Note: Descriptions are shown in the official language in which they were submitted.
~ W094118800 2 1 ~ 5 ~ 3 7 PCT~S94/0130~
VIDEO DENSITOMETER WITH DETERMINATION OF
COLOR COMPOSITION
BACKGROUND OF THE INVENTION
l. Field of the Invention
The present invention relates generally to thin
layer chromatography (TLC) and in particular to a
computer enhanced video area densitometer and its
application in determining the color composition
concentrations of chemical and biological compounds
deposited on a variety of different chromatographic and
electrophoretic media.
2. Description of Related Technology
Chromatography is one of the most widely used
methods of performing specific quantitative analysis in
chemistry and biology. In the past, using thin layer
chromatography (TLC), the concentrations or densities
of compounds present as spots and bands of
light-absorbing, fluorescent, or chemiluminescent
materials on transparent or translucent supports, such
as thin-layer plates, radioautograms, paper
chromatograms, electrophoresis gels, etc., have been
analyzed using a light intensity scanner. This scanner
was typically a mechanical device which moved the
transparent or translucent support containing the
material under analysis across a light sensor such as a
photomultiplier or photocell. The support, holding the
material being analyzed, was placed either between a
light source and the light sensor, or the light source
was placed on the same side as the light sensor for
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absorbance or reflectance measurements of the material.
A mechanical slit was used to focus the light source
into a narrow beam.
More recently, the density of a spot of a specimen
compound has been determined by using a video camera,
video image digitizer, and digital computer to create a
videodensitometer. Such videodensitometers did not
require mechanical support movement mechanisms, nor
focusing apparatus for light beam definition, and
enabled an entire field of spots to be measured within
a single video scan with a high degree of resolution.
Digital capture of video information has been used
for image enhancement and analysis, however, its
application in densitometry and analytical biochemistry
has been limited due to the relative complexity and
high cost of known systems necessary for digital
capture of a video image. Previous systems required
multiple frames collected at different video scan times
to construct the video image. Prior art systems were
therefore not well suited for image capture in
circumstances where the image quality deteriorated
quickly.
SUMMARY OF THE INVENTION
Recently, the development of low cost high speed
analog-to-digital integrated circuit converters and
personal computers with enhanced display capabilities
makes it possible to develop a low cost video
densitometer system from commercially available
components. The system described in this application
utilizes a home video camcorder, a composite video
monitor, a commercially available personal computer
with a high resolution color monitor, a programmable
high speed analog-to-digital converter to facilitate
~ WO94/18800 21~ 5 ~ 3 7 PCT~S94/013Q~
density calculations, and a unique computer program to
facilitate the analysis and display of color
composition density and gray scale integrated density
information concerning a subject specimen.
In the system of the present invention, up to all
gray levels of the video image are converted into
digital form during a single video frame of l/60th
second duration by the use of a high speed flash
analog-to-digital converter. This improved data
cap~ure speed makes it possible to capture and analyze
data for specimens using volatile stains such as iodine
vapor and to simultaneously collect an image of
standards and samples without significant loss of data
integrity. In the system of the present invention, the
black and white levels of transmission or reflectance
for each sample are capable of being set directly and
interactively rather than using fixed levels, making it
possible to increase sensitivity and decrease the
influence of background video density on the accuracy
of the measurements.
The system of the present invention enables the
user to determine an integrated density of irregularly
shaped light absorbing areas of a subject specimen by
interactively selecting individual spots for
determination of color composition density and for
integration into a two dimensional density
representative of the spot area density. The results
can be displayed on an output display means such as a
printer or CRT monitor.
The system of the present invention also provides
the user with accurate data representing the percentage
color composition as well as the total integrated
o density of irregularly shaped spots formed by a
compound after separation and visualization using thin
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layer chromatography or polyacrylamide gel
electrophoresis. This data can therefore be used to
accurately determine the concentration of the compound
applied to the separation media and thus provide a
rapid methodology for analysis of biological and
chemical compounds and the progress of a disease
through tissue using the change in color composition
density over time.
The system of the present invention can also be
used to provide accurate analyses of the clarity of an
optical device by determining the amount of light
transmitted and absorbed through the optical area of
the device.
An aspect of the present invention is the
interactive selection of spot areas and associated
color composition of the spot areas for density
analysis by displaying a digital video representation
of the optical density of the subject specimen on the
computer system and selecting the display coordinates
for analysis.
An additional aspect of the present invention is
the interactive selection of graphically displayed one
dimensional densities within a specified column or row,
containing spots of the subject specimen, for more
exact determination of where density peaks,
representative of spot areas, begin and end thus
enabling more accurate and repeatable density analysis.
Thus, in accomplishing the foregoing objects the
present invention with its combination of home video
camcorder, programmable high speed analog-to-digital
converter, computer system and software provide a
system capable of accurately measuring the total
integrated density of absorbing areas, and color
composition density of the areas, on any media which
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can be sufficiently illuminated by reflected or
transmitted light, such as chromatographic plates
illuminated by either white or ultraviolet light,
photographs or photographic negatives, radioautograms
and visually stained polyacrylamide gels.
The above-noted and other objects and advantages
of the present invention will become more apparent from
a detailed description of the preferred embodiment when
read in conjunction with the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic block diagram of a
preferred embodiment of the present invention.
Figures 2 through 21 are schematic block diagrams
of the logic sequences which form a part of the present
invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Referring now to Figure 1, the letter S designates
generally a system according to the present invention
which is illustrated in block diagram form. The system
S includes light source 50 which is used to illuminate
a subject specimen 52 so that a video image gathering
means 54 may collect a video image of the specimen.
The video image gathering means 54 converts the video
image of the specimen into an analog electronic signal
representative of this image. Light source 50 may be
fluorescent white light or ultraviolet light. This
light source may be positioned to shine through the
subject specimen 52 or may be placed on the same side
of the subject specimen 52 as is the video image
gathering means 54. Thus, light from light source 50
either shines through the subject specimen 52 or is
reflected off of the surface of the subject specimen 52
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facing the video image gathering means 54.
The video image gathering means 54 of the present t
invention may be for example, a video camera, a charge
coupled device, or an area detector device similar to
what is used in low-light military snooper scopes. For
the purposes of the present invention, the video image
means 54 may be any suitable technology which receives
light as an input and provides a standard analog
television video frame formatted output signal. The
standard analog television video frame format signal
from the video image gathering means 54 is provided to
the input of a programmable analog-to-digital
converter 58. Converter 58 may be chosen from any of
the suitable, commercially available devices.
Converter 58 converts the analog video signal from
the video image gathering means 54 into digital values.
Using present television technology a video frame is
completed in 1/60th of a second. One advantage of the
present invention is that up to an entire frame of the
video image of the subject specimen is capable of being
captured and converted into a digital representation of
the irregularly shaped light-absorbing areas of the
subject specimen within 1/60th of a second. The
present invention's rapid conversion of the subject
specimen optical light intensities allows greater
measurement accuracy because equipment drift is not as
significant a factor as it was in the prior art, and
the short video frame conversion time, similar to a
photographic camera snapshot, allows capture of data
representative of rapidly decomposing subject
specimens.
System S may also include a video monitor 56 which
is useful in monitoring the position of the subject
specimen for proper alignment. For the purposes of the
~ WO94/18800 215 ~ 4 3 7 PCT~S94/0130~
present invention, video monitor 56 may be any standard
analog television monitor suitably connected to the
output of the video image gathering means 54.
To obtain optical accuracy for calculation of spot
densities, the system of the present invention includes
a means for calibrating bright and dark (white and
black) video image intensity levels prior to digital
conversion. By using the greatest video optical
intensity resolution possible, the subject specimen
video image gives the most accurate information for
calculation of color composition densities and
calculation of the spot irregular absorbing area
integrated densities.
To provide optimal calibration of the video image
intensity, system S includes a black level adjustment
means 60 which provides an analog voltage bias
representation of the darkest desired video optical
intensity signal for the specific sample. Likewise, a
white level adjustment means 62 provides an analog
voltage bias representative of the brightest desired
video optical intensity signal. The black level and
white level adjustment means may be either manually
adjusted potentiometers or program controlled
digital-to-analog converters. Either means of
adjusting dark and bright video optical intensity
signal levels are practical and may be provided using
devices or programmably controlled systems that are
well known in the art.
once the black and white level video optical
intensity signal level adjustments are set, the
programmed computer system 64 is used to store the
digital signal values representative of video image
optical intensity generated by the analog-to-digital
video converter 58. Up to a frame of digitized data,
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typically comprising 62,464 bytes of information, is
capable of being stored by computer system 64 in its
memory. Thereafter, computer system 64 under program
control processes this digital intensity data to
provide a calibrated optical intensity video image of
the subject specimen 52.
The system of the present invention includes
software which permits the laboratory technician to
interactively initialize system variables and select
from the various program options available. This
interactive selection is provided via the computer
video monitor 66 and the keyboard of computer 64. The
results of the requested computations are displayed in
both tabular and graphic form via display means 68.
The system of the present invention includes a
menu driven computer program which utilizes a novel set
of instructions to accomplish the following procedures.
The present invention provides for interactive
adjustment of the digital video conversion to give a
value of zero on a given number of bytes of video
information when a black object is present in the video
image, and interactive adjustment of the digital video
conversion to give a maximum digital value when a white
object is present in the video image. Capture of the
video image of the subject specimen in view is capable
of being completed within 1/60th of a second. The
image in view is then converted under program control
into a digital representation of the video image
optical intensity. This digital information is saved
to a non-volatile memory means of the computer 64.
This memory means may be hard disk, floppy disk, tape,
or other appropriate storage medium.
The program of the present invention further
causes the computer 64 to store digital data
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representative of the video image optical intensity ,
convert the intensity data to optical density data and
display the density data on a high resolution color
monitor typically in 16 levels of gray. The program is
also capable of converting optical intensity data from
a color video camera into color composition density
data. The system program allows the operator to
interactively select individual spots, color
composition, vertical lanes of spots, or horizontal
rows of spots for analysis.
The system of the present invention converts the
raw digital data representative of optical intensities
to digital data representative of optical and color
composition densities for up to 62,464 pixels in
accordance with Beer's Law. Beer's Law states that
optical density is proportional to the log10of the
reciprocal of the optical intensity.
Each one of these digital density values contains
a gray level value for one pixel of the digital density
image displayed on the computer video monitor 66. The
digital density values representative of the conversion
of each of the digital intensity values to density
values will henceforth be referred to as "point
densities". The color composition values
representative of the conversion of each of the color
composition density values will henceforth be referred
to as "color densities". A "line density" is the sum
of the point densities contained on a given vertical or
horizontal line, and an "area density" is the sum of
the point densities contained within a given area.
Line density is used in the case of row or lane
procedures which partially integrate the point
densities as a function of either horizontal or
vertical line position respectively. The line
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densities are displayed as a function of line position
within the selected row or lane on the computer video
monitor 66. This graphical display of line densities
clearly shows where a spot density peak begins and ends
thereby enabling more accurate selection of given spot
areas for density analysis.
After the interactive selection of color
composition or individual spots using either an
operator selected area or the row or lane selection
method, the program of the present invention causes
computer system 64 to perform an integration of the
color composition density and point density data for
the selected spots into a two dimensional density.
Thereafter the program causes the computer 64 to
calculate the appropriate background densities and
display the results on an output display means such as
a CRT monitor, or a printer. The system of the present
invention provides a system capable of accurately
measuring the total integrated density of absorbing
areas and associated color composition density on any
media which can be sufficiently illuminated by
reflected or transmitted light, such as chromatographic
plates illuminated by either white or ultraviolet
light, photographs or photographic negatives,
radioautograms and visually stained polyacrylamide
gels.
The present system provides the ability to
accurately obtain the color composition density and
total integrated density of irregular spots formed by a
compound after separation and visualization using thin
layer chromatography or polyacrylamide gel
electrophoresis. In accordance with known analytical
methods this data can be readily used to accurately
determine the color compositiGn and concentration of
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the compound applied to the separation media and thus
provide a rapid methodology for analysis of biological
and chemical compounds and the advancement of a disease
through tissue.
The present system also provides the ability to
accurately determine a percentage color composition for
a given area of the specimen.
The present invention is an improvement over the
prior art in that up to all gray levels of the video
image are capable of being converted into digital
format during a single video frame of l/60th second
duration by the use of a high speed flash
analog-to-digital converter. Previous systems required
multiple frames collected at different video scan times
to construct the video image. This improved image
capture time makes it possible to use volatile stains
such as iodine vapor and to simultaneously collect an
image both of standards and samples.
The following description is a preferred
embodiment of the program instruction sets of the
invention. Referring now to the drawings, the sequence
of instructions utilized in the present invention to
cause the computer 64 to interactively process the
incoming digitally stored intensity information and
calculate a density for the irregularly shaped
absorbing areas and associated color composition
densities of the subject specimen will be described in
detail.
Referring now to Figure 2, the computer 64 begins
execution of the main routine at step 100. Step 100
~ causes computer 64 to determine if the computer system
is in the low resolution display mode. If not, control
- is transferred to step 102 which causes the computer to
send an appropriate message, and thereafter to steps
WO94/18800 PCT~S94/0130~ ~
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120 and 122 which enable the mouse subroutine, set the
computer system to graphic mode, close the program
window and then exit.
If, at step 100, the computer system is in the low
resolution display mode then control is transferred to
step 104 which causes the computer 64 to initialize its
memory locations by specifying dimension arrays which
store the incoming digital information representative
of intensity, store the default floppy or hard disk
drive to be used for permanent data storage, and set up
parameters for a serial port to receive the digital
video intensity data information from the video
analog-to-digital converter. Step 104 also causes
computer 64 to initialize software routines to read the
lS video information into memory, open an information
window on the computer video monitor 56 and display a
title page for the lab technician operator to
interactively control the various program options.
Control of computer 64 then transfers to step 106.
Step 106 causes computer 64 to activate the
program menus selection display steps 108 and 110 which
re-initialize the video monitor screen and begin a menu
selection subroutine. Control is then transferred to
steps 112, 114, and 116 which cause computer 64 to idle
until a menu selection is made, exit the main routine
to execute the selected subroutine and return when all
subroutine execution is completed. Depending on what
menu option is selected the program will cause computer
64 to execute a particular subroutine as illustrated in
Figure 3. Step 116 of Figure 2 causes computer 64 to
check for completion of the selected subroutine and
return control back to the main routine of Figure 2.
Thereafter, steps 118 and 120 cause computer 64 to
restore the previous video screen colors, enable the
~ W09~/18800 21~ 5 4 3 7 PCT~S94/0130~
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mouse control, set the graphic mode back and close the
program information window. When the operator
completes utilization of the present invention, exit
step 122 causes control of computer 64 to return to the
operating system of the computer.
Referring now to Figure 3, the menu handling
subroutine is used to activate operator selected
program subroutines. The available program subroutines
are: analyze data by selection of spots, step 124;
analyze data by selection of columns, step 128; analyze
data by selection of rows, step 132; collection of
digitized video information, step 136; setting the
white video level intensity, step 140; setting the
black video level intensity, step 144; analyze RGB
color composition, steps 145, 147 and 148; load digital
video intensity values into a disk file for analysis,
step 149; and exiting the program when finished, steps
152 and 156.
When a subject specimen data file is to be
analyzed, the operator selects the menu option of
step 149. Step 149 enables step 150 which causes
computer 64 to begin execution of the files subroutine.
Referring now to Figure 4, the files subroutine steps
200, 202 and 204 causes computer 64 to enable an error
routine, select disk drive 2, and retrieve the file
specified by the operator. If the file name specified
is an existing valid data file and the operator has
correctly indicated that the file is a color file, then
steps 205, 207, 208 and 211 allow computer 64 to load
data into the working TLC (thin layer chromatography)
~ memory array and the working RGB color array. If the
file name specified is an existing valid data file and
~ the operator has correctly indicated that the file is
not a color file, then steps 205, 207, 209 and 212
WO94/1~00 PCT~S9410130~ ~
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allow computer 64 to load data into the working TLC
memory array, only.
If the operator has erroneously indicated that the
file is not a color file, then steps 209 and 213 cause
computer 64 to display a message to inform the operator
that an incorrect file type has been selected. If the
operator has erroneously indicated that the file is a
color file, then steps 208 and 210 cause computer 64 to
display a message to inform the operator that an
incorrect file type has been selected.
Next, step 214 sets the name of the working memory
array variable to the name of the selected input file.
If, however, the file name specified is not a
valid data file then step 205 does not allow computer
64 to load data into the working TLC (thin layer
chromatography) memory array, rather, step 206 enables
the file error subroutine illustrated in Figure 8. Now
referring to Figure 8, steps 280, 282 and 284 cause the
computer 64 to alert the operator of a system error,
and then return program control to the files
subroutine. Steps 215 and 216 then cause computer 64
to reset the file loading program logic and turn off
the file error handling subroutine. Steps 217 and 218
cause the computer 64 to enable the originally selected
disk drive and return control to the menu handling
subroutine.
Referring back to Figure 3, steps 140 and 142
cause the computer 64 to execute the white level
subroutine as illustrated in Figure 5. Referring now
to Figure 5, steps 220 and 222 cause the computer to
clear the screen information on the computer video
monitor 66 and to collect data using a low resolution
collection mode. Faster calibration of the video image
collection system is obtained in the low resolution
WO94tl8800 PCT~S94/0130~
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collection mode because of the reduced number or pixels
that must be stored and displayed. The low resolution
mode is not mandatory for proper operation of the
present invention, but greatly facilitates the speed of
digital video data collection using present computer
technology. As future computer technology becomes more
powerful this low resolution mode may not be needed.
Step 224 causes the computer 64 to print
instructions on the video monitor screen 66 thereby
enabling the operator to interactively control the
calibration of the video white level. Steps 226, 228,
and 230 cause computer 64 to collect digital video data
from the video analog-to-digital converter 58 and store
in a memory array, count the number of digital data
values equal to a binary value of sixty three, print
this number on the computer video monitor 66, then
request further input from the operator. The number
sixty three is representative of the maximum binary
value of a six bit binary number, however, another
embodiment uses eight bit binary data allowing a
maximum binary value of two hundred and fifty five.
The system of the present invention adjusts the
upper limit of the white video level intensity to
optimize the bright intensity video image resolution.
During this optimization procedure, the white level
adjustment means 62 is varied to produce an operator
specified number of six bit digital video data values
equal to binary sixty three. If the resulting number
of binary values equal to binary sixty three are not
satisfactory, then the operator or computer system
- under program control may make an adjustment to the
white level adjustment means 62 in order to bring the
bright intensity video level into the desired range.
For example, if the number of data values equal to
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binary sixty three is less than the optimum specified
number, then the video image is too dark and the bright
resolution may be increased through the white level
adjustment means 62. Conversely, if the number of
values equal to binary sixty three is greater than the
optimum specified number, then the video image is too
bright and the bright resolution should be decreased.
When the optimum specified number of binary values
equal to sixty three is optimum, then steps 232, 234
and 236 cause computer 64 to reset the video screen and
return control back to the menu handling subroutine of
Figure 3.
Referring back to Figure 3, steps 144 and 146
cause the computer 64 to execute the black level
subroutine as illustrated in Figure 6. Referring now
to Figure 6, steps 240 and 242 cause the computer to
clear the screen information on the computer video
monitor 66 and to collect data using a low resolution
collection mode. Setting to a low resolution mode is
for the same purposes as described above in the white
level subroutine.
Step 244 causes the computer 64 to print
instructions on the video monitor screen 66 thereby
enabling the operator to interactively control the
calibration of the video black level. Steps 246, 248,
and 250 cause computer 64 to collect digital video data
from the video analog-to-digital converter 58 and store
in a memory array, count the number of digital data
values equal to a binary value of zero, print this
number on the computer video monitor 66, then request
further input from the operator. The number zero is
representative of the minimum value of a binary number.
The system of the present invention adjusts the
lower limit of the black video level intensity to
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optimize the dark intensity video image resolution.
During this optimization procedure, the black level
adjustment means 60 is varied to produce an operator
- specified number of digital video data values equal to
binary zero. If the resulting number of binary values
equal to binary zero are not satisfactory, then the
operator or computer system under program control may
make an adjustment to the black level adjustment means
60 in order to bring the dark intensity video level
into the desired range.
For example, if the number of data values equal to
binary zero is less than the optimum specified number,
then the video image is too bright and the dark
resolution may be increased through the black level
adjustment means 60. Conversely, if the number of
values equal to binary zero is greater than the optimum
specified number, then the video image is too dark and
the dark resolution should be decreased. When the
number of binary values equal to zero is optimum, then
steps 252, 254 and 256 cause computer 64 to reset the
video screen and return control back to the menu
handling subroutine of Figure 3. The above bright and
dark video intensity calibration of the system of the
present invention maximizes the accuracy of the video
intensity data by utilizing the best resolution of the
system components.
The operator initiates data collection by
selecting the data collection subroutine as illustrated
in Figure 3. Steps 136 and 138 cause computer 64 to
begin collecting digitized video data. Referring now
to Figure 7, steps 260 and 262 cause computer 64 to
clear the screen of the video monitor 66 and define the
video resolution of the screen, using present
technology, to 256 by 244 pixels of information. Step
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264 causes the computer 64 to print instructions on
video monitor 66 which enable the operator to
interactively interface with the data collection system
of the present invention. Step 265 causes computer 64
to prompt the operator regarding whether color data is
to be collected.
If the operator chooses to collect color data,
steps 265 and 267 cause computer 64 to execute a
program to collect color data from the digitizer and to
define a working RGB array in memory to sequentially
store each digital value. If the operator chooses to
collect gray level data, steps 265 and 266 cause
computer 64 to execute a routine to collect gray level
data from the digitizer and to define a working TLC
array in memory to sequentially store each digital
value.
Next, steps 268, 270, 271 and 272 cause computer
64 to select disk drive 2, request a file name from the
operator, mark the file as color data or gray level
data, then store the digital data on disk drive 2 under
the specified file name. Steps 274 and 276 cause
computer 64 to reselect drive 1 and restore the
previous menu information to the screen of video
monitor 66, then return control to the menu handling
subroutine of Figure 3.
Referring back to Figure 3, steps 124 and 126
cause the computer 64 to execute the spots subroutine
as illustrated in Figure 9. Referring now to Figure 9,
steps 300 and 302 cause the computer to set its color
registers for gray level video display and clear the
screen of the video monitor 66. Steps 304, 306 and 308
cause the computer to start an iterative loop which
maps the digital video data to the screen of the video
monitor 66, retrieve the digital data from the memory
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array, and define the digital data as various levels of
gray on the screen of video monitor 66.
Step 310 causes the computer 64 to check each
- binary value of digital density data. When a digital
density data value is less than binary eight, then step
312 causes the computer to set each corresponding video
screen pixel color to black. Likewise, step 314 causes
the computer to check for a binary value equal to zero,
if so, then step 316 causes the computer to set each
corresponding video screen pixel color to red. Setting
the low intensity level pixels to red and black more
readily depicts these pixels in relation to the usable
video intensity data.
Steps 318 and 320 cause the computer 64 to plot on
the screen of the video monitor 66 all digital video
data values equal to or greater than binary eight.
Steps 322 and 324 cause the computer to initialize the
menu bar on the video monitor 66, set the spot counter
to zero, and look for a mouse event to happen. The
operator may interactively define the parameters for
spo~ area density calculation by the use of, for
example, a mouse. A mouse as used in the present
invention is a computer device which interactively
controls the location and direction of a cursor on a
computer screen. A mouse is well known in the art and
no further explanation of it will be made.
The mouse is used to set the boundaries of the
area of interest containing the spot area density to be
calculated. Steps 324 and 326 cause computer 64 to
wait for a mouse event that is representative of cursor
- position, then draw a rectangular box on the screen of
video monitor 66 which encompasses the desired spot
area. Once the box is drawn to the satisfaction of the
operator, the coordinates of the displayed box are
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calculated to determine the digital values to be used
in calculation of spot density.
Now referring to Figures 9 and 10, steps 328, 330
and 332 cause the computer 64 to check if the
coordinates of the box are within the video display
area, contains more than one pixel, and if so, then
draw the box on the screen of video monitor 66. The
present invention is capable of calculating the area
densities of up to 100 selected spots. If more than
100 spots are selected for area density calculation,
then steps 334 and 336 will cause the computer to
terminate the program. However, if less than 100 spots
are selected then step 338 causes the computer to store
the coordinates of the selected rectangles for
subsequent computation of spot area densities. The
operator uses the mouse to select spot areas to be
analyzed until the "alt" key is pressed.
After the "alt" key is pressed, if color data has
been selected to be analyzed by the operator, steps
340, 342, 343 and 345 cause the computer to restore and
reset the menu on the screen of the video monitor 66,
and print a header for subsequently calculated color
data on the output display means 68, for example, a
printer. Step 347 then causes computer 64 to convert
all of the digital intensity values, lying within the
selected box rectangular coordinates, to RGB density
values. The computer utilizes Beer's Law to convert
the digital intensity data to digital density data.
Beer's Law states that the optical density is equal to
the log10 of the reciprocal of the optical intensity.
Thus each digital intensity value is converted into a
corresponding digital density value.
Background area RGB density is first calculated by
summing the digital density values located on the left
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and right edges of the rectangle enclosing the spot for
each of the red, green and blue color arrays. The edge
density sum is then stored in memory as RGB background
sum. The background average is calculated by dividing
that sum by the number of density values used in
computing the sum. The density values within the
rectangle for each of the red, blue and green arrays
are summed and stored in memory as red sum, green sum
and blue sum.
Next, spot area density for RGB data is calculated
by subtracting the RGB background sum from the total of
the red, blue and green sums. Steps 347, 348 and 350
cause the computer 64 to calculate the spot area
density for RGB, store the results in memory, and when
all spot area density calculations are complete, print
the results on the output display means 68. Now
referring to Figure 11, steps 352, 354, 356, and 358
cause the computer to restore the program menu, restore
and reset the original video screen colors, and return
control back to the menu handling subroutine of Figure
3.
After the "alt" key is pressed, if gray level data
has been selected to be analyzed by the operator, steps
340, 342, 343 and 344 cause the computer to restore and
reset the menu on the screen of the video monitor 66,
and print a header for subsequently calculated gray
scale data on the output display means 68, for example,
a printer. Step 346 then causes computer 64 to convert
all of the digital intensity values, lying within the
selected box rectangular coordinates, to gray scale
density values. The computer utilizes Beer's Law to
convert the digital intensity data to digital density
data. Beer's Law states that the optical density is
equal to the log10 of the reciprocal of the optical
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intensity. Thus each digital intensity value is
converted into a corresponding digital density value.
8ackground area gray scale density is first
calculated by summing the digital density values
located on the left and right edges of the rectangle
enclosing the spot. The edge density sum is then
stored in memory as gray scale background sum. The
background average is calculated by dividing that sum
by the number of density values used in computing the
sum. The gray scale density values within the
rectangle are summed and stored in memory as gray scale
sum.
Next, spot area density for gray scale data is
calculated by subtracting the gray scale background sum
from the gray scale sum. Steps 346, 349 and 350 cause
the computer 64 to calculate the spot area density for
gray scale, store the results in memory, and when all
spot area density calculations are complete, print the
results on the output display means 68. Now referring
to Figure 11, steps 352, 354, 356, and 358 cause the
computer to restore the program menu, restore and reset
the original video screen colors, and return control
back to the menu handling subroutine of Figure 3.
The operator may analyze area density by selecting
vertical columns of spots. Referring back to Figure 3,
steps 128 and 130 cause the computer 64 to execute the
columns subroutine as illustrated in Figure 12.
Referring now to Figure 12, steps 400, 402 and 404
cause the computer to set its color registers for gray
level display, clear the screen of the video monitor
66, then display the digital density data on the video
monitor in shades of gray representative of each point
density. Steps 406 and 408 cause the computer to
retrieve each digital density value stored in the
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memory array and sets each pixel color corresponding to
these values.
Step 410 causes the computer 64 to check each
binary value of digital density data. When a digital
density data value is less than binary eight, then step
412 causes the computer to set each corresponding video
screen pixel color to black. Likewise, step 414 causes
the computer to check for a binary value of zero, if
so, then step 416 causes the computer to set each
corresponding video screen pixel color to red.
Characterizing dark level pixel values in this manner
facilitates greater accuracy in the selection of useful
area density evaluation boundaries.
Steps 418 and 420 cause the computer 64 to plot on
the screen of the video monitor 66 all digital density
data values equal to or greater than binary eight.
Steps 422 and 424 cause the computer to initialize the
menu bar on the video monitor 66, set the column (lane)
counter to zero, and look for a mouse event to happen.
After the mouse event happens, step 426 causes the
computer to draw a rectangular box on the screen of the
video monitor 66 which encompasses the desired column
(lane) area, then return the box coordinates to the
program.
Now referring to Figure 13, steps 428, 430 and 432
cause computer 64 to check if the box coordinates are
within the display area, encloses more than one pixel,
and if so, then draw the chosen column on the screen of
video monitor 66. The present invention is capable of
handling spot area density calculations for up to lO
- columns (lanes) of spots. If more than lO columns are
selected for lane density calculation, then steps 434
and 436 will cause the computer to terminate the
program. If lO or less columns are selected then step
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438 causes the computer to store the coordinates of the
selected columns in memory as a function of each left
and right horizontal boundary of each column as
displayed on the screen of the video monitor 66. The
operator uses the mouse to select lanes to be analyzed
until the "alt" key is pressed. After the "alt" key is
pressed, steps 440, 442, and 444 cause the computer to
clear the screen of the video monitor 66, and print
"wait for integration" on the screen of the video
monitor 66.
Step 446 causes computer 64 to set the column
counter to one and start the one dimensional line
density calculations on the selected column digital
density data. The purpose in calculating one
dimensional line densities is to enable more accurate
selection of the area boundaries used in determining
each spot area density. All digital density values on
a given horizontal line within the selected vertical
lane (column) are summed to give a one dimensional line
density as a function of vertical position.
Repeatedly, the digital density values are summed for
each individual horizontal line until all, for example,
244 horizontal lines within the vertical lane are so
calculated. Then a graph of the line densities as a
function of vertical position within the lane is
plotted on the screen of the video monitor 66. This
graph depicts line density peaks which are
representative of the spot density boundaries within
the lane. Thus the start and finish of the
y-coordinates representing spot location are more
easily and repeatably determined.
Step 450 causes the computer to increment the line
density calculations to the next horizontal line. Now
referring to Figure 14, the line density calculations
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continue to increment to a subsequent line until step
452 causes the computer to determine that the last
horizontal line density was calculated. Steps 454 and
- 456 cause the computer to increment the column counter
and calculate subsequent lane line densities until the
last line density is determined.
Steps 458 and 460 cause the computer 64 to clear
the screen of video monitor 66 and prompt the operator
for the desired column number of the desired density
graph. Step 462 causes the computer to check for a
legitimate column number, if so, then step 468 causes
the computer to prompt the operator for a scaling
factor to be used in plotting the lane density graph.
If, however, the column number input is 99 than steps
462, 464 and 466 cause the computer to return control
back to the menu handling subroutine of Figure 3.
After the operator specifies the requested scaling
factor, step 470 causes the computer to print header
information on the output display means 68. Steps 472
and 474 cause the computer to plot a graph of the line
densities as a function of vertical position within the
lane (column) on the video monitor 66, and print this
line density graph on the output display means 68
(printer). Steps 476, 478 and 480 cause computer 66 to
initialize the peak counter to zero, change the screen
display cursor to a cross bar, initialize coordinate
variables, and request the operator to select a start
point for the beginning of a peak density
representative of the first spot area within the
selected lane (column).
~ Now referring to Figure 15, step 482 causes the
computer 64 to wait for a mouse event to happen. If a
mouse event happens without the "alt'l key being
pressed, then step 486 causes the computer to wait for
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the left hand button on the mouse to be pressed. When
the left hand button is pressed, steps 488 and 490
cause the computer to draw a line on the video
monitor 66 indicating the start of a spot area density
peak within the lane and store its y-coordinate
position. Step 492 causes the computer to request the
operator to select an end point for the termination of
the peak density of the spot area. Steps 494, 496,
498, and 500 cause the computer 64 to wait for a mouse
event, then when the right hand button on the mouse is
pressed draw lines on the screen of video monitor 66
indicating the peak width and end of the peak, and
store the end point of the peak in memory.
If the operator has selected color values to be
analyzed, steps 502, 503, 505 and 507 cause the
computer 64 to convert the y-coordinates interactively
defined for the start and end of each density peak, map
the TLC coordinates to the RGB array rectangle, then
calculate the red, blue and green background densities
for the selected peak. Now referring to Figure 15,
steps 509 and 510 cause the computer 64 to convert the
digital values within the mapped rectangle to density
values, to integrate the area density of the red, blue
and green rectangles by summing the horizontal line
densities between the start and end of the peaks, to
store the respective densities in memory as red sum,
green sum and blue sum and to print the results to the
output display means 68, for example, a printer.
Step 484 of Figure 15 causes the computer 64 to
continue to calculate and print the lane (column)
densities until the "alt" key is pushed by the
operator. If the column number equals 99 then step 462
of Figure 14 causes the computer to return control back
to the menu handling subroutir.e program of Figure 3.
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If the operator has selected gray scale va~ues to
be analyzed, steps 502, 503, and 504 cause the computer
64 to convert the y-coordinates interactively defined
for the start and end of each density peak, then
calculate the gray scale background density for the
selected peak or spot area. Spot area density is
calculated as described above where the area boundaries
have been defined by selection of the start and end of
the corresponding peak in the lane. Now referring to
Figure 16, steps 506 and 508 cause the computer 64 to
convert the digital values within the mapped rectangle
to density values, to integrate the area density of the
rectangle by summing the horizontal line densities
between the start and end of the peaks, to store the
density value in memory as gray scale density and to
print the results to the output display means 68, for
example, a printer. Step 484 of Figure 15 causes the
computer 64 to continue to calculate and print the lane
(column) densities until the "alt" key is pushed by the
operator. If the column number equals 99 then step 462
of Figure 14 causes the computer to return control back
to the menu handling subroutine program of Figure 3.
In a similar fashion to the above mentioned lane
density analysis, the operator may analyze area density
by selecting horizontal rows of spots. Referring back
to Figure 3, steps 132 and 134 cause the computer 64 to
execution the rows subroutine as illustrated in 17.
Referring now to Figure 17, steps 600, 602 and 604
cause the computer to set its color registers for gray
level display, clear the screen of the video monitor
66. then display the digital density data on the video
monitor in shades of gray representative of each point
A density. Steps 606 and 608 cause the computer to
retrieve each digital density value stored in the
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memory array and sets each pixel color corresponding to
these values.
Step 610 causes the computer 64 to check each
binary value of digital density data. When a digital
density data value is less than eight, then step 612
causes the computer to set the pixel color to black.
Likewise, step 614 causes the computer to check for a
binary value of zero, if so, then step 616 causes the
computer to set the pixel color to red. Characterizing
dark level pixel values in this manner facilitates
greater accuracy in the selection of useful area
density evaluation boundaries.
Steps 618 and 620 cause the computer 64 to plot on
the screen of the video monitor 66 all digital density
data values equal to or greater than binary eight.
Steps 622 and 624 cause the computer to initialize the
menu bar on the video monitor 66, set the row counter
to zero, and look for a mouse event to happen. After
the mouse event happens, step 626 causes the computer
to draw a rectangular box on the screen of the video
monitor 66 which encompasses the desired row area, then
return the box coordinates to the program.
Now referring to Figure 18, steps 628, 630 and 532
cause computer 64 to check if the box coordinates are
within the display area, encloses more than one pixel,
and if so, then draw the chosen row on the screen of
video monitor 66. The present invention is capable of
handling spot area density calculations for up to 10
rows of spots. If more than 10 rows are selected for
row density calculation, then steps 634 and 636 will
cause the computer to terminate the program. If 10 or
less rows are selected then step 638 causes the
computer to store the coordinates of the selected rows
in memory as a function of each top and bottom vertical
~ WO94/18800 PCT~S94/0130~
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boundary of each row as displayed on the screen of the
video monitor 66. The operator uses the mouse to
select rows to be analyzed until the "alt" key is
- pressed. After the "alt" key is pressed, steps 640,
642, and 644 cause the computer to clear the screen of
the video monitor 66, and print "wait for integration"
on the screen of the video monitor 66.
Step 646 causes computer 64 to set the row counter
to one and start the one dimensional line density
calculations on the selected row digital density data.
The purpose in calculating one dimensional line
densities is to enable more accurate selection of the
area boundaries used in determining each spot area
density. All digital density values on a given
vertical line within the selected horizontal row are
summed to give a one dimensional line density as a
function of horizontal position. Repeatedly, the
digital density values are summed for each individual
vertical line until all, for example, 256 vertical
lines within the horizontal row are so calculated.
Then a graph of the line densities as a function of
horizontal position within the row is plotted on the
screen of video monitor 66. This graph depicts line
density peaks which are representative of the spot
density boundaries within the row. Thus the start and
finish of the x-coordinates representing spot location
are more easily and repeatably determined.
Step 650 causes the computer to increment the line
density calculations to the next vertical line. Now
referring to Figure 19, the line density calculations
continue to increment to a subs quent line until step
652 causes the computer to determine that the last
vertical line density was calculated. Steps 654 and
656 cause the computer to increment the row counter and
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calculate subsequent row line densities until the last
line density is determined.
Steps 658 and 660 cause the computer 64 to clear
the screen of video monitor 66 and prompt the operator
for the desired row number of the desired density
graph. Step 662 causes the computer to check for a
legitimate row number, if so, then step 668 causes the
computer to prompt the operator for a scaling factor to
be used in plotting the row density graph. If,
however, the row number input is 9~ than steps 662, 664
and 666 cause the computer to return control back to
the menu handling subroutine of Figure 3.
After the operator specifies the requested scaling
factor, step 670 causes the computer to print header
information on the output display means 68. Steps 672
and 674 cause the computer to plot a graph of the line
densities as a function of horizontal position within
the row on the video monitor 66, and print this line
density graph on the output display means 68 (printer).
Steps 676, 678 and 680 cause computer 66 to initialize
the peak counter to zero, change the screen display
cursor to a cross bar, initialize coordinate variables,
and request the operator to select a start point for
the beginning of a peak density representative of the
first spot area within the selected row.
Now referring to Figure 20, step 682 causes the
computer 64 to wait for a mouse event to happen. If a
mouse event happens without the "alt" key being
pressed, then step 686 causes the computer to wait for
the left hand button on the mouse to be pressed. When
the left hand button is pressed, steps 688 and 690
cause the computer to draw a line on the video
monitor 66 indicating the start of a spot area density
peak within the row and store its x-coordinate
~ W O 94/18800 PCTrUS94/0130~
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position. Step 692 causes the computer to request the
operator to select an end point for the termination of
the peak density of the spot area. Steps 694, 696,
698, and 700 cause the computer 64 to wait for a mouse
S event, then when the right hand button on the mouse is
pressed draw lines on the screen of video monitor 66
indicating the peak width and end of the peak, and
store the end point of the peak in memory.
If the operator has selected to analyze color
values, steps 702, 703, 705 and 707 cause the computer
64 to map the TLC coordinates to the RGB array
rectangle, convert the x-coordinates interactively
defined for the start and end of each density peak,
then calculate the red, green and blue background
densities of the selected peak, or spot area. Spot
area density is calculated as described above where the
area boundaries have been defined by selection of the
start and end of the corresponding peak in the row.
Now referring to Figure 20, steps 709 and 710 cause the
computer to ccnvert the digital values within the
mapped rectangle to densities, to integrate the red,
green and blue rectangles and to store the density
values in memory respectively as red sum, green sum and
blue sum. Steps 709 and 710 further cause the computer
to integrate the area density of the selected peaks by
summing the vertical line densities between the start
and end of the peaks, then to print the results to the
output display means 68, for example, a printer.
Step 684 of Figure 20 causes the computer 64 to
continue to calculate and print the row densities until
the "alt" key is pushed by the operator. If the row
number equals 99 then step 662 of Figure 19 causes the
computer to return control back to the menu handling
subroutine program of Figure 3.
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If the operator has selected to analyze gray scale
values, steps 702, 703 and 704 cause the computer 64 to
convert the x-coordinates interactively defined for the
start and end of each density peak, then calculate the
background density of the selected peak, or spot area.
Spot area density is calculated as described above
where the area boundaries have been defined by
selection of the start and end of the corresponding
peak in the row. Now referring to Figure 21, steps 706
and 708 cause the computer to integrate the area
density of the selected peaks by summing the vertical
line densities between the start and end of the peaks,
then print the results to the output display means 68,
for example, a printer. Step 684 of Figure 20 causes
the computer 64 to continue to calculate and print the
row densities until the "alt" key is pushed by the
operator. If the row number equals 99 then step 662 of
Figure 19 causes the computer to return control back to
the menu handling subroutine program of Figure 3.
Thus, it will be appreciated that a new and
improved video area densitometer has been described
which achieves faster acquisition of video information
from a thin layer chromatographic slide. Data
acquisition by a preferred embodiment of the invention
is accomplished within 1/60th of a second. This rapid
acquisition time of a complete video frame reduces the
probability of measurement equipment drift and/or
subject specimen degradation due to factors beyond the
control of the measurement technician.
In addition, the present invention allows the
maximum resolution of a subject specimen by presetting
absolute values of white level video intensity and
black level video intensity so as to maximize the video
resolution of the subject specimen. The present
_ WO94/18800 PCT~S94/013Q~
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invention by use of a digital computer may store high
resolution video digital data representative of the
original analog video signal. Once the analog video
signal has been captured in computer memory, the
representative video digital data may be mathematically
manipulated to disclose useful information. The
present invention enables reliable and repeatable test
results. As mentioned above, the tests performed and
experiments run gave extremely reliable and repeatable
results.
Although several preferred embodiments are
described in a fair amount of detail, it is understood
that such detail is for the purpose of clarification
only. Various modifications and changes will be
apparent to one having ordinary skill in the art
without departing from the spirit and scope of the
invention as hereinafter set forth in the claims.
What is claimed is:
