mounted on top of
the microscope adjacent the eyepieces 23A and herein is a
commercially available CCD camera although other cameras or sensors
could be used. As best seen in FIG. 1, the cell image at the face
of the CCD sensor 18 is seen by the operator of the apparatus as an
image 40 (FIG. 1) on the display monitor 37. The image 40 is
generated by the computer 41 and a second look-up table 51 which is
connected to the display monitor 37. It will be appreciated that
the background light I.sub.0 which has passed through the
transparent portion of the slide where there is no specimen and has
passed through the optics may because of light scattering and
imperfection in the lenses and in the tubes at an angle such that
some scattered photons in the background light impinge at the area
of the image 38 and thereby lessen the optical density at a pixel
so that the pixel analog signal is in reality related to a less
thick object or less mass than is actually present at the specimen
20. Because of the scattering and glare, it is most important that
the light intensity be carefully controlled and that the glare be
kept to a minimum and that the amount and intensity of glare or
scattered light be kept relatively constant from machine to machine
and from operation to operation on the same machine by the same or
different operators.
An additional problem arises from the fact that not all of the
wavelengths of light are at the peak wave e.g. 410 or 630
nanometers because the band pass filters used pass additional
wavelengths of light. That is, the light I.sub.0 and I.sub.T which
provides the image 38 on face 39 of the CCD sensor 18 is filtered
by a filter 43 which is a narrow bypass band filter of a particular
wavelength, for example a 10 band wavelength pass filter for a 410
peak band wavelength Thus, the light impinging on the face 39 of
the CCD sensor is not of all the same wavelength but may vary
between 405 and 415 nanometers with the primary absorption for
hemoglobin spectrum being at 410 nanometers. As diagrammatically
illustrated in FIG. 7, the CCD sensor 18 when measuring hemoglobin
has a relatively flat response line for generating light voltage,
for light having wavelengths is within the range of 400 to 800
nanometers. Thus, whatever wavelengths of light are coming through
in the 10 nanometer range filter will be effective. Herein, the ten
nanometer filter width is measured as shown in FIG. 7 for a peak
410 nanometer wavelength at one-half of the peak 410 (FIG. 6) as
indicated by the marks 411 in FIG. 6. Likewise for the feulgen blue
azure stain and the DNA measurements, the filter is a 630 peak band
pass filter with a 10 nanometer wide width.
In accordance with the present invention, there is provided a new
and improved method and apparatus for reducing the amount of
scattered light and providing a relatively constant amount and
intensity of light at the face 39 of the CCD sensor 18 to produce
more accurate and more consistent actual mass measurements from an
optical density analysis of specimens 20 on a slide. This is
achieved by optimizing light conditions by having the amount of and
intensity of light relatively constant and reduced to that needed
to do the specimen cell measurements and, in particular, without a
large amount of extra light which results in more glare and more
scattered light which adversely affects the optical densities and
thereby the mass measurements which are preferably in real units
such as picrograms. Of the four different constants which can be
changed, each of the constants is regulated in a manner to be
hereinafter explained and particularly the condenser iris or
aperture is controlled closely to provide a constant amount of
light which is passing as background light through the slide 14.
Furthermore, this constant amount of light is closely matched to
the amount of light that was used when a glare correction was made
for the values in the look-up tables, as will be explained.
The preferred manner of control of the condenser iris and the
amount of light passing through the condenser iris is by a
non-invasive technique to the light microscope such that the light
microscope may be used in its normal manner; and, in so doing, one
may adjust the size of the condenser iris between its normal fully
opened to its fully closed position. The preferred non-invasive
technique includes an attachment means or device which can be
readily attached to the instrument to provide it with a fixed
constant iris or aperture 23 to limit the amount of background
light to that amount correlated to the particular condenser lenses
being used. Without the attachment, the previously used adjustment
procedure was to turn the knob 32 to the fully opened position so
that the variable iris aperture 28 was at its largest diameter and
then to turn the ring 27 in the opposite direction to reduce that
aperture size by about one-third. A significant problem occurs that
when the operator manually turns the knob to reduce the size of the
aperture by one-third since that varies significantly in size which
is not readily detected by the human eye and this causes a
considerable difference in the background light and eventually
scattered light which reaches the face 39 of the CCD sensor 18.
A very inexpensive and easily attachable and detachable means to
provide a fixed iris 23 may be in the form of an attachable member
or means 60 (FIG. 9) which includes a central cylindrical wall 61
which defines a fixed sized aperture 23. The fixed aperture 23 has
a size which is reduced one-third from the maximum opening achieved
for the variable condenser iris 28 when the leaves 29 are shifted
to their fully opened position as shown in FIGS. 9 and 10. Thus, in
operation the operator when adjusting the condenser iris will fully
open the leaves 29 so that the leaves are no longer effective and
the size of the iris opening 23 is then the size of the fixed iris
opening in the member 60. Because the fixed iris and the variable
iris are so closely adjacent as to almost be in the same horizontal
plane, the difference in location of the fixed and variable iris
does not make a substantial difference in this invention.
The method of adjusting the light for the four variables in
accordance with the invention will now be described. The first
adjustment that is made is that the operator will adjust the field
iris 18 to make the cone of light as small as possible when viewing
so that the amount of light which will ultimately end up on the
face of the CCD sensor is kept to a reasonable value. The operator
of the equipment will look through the microscope lens and adjust
the knob 21 at the front of the microscope while viewing the cone
of light to make it small as possible and still see the leaves of
the diaphragm and then will stop adjusting with the knob 21. Then
the operator will adjust the knob 32 to fully open the leaves 29 of
the condenser variable iris 28 so that the fixed iris 23 becomes
the controlling iris for the amount of light which is to pass
through the condenser lenses 26. Next, the operator will turn the
adjustment ring 27 to properly focus the condenser lenses while
viewing the image 40 on the monitor 37. Also, the operator will
adjust the light intensity by turning the knob 33 to provide a
digital read-out at 65 (FIG. 4) at a predetermined voltage
read-out, e.g. 8.3 volts. The operator then will view the image on
an instruction display monitor 62 and displayed on the monitor will
be a digital reading value for I.sub.o. In this instance, it is
desired that the I.sub.0 be closely controlled the operator may
again turn the light intensity knob 23 until I.sub.o reads 130 (or
any predetermined value consistent with the input look up table) on
the display monitor. The values in the look-up tables have been
previously adjusted by using specimens which are dark objects of a
known optical density and with the equipment adjusted to provide
I.sub.o of 130, a reading is taken of the dark objects optical
density from the CCD camera 18 and this optical density value is
then compared to the known optical density value. Because scattered
light photons at an I.sub.o =130 will be effecting image 38 at the
face 39, the optical density value read will be less than the true
optical density. After this analysis, the look-up tables are
calibrated to give the true optical density value for when I.sub.o
=130. Thus, the apparatus will be adjusted for light control and
for reading out actual true mass with a minimum amount of error
caused by scattered light or variations in light amount and
intensity. Thus, this invention provides a true optical density
read-out for each pixel in real time.
Referring now in greater detail to the illustrative and preferred
embodiment of the invention, as shown in FIG. 2A, will now be
described in greater detail.
Briefly, the apparatus 11 functions as a digital image processing
system 113, FIG. 2A, and includes the conventional, commercially
available, high resolution microscope, with which an operator can
view specimens 20 on a support 14. As best seen in FIG. 2, the
microscope stage 11 is movable incrementally by means 112 and 112A
in X and Y directions for viewing various areas of the slide on the
microscope platform or stage. The specimens or material on the
slide are further viewable by the imaging system 13 which is
controlled by a system control 122 in the form of a digital
processor such as a personal computer 41. An operator can
communicate with the system control 122 via keyboard 136 (FIGS. 1
and 2) and interacts with an apparatus 11 for viewing two displays.
The first display, image display 37 is an RGB monitor which
displays through the system control 22 the same image as seen
through the microscope 15. A second display, instruction monitor
62, is another RGB monitor and is used to provide the operator with
interactive prompts, messages, information, and instructions from
the program controlling system control 122. A printer 138 is
provided to produce a reliable hard copy output of the data
produced by the apparatus 11.
The optics form an optical image of each of the cells on the slide
and transmit them to an image splitter 125 which can take the form
of a prism. On one side of the splitter 125, as seen in FIG. 2A,
the television camera 18 or other detector converts the optical
images point-by-point into a scanned electronic signal representing
the optical intensity of each point in the image. The output of the
camera 18, which is a standard NTSC analog video signal is applied
to an analog to digital convertor of an image processing interface
121. The image processing interface 121 converts the image signal
from the television camera 18 to a digitized signal which is
received and stored by the system control 122. Because of the
continuous scanning, a real time image of the area of the optics
are focused on is provided by the image display 37. In general, the
image is a 512 .times. 512 array of pixels, each having a measured
light intensity.
On the other side of the image splitter 125, are located the
viewing optics 123 of the microscope 15. This parofocal arrangement
allows the same image to be displayed on the image display 37. This
feature allows the positioning of the platform 11 by manual X and Y
adjustments means 12 and 12A until the operator views a field of
interest on the slide 14. At that time, a computer enhanced
digitized image of the selected field is displayed on the image
display 37 for further analysis.
Both of the image displays 37 and 62 are controlled by a system
control 122 through standard video interface circuitry 139 and 161,
respectively. Similarly, the keyboard 136 and printer 138
communicate with the system control 122 through conventional
interface circuitry 135 and 141, respectively. In addition, the
system control 122 controls a random access memory 73 and bulk
storage in the form of floppy and hard disk drives through memory
control 71.
All of the interface circuits 121, 135, 139 and 141, 161, 71, and
106 can be selectively embodied on their own printed circuit boards
which are mounted in the back plane or card connector of a
conventional personal computer forming the system control 122.
Preferably the personal computer can be one manufactured by the IBM
corporation having a model designation AT. Such system control can
be run under a disk operating system such as PC DOS version 3.1 or
later. The system software for the image analysis is called an
application program from the disk drive 75, and could, for example,
be supplied on a floppy disk 77. The system software is read from
the disk 77 and loaded into RAM 73. After loading program control
is transferred to the analysis to regulate the various hardware
elements previously set forth in a known manner and further
description of this details of the invention may be found in the
aforesaid co-pending patent application serial No. 927,285.
Referring to the fixed iris member 60 which is in the form of a
cup-shaped member shown in FIGS. 8 and 9, it is generally a one
piece body 79 of metal which has a bottom annular flange 80 with a
hole 81 therein which receives a screw 82 which is threaded into a
threaded opening or hole 83 in a depending circular member 84 on
the microscope. The screw 82 may be readily unthreaded and then the
cup shaped member 60 may be quickly slipped from engagement within
a cylindrical wall 86 in the bottom of the cylindrical member 84.
Herein, the cup shaped member 60 includes an upstanding cylindrical
sidewall 87 mated in size to fit against the cylindrical wall 86. A
small stepped shoulder 88 is formed on the cup-shaped member to fit
against a stepped shoulder formed in the cylindrical 86. The fixed
iris 23 in the upper wall 90 of the cup-shaped member 60 is sized
to be the fixed diameter iris needed for the particular microscope
condenser lenses. The size of the iris 23 has been calculated to be
one-third less in area than the widest opening area for the
variable iris 28 when the aperture is totally opened by opening the
leaves 29 as far as possible, as shown in FIGS. 9 and 10. The cup
has the effect that the fixed iris opening sets in approximately
the same optical plane that the variable iris opening is in. It has
been found that this one-third reduction provides about the best
results for using the particular condenser lenses provided with the
Reichert microscope.
The illustrated cup 60 is of one piece metal material and it is
very inexpensive to manufacture and yet can be readily attached and
detached with the one screw 82 so that it may not be viewed as an
invasive member which will adversely affect the operation of the
Reichert light microscope. The microscope, with the cup 60 removed,
may function in its normal manner. Likewise, if there is any need
for repair or assembly of the microscope, the microscope company
will not have the excuse that it is not responsible for the cost of
repair because of an invasive technique which has altered or
modified the microscope.
Manifestly, the present invention is not limited to any particular
microscope and various types of microscopes may be used from that
disclosed. The Reichert microscope is used by way of example only
and not by way of limitation. Rather than using the simple cup with
the fixed aperture 23, other devices such as knobs, fixed stops for
the variable leaves, or other devices could be used to allow for
the variable apertures to be sized to a constant given value at the
time of the cell analysis. However, such other equipment will
either be invasive of the microscope or it will be substantially
more costly than the attachment member 60 disclosed herein.
Thus, it will be seen from the foregoing that the present invention
provides a new and improved method and apparatus for controlling
the light where there are four variables which can be adjusted and
which need to be adjusted, to optimize the actual true values or
true densities being measured of a given material such as
hemoglobin or DNA in actual cell specimens or in control cell
objects as described in the aforementioned patent application. The
controlling of the four light variables for the microscope has
resulted in practically identical optical densities being obtained
in real time for the same specimens being measured at different
times by different operators on the same or different apparatus
from the owner of this invention.
While a preferred embodiment has been shown and described, it will
be understood that there is no intent to limit the invention by
such disclosure, but, rather, it is intended to cover all
modifications and alternative constructions within the spirit and
scope of the invention as defined in the appended claims.
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