ANALYTICAL
BIOCHEMISTRY
163,446-457
(I 987)
Electronic Imaging System for Direct and Rapid Quantitation
Fluorescence from Electrophoretic
Gels: Application to
Ethidium Bromide-Stained
DNA’
of
JOHNCLARKSUTHERLAND,'BOHAILIN,~
DENISEC.MONTELEONE,JOANNMUGAVERO,
BETSYM.SUTHERLAND,ANDJOHNTRUNK
Biology
Department,
Brookhaven
National
Laboratory,
Upton,
New
York
11973
Received December 8, 1986
We have built an electronic imaging system based on a modified charge-coupled-device
television camera that directly quantitates the distribution of fluorescence from electrophoretic
gels, chromatograms, and other stationary sources. Exposure times can exceed 1 min. Unlike
the photographic system that it replaces, the response of the camera is directly proportional to
the intensity of incident fluorescence, and image data are digitized and stored in computer
memory ready for analysis immediately upon completion of an exposure. We describe procedures for the display, normalization, and archival storage of image data and programs that use
images of ethidium bromide-stained DNA in alkaline agarose gels to quantitate single-strand
breaks in DNA.
Gel electrophoresis of DNA is one of the
most widely used methods in molecular biology. At present, most applications use only
the spatial information
from a gel, i.e., the
mobilities
of DNA molecules of different
lengths or conformations.
Information
obtainable from the relative amounts of DNA
at different positions on a gel is usually used
only in a qualitative manner or, more often,
ignored completely. Use of this “amplitude”
data, obtained by staining the DNA with a
dye such as ethidium bromide and quantitating the fluorescence of the dye induced by
ultraviolet excitation, can increase significantly the information
obtained from an
electrophoretic
analysis. One application
that requires the measurement of the distribution of DNA along the direction of migra’ This research was sponsored by the Office of Health
and Environmental Research, U.S. Department of Energy and by a grant from the National Institutes of
Health (GM-35662) to J.C.S.
’ To whom correspondence should be addressed.
3 Present address: Institute of Biophysics, Academia
Sinica, Bejing, China.
0003-2697187
$3.00
446
tion is the quantitation of radiation-, chemical-, and enzyme-induced
single-strand
breaks in DNA using alkaline gel electrophoresis (1,2). This method has been used to
measure pyrimidine dimers produced by uv
irradiation of DNA in human skin in situ
(3-7). After electrophoresis, the agarose gel is
neutralized and stained with ethidium bromide and the spatial distribution
of uv-induced fluorescence is recorded to compute
the number of single-strand breaks per thousand base pairs (1).
Until now, the gel electrophoresis method
for measurement of strand breaks in DNA
used photography to record the pattern of
fluorescence from a gel (l-8). However, the
optical density of a photographic negative is
a nonlinear function of the incident visible
light that exposed it (see e.g., Ref. (9)). A
number of procedures for converting the
density of a photographic negative to a value
proportional
to gel fluorescence have been
described (8,l O-l 3). The early methods were
laborious (lo- 12) and the accurate calculation of fluorescence was difficult because of
ELECTRONIC
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the nonuniform distribution of DNA across
the width of a lane (8). Another disadvantage
of photographic
film is the limited range,
typically less than two orders of magnitude,
of incident intensities that can be recorded
between the threshold and saturation levels
of the emulsion (10,ll). Finally, the processing of film data, developing, washing, drying,
and digitizing, is labor intensive and time
consuming.
We have built an electronic imaging system that uses a modified CCD4 TV camera
to record, digitize, and store in computer
memory the fluorescence from a gel. Cameras using solid-state CCD light detectors,
unlike older vacuum tube TV cameras, respond linearly to incident light. The range of
intensities measurable in a single exposure
with our system is limited to 256 gray levels
by the resolution of an analog to digital converter (ADC) but can be extended with multiple exposures of different durations. We
have also performed preliminary studies with
a slow-readout CCD camera that can respond linearly over a wider range of incident
intensities.
The time elapsed between termination
of
an exposure of a gel containing 15 lanes until
the completion of the storage of the resulting
digital data, a process requiring 2-3 h with
our electromechanical
scanner (8) drops to
33 ms for our TV camera-based electronic
imaging system. Each image produced by the
new system contains about 370,000 pixels,
thus making practical more sophisticated
procedures for the subtraction
of background fluorescence and the normalization
of image data for spatial variation in the sensitivity of the imaging system.
The measurement of single-strand breaks
usually requires electrophoretic separation of
the sample in only one dimension. Since the
imaging system acquires a complete two-dimensional image of the gel, it can also be
4 Abbreviations used: ADC, analog to digital converter: CCD, charge-coupled device; DAC, digital to analog converter: RGB, red-green-blue; TV, television.
FOR GEL FLUORESCENCE
447
used to analyze the distribution
of fluorophore-labeled DNA that has been dispersed
in two dimensions. In addition to DNA, it
can be used to record the distribution
of
other fluorophore-labeled
macromolecules
such as proteins separated on gels or even
intrinsic fluorophores separated on chromatograms.
DESIGN
AND OPERATION
The major components of the electronic
imaging system are shown schematically in
Fig. 1. The fluorophore-stained
gel is placed
atop a source of uv radiation. The gel can
also be illuminated
from above by an incandescent spotlight for alignment. The sense
head of the camera is directly above the gel.
A lens forms an image of the gel on the CCD
sensor. The sense head is mounted in a refrigerated housing to inhibit the buildup of
thermal “noise” signals during timed exposures that may last over 100 s. The interior of
the refrigerated housing is continuously
purged with dry nitrogen gas to prevent condensation.
The monochrome
CCD-TV camera system (Model CCD 3000, Fairchild Weston
Systems, Sunnyvale, CA) consists of three
parts, sense head, camera control unit, and
INTERFACE++
GEL ,\
f-MODIFY
++BUILD
FIG. 1. Schematic diagram of the components of the
CCD-TV camera-based imaging system. Components
that were modified are indicated by #. Components that
we designed and built are indicated by ##.
448
SUTHERLAND
power supply-control
interface, which are
connected by multiconductor
cables. The
sense head must be located away from the
control unit since the latter is too big to fit
into the refrigerated housing and may operate improperly at temperatures below 0°C.
Analog image data from the camera is
coded in a closed-circuit TV format. This
signal is connected to the monochrome input
of a color monitor (Model CT- 1400 MG,
Panasonic Industrial Co., Secaucus, NJ) and
also goes to a frame grabber (Model RTPQVG-123-2, Datacube Inc., Peabody, MA)
via a video interface which we constructed.
The RGB (color) signal from the frame grabber is connected to the color input of the
monitor,
also via our video interface.
Switches on the front of the monitor select
the input signal to be displayed.
The critical function of the video interface
is to buffer and decode logic signals from the
frame grabber that we use to inhibit the readout of the CCD sensor and thus permit extended exposures of the CCD sensor to the
weak fluorescence from the gel. Obtaining
the necessary logic signals required modifications to the frame grabber. The control interface of the camera was also modified to
accept the readout-inhibit
signal from the
video interlace.
The frame grabber is connected to the
input/output
bus of a MicroVAX
II computer (Digital Equipment Corp., Maynard,
MA). The operator controls the frame grabber from a graphics terminal that also displays plots of row, column, and lane profiles,
as discussed below. Images in digital form are
transferred to an optical disk for archival
storage About 1300 images can be stored on
each side of each removable disk.
The system can acquire images in three
modes that we call VIEW, GRAB, and INTEGRATE.5 In the VIEW mode, the frame
grabber digitizes each of the 30 images generated per second by the camera. Each suc5 Command keywords used in our programs are
printed in capital letters.
ET AL.
cessive image overwrites the previous image
in the memory of the frame grabber. The
digitized values are also routed to the color
monitor. VIEW mode is used to align the
wells of a gel with respect to the camera’s
field of view using reflected light from an
incandescent spot lamp. The advantage of
using the RGB image generated by the
VIEW mode rather than the monochrome
signal from the camera is that we can superimpose an alignment grid on the digitally
generated image. The GRAB mode causes
the frame grabber to digitize a single image.
The RGB output continues to display the
stored image while the stream of new images
from the camera is ignored by the frame
grabber. Image acquisition modes similar to
VIEW and GRAB are standard features of
most frame grabbers.
We use the INTEGRATE
mode to acquire
the image of fluorescence from a gel. This
special mode is made possible by the video
interface that we built and by modifications
made to the frame grabber and camera control interface. During an integration
the
video interface generates a signal that inhibits the readout of the CCD sensor. Lightinduced electronic charge accumulates in
each pixel area of the sensor while the readout is not operating. After a predetermined
exposure time, the video interface automatically restores sensor readout. A single video
frame that contains all of the signal resulting
from light incident on the CCD during the
period of integration is generated by the camera and digitized by the frame grabber.
The frame grabber can superimpose an
alpha-numeric overlay on the image. We use
this feature to generate a grid to aid in the
alignment of gels. The lower edges of the
wells are aligned with the horizontal dashed
grid line and the wells are positioned midway
between the vertical grid marks as shown in
Fig. 2A. The alpha-numeric
overlay also
labels each of the 15 lanes of the gel, but it
does not change any of the image data. The
operator can turn the overlay on and off as
desired.
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FIG. 2. Photographs of monochrome displays of
images of a pulsed alkaline 0.4% agarose gel (2). Pulses of
1.1 V/mm lasting 0.3 s were applied every 10 s for 17 h.
The image of the gel shown in (A) was acquired in 10 s
while the image in (B) was acquired in 20 s. (A) Shows
the alignment grid overlay plus row 75, and columns
167, 212, 257, and 685 in white. Lanes 1, 8, and 15
contain a mixture of DNAs of defined length (see Fig. 5).
All other lanes contain about 100 ng of DNA extracted
from cultured human cells and irradiated with 254 nm
uv light: lanes 2 and 3, no uv light; lanes 4 and 5, 0.2
J/m’; lanes 6 and 7, 0.5 J/m2; lanes 9 and 10, 1 J/m2;
lanes 11 and 12, 2 J/m’; lanes 13 and 14, 3 J/m2. For
each uv exposure the sample in the higher numbered
lane was treated with the uv endonuclease from Micrococcus luteus prior to electrophoresis (I). The other sample was not treated with endonuclease. The diffuse fluorescence between rows 350 and 400 is due to ethidium
bromide-stained RNA. Curvature of rows and columns
near the edges of the images results from photographing
the curved surface of the video monitor and not from
distortions in the digital image (c.f. Fig. 7).
The output of the frame grabber can be
displayed as a monochrome
(black and
white) image as shown in Fig. 2 or as a color-
FOR GEL FLUORESCENCE
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enhanced image in which different levels of
fluorescent intensity are displayed as different colors. Color enhancement facilitates the
detection of slight changes in intensity since
the human eye can distinguish more hues of
color than shades of gray. We use a modified
“rainbow” transformation to convert intensity values into colors. Low pixel values appear blue, midrange values are green, and
high values are red. There is a smooth variation in hue with changing data values. The
color-enhanced displays of the same images
that produced Fig. 2 are shown in Fig. 3. We
depart from the strict rainbow transformation for data values of zero and 255. These
are the minimum and maximum values that
can be generated by the &bit ADC. They are
also the values returned if the input analog
signal is respectively less than or greater than
the input range of the ADC. We make areas
of ADC underflow or overflow conspicuous
on the color monitor by causing areas of underflow to appear black and areas of overflow to appear white, thus distinguishing
them from all intermediate values. For example, saturation of several bands is immediately apparent in the pseudo-color display
of the image shown in Fig. 3B, while in the
monochrome
display of the same image
shown in Fig. 2B it is difficult to determine
that some of the bands are saturated while
others are not.
Our programs have three modes for displaying subsets of the data from an image:
ROW, COLUMN,
and LANE. The ROW
and COLUMN
functions extract the picture
elements from a specified row or column of
an image and plot them on the screen of the
graphics terminal. The row or column being
displayed is indicated on the color monitor
as a white line as shown in Fig. 2A. The corresponding row and column plots are shown
in Figs. 4 and 5. Note that for a plot of image
data across a row the independent variable
is column number, while for a plot of column data, the independent variable is row
number.
The LANE function generates an average
450
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ET AL.
FIG. 3. Photographs of the pseudo-color display of the same two images shown in Fig. 2. The white
centers of several of the bands in the 20-s image (B) indicate that the detector is saturated, a condition that
is not immediately obvious in the corresponding monochrome display.
of tl re values of all the image elements along
eacf 1row between two specified columns that
are usually selected by the operator to be
mid lway between adjacent wells. Figure 6
shoj w’sthe profiles of lanes 4 and 5 in Fig. 2A.
The averaging process reduces the scatter
the lane plot compared to a single column
We can also use the two extreme colum
that define a row to compute the portion
the lane profile above the flanking bat
in
ns
of
k-
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I...,..........1
150
300
COLUMN
450
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600
60
750
FIG. 4. Profile of row 75 from the image shown in Figs.
2A and 3A demonstrating the variations in the distribution of DNA that can exist across a iane. Endonucleaseproduced single-strand breaks result in progressive reduction in the amount of DNA at row 75 for lanes 5, 7,
10. 12, and 14.
ground level, as described previously (8). The
20- to 30-fold increase in the time required to
average across a lane with our electromechanical scanner (8) resulted in this mode
being used infrequently. With the electronic
imaging system, however, lane averaging requires little additional time and is performed
routinely, hence improving the accuracy of a
measurement. A more sophisticated procedure for background subtraction and image
normalization
is presented in the discussion.
250
I
200
z
(D
150
Z
100
f
i
0
50
O Oi60
ROW
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NUMBER
FIG. 5. Profile of column 685 from the image shown in
Figs. 2A and 3A showing the separation of DNA length
standards achievable with unidirectional pulsed-field
electrophoresis (2). In order of increasing distance of
migration the length standards are phage T4 DNA ( I SO
kb), phage X DNA (48.5 kb), phage T7 DNA (40 kb), and
the three fragments of T7 resulting from digestion with
the endonuclease Bgll (22.5, 13.5, and 4 kb, respectively).
25
I
so
75
ROW
100
NUMBER
125
150
FIG. 6. The averaged profiles of lanes 4 and 5 from the
image shown in Figs. 2A and 3A. These two lanes contain human DNA that was irradiated with 0.2 J/m2 of
254~nm uv radiation. The sample in lane 5 was treated
prior to electrophoresis with the endonuclease from Micrococcus luteus that produces single-strand breaks adjacent to pyrimidine dimers. The sample in lane 4 was not
treated with endonuclease. Using the procedures described in Refs. (1) and (2), we calculated that the number average molecular lengths for these samples are 137
kb (lane 4) and 129 kb (lane 5) corresponding to 0.45
pyrimidine dimers per million bases.
PERFORMANCE
The digital image should record accurately
the intensity of fluorescence as a function of
position in the plane of the gel. We thus investigated both the spatial fidelity and photometric performance of our system.
Spatialjdelity.
We tested the spatial fidelity of the camera system by placing a piece of
graph paper ruled with 10 lines per inch in
the object plane, i.e., the position of the gel.
The graph paper was oriented with its axes
parallel to the horizontal and vertical axes of
the camera, respectively. An image was recorded with the graph paper illuminated
from above by an incandescent spot lamp
(instead of from below with the uv source
used for quantitation of fluorescence). Nine
COLUMN profiles of this image, taken from
different regions of the image, are shown in
Fig. 7. The profiles have been displaced vertically for clarity. The periodic dips in the
profiles are due to the grid lines of the graph
paper. These data show that the separation of
the grid lines are the same for all parts of the
452
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:
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600
500
Z
400
300
5
200
i
100
0
0
0
100
200
ROW
300
NUMBER
400
500
FIG. 7. Column profiles from an image of a piece of
graph paper. The data have been displaced vertically for
clarity. The numbers at the left indicate the column of
pixels displayed. The regular dips every 25 pixels in the
column profiles reflect the grid lines of the graph paper,
which are spaced one-tenth of an inch apart. Thus, for
this image (and all other images shown in this paper)
there are about 10 pixels/mm. The spatial resolution is
easily changed by moving the camera vertically with
respect to the gel and refocusing the lens.
image. Similar data (not shown) were obtained for a set of ROW profiles. Thus we
conclude that the images produced by the
camera system are accurate representations
of the location of points in the object plane.
Photometric performance. The photometric properties of the system can be influenced
by the uv illuminator,
the lens, and the CCD
detector. Ideally, the response of the detector
elements should be a linear function of the
fluorescence emitted from the corresponding
location in the object plane. If the fluorescence intensity is constant in time, then the
signal accumulated by the detector element
should be a linear function of the exposure
time. The intensity of the uv excitation light
should be uniform over the object plane and
the sensitivity of detection of fluorescence
should be uniform for all pixel detector elements in the image plane. Finally, the integrated fluorescence produced by a band of
fluorophore-stained
DNA in a gel should be
proportional to the quantity of DNA loaded
prior to electrophoresis. Linearity of fluorescence as a function of DNA is not, however,
an automatic consequence of satisfactory
performance characteristics of the individual
ET AL.
optical and electronic components of the
system.
Tests of the individual
components required a fluorescent reference that can cover
the field of view in the object plane with uniform efficiency at all points and cannot be
changed by age or bleached by exposure to
uv light. We chose as our standard fluorescent object an orange glass filter 165 by 165
by 2.5 mm (Hoya Glass Works, Freemont,
CA). The fluorescence from this glass is in
the same wavelength region as ethidium bromide (14), and it absorbs all of the incident
uv light for all wavelengths in the spectrum
of our illuminator.
We demonstrated that
the efficiency of fluorescence of the filter was
constant at all points by recording the images
produced when the filter was placed on the
uv illuminator
in place of the gel. The image
intensities produced by a given period of exposure were the same when the filter was rotated through various angles about the vertical axis (data not shown).
We tested the response of the camera as a
function of fluorescence intensity by placing
neutral density filters on top of the fluorescent filter. The intensity recorded by the
camera for a fixed time of integration is a
256-
0
0
Y
20
PERCENT
40
60
TRANSMISSION
60
I
I 00
FIG. 8. Values of fluorescence from an orange glass
filter excited by uv light as a function of the transmission
of neutral density filters (Model 202, Gilford Instrument
Laboratories, Oberlin, OH). The transmission of the
filters were 76.9 and 8.8% and were checked with a spectrophotometer. The point for 100% transmission was
obtained using a blank filter holder to define the area of
the image to be sampled. A separate 5-s image was used
to measure each point.
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_
0
I
v.
0
2
INTEGRATION
4
6
TIME
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<SEC>
FIG. 9. Values of fluorescence from an orange filter as
a function of the time of integration used to produce an
image. The linearity of this response means that we can
compare data from different images of a gel recorded
with different exposure times, thus increasing the range
of intensities that can be measured with the camera.
linear function of the transmission
of the
neutral density filters, which were determined at 600 nm using a Cat-y 118 spectrophotometer (Varian Associates, Palo Alto,
CA). These results are shown in Fig. 8.
Response as a function of time of integration was tested by plotting the response obtained at a point on the image as a function
of exposure time. The response is linear, as
shown in Fig. 9. The data in Fig. 9 extrapolate to the origin as a result of careful adjustment of the “video output black level” of the
camera, a procedure described in the instruction manual supplied by Fairchild. This
adjustment suppresses the thermally generated noise signal and thus is sensitive to the
temperature of the CCD sensor. If the sensor
is cooled after the adjustment is made, the
response, while linear, may extrapolate to
zero for some finite fluorescence intensity,
resulting in spurious quantitation.
Our programs alert the operator to areas of the image
where fluorescence does not register by displaying pixel values of 0 as black in the
pseudo-color display. When the background
level is properly adjusted and the sense head
is held near -30°C
thermally
generated
noise saturates the camera’s output for integration times of about 15 min. Thus, for ex-
posures lasting 90 s, thermal noise contributes about 10% of the total signal.
Response as a function of position in the
image is determined by the combined effects
of the uv illuminator,
the camera lens, and
the CCD detector. Fairchild specifies a typical photoresponse nonuniformity
of 1% and
a maximum
photoresponse nonuniformity
of 10% of the average response of the detector. The camera lens can contribute to nonuniform photometric
response due to vignetting and other geometrical factors (see
e.g., Ref (15)). Finally, the intensity of the
exciting uv light from the illuminator
may
vary across the surface of the object plane.
We constructed a uv illuminator
to minimize variations in source intensity. The necessity for a special illuminator
is demonstrated in Fig. 10A which shows a column
250
A
250
B
0
?I
200.
150,
:
1
100.
i0
50
0
_
0
t
100
200
ROW
300
400
F JO
NUMBER
FIG. IO. Column profiles of images of fluorescence
from an orange glass filter recorded by the CCD-TV
camera. (A) Fluorescence excited with a commercial illuminator. (B) Fluorescence excited with the illuminator
that we constructed.
454
SUTHERLAND
profile from an image of the fluorescence
from the orange filter excited by uv light
from a commercially available gel illuminator. The figure represents a section of the
image perpendicular to the orientation of the
uv light bulbs. (When this illuminator is used
to photograph gels, the lanes are usually oriented parallel to the axis of the bulbs, so variations along the length of a lane will be less
dramatic than the profile shown.) The inhomogeneity
of this exciting source would
complicate the extraction of quantitative
data from an image. The spatial inhomogeneity shown in Fig. 1OA is typical of several
commercial
units from different vendors
that we tested. The corresponding column
profile recorded with the illuminator
that we
built is shown in Fig. 10B. The illuminator
was also oriented with the long axis of the
bulbs perpendicular
to the section of the
image displayed. Analysis of column and
row profiles from different regions of the gel
indicate that we can achieve spatial uniformity of better than 5% over the region of a
gel occupied by a given lane. The accuracy of
determining the distribution of DNA along a
lane can be improved further by normalizing
the experimental data by the reference image
obtained by recording fluorescence from the
orange filter (vide infiu).
Linearity of response as a function of the
quantity of DNA in a band is demonstrated
by the data in Fig. 11. Increasing amounts of
DNA were loaded into the wells of a gel and
electrophoresed into the agarose matrix. To
obtain the entire range, images of the gel
were acquired for 2,6, and 20 s and the integrated fluorescence under the peaks was
scaled accordingly. The quantities of DNA
loaded into the wells spanned a range of 3
decades (0.5 to 500 ng) and the average response of the camera was a monotonically
increasing function over this range. However, a range this large cannot be plotted satisfactorily using linear scales. Thus, in Fig. 11
we plot the logarithm of fluorescence versus
the logarithm
of the quantity
of DNA
loaded. The solid line through the data from
ET AL.
*
loo-
200
/’
B 50E
/:
/’
p
E 202 lo2
52-
.
l-
a
/
.
4:
l
l
.
.5.
I
.2
I
.5 t
1
2
I
1
1
5 10 20
1
’
’
50 100200
’
500
1000
FIG. 11. Integrated fluorescence in a lane as a function
of the amount of DNA loaded into each well prior to
electrophoresis. The samples of T7 DNA were obtained
by serial dilution from a single stock. They were electrophoresed on a 0.4%, pH 6.4, agarose gel at 4°C for 17 h
with a field of 0.5 V/cm. The gel was stained with ethidium bromide solution for 15 min and distained in distilled water for 150 min. Three exposures of 2,6, and 20
s were required to record the fluorescence of all samples,
and fluorescence intensities have been scaled accordingly. The straight line has a slope of unity indicating
that integrated fluorescence is a linear function of the
amount of DNA loaded from 2 to 120 ng per band.
2 to 120 ng of DNA has a slope of unity on
the log-log plot. The slope of unity on the
log-log plot indicates that over this range fluorescence is directly proportional
to the
quantity of DNA loaded on the gel. The deviation from the unitary slope for smaller
amounts of DNA may be due to increased
scatter in the data and difficulties in separating the DNA-specific fluorescence from the
fluorescence of unbound ethidium remaining in the gel. Above 200 ng, the deviation
from linearity may reflect the inner filter effect as well as problems associated with overloading the gel. The range of the linear response is greater than we reported previously
using photographic film (8).
DISCUSSION
Our electronic imaging system, based on a
modified CCD television camera, improves
ELECTRONIC
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SYSTEM
the accuracy and speed of the gel electrophoresis method for measuring DNA strand
breaks and may find uses in other areas of
molecular biology. Further improvements in
spatial resolution and photometric
performance are possible using available slow-readout CCD cameras, but these devices are significantly more expensive.
Comparison of the electronic imaging sys-
tem with photographic detection of gel jluorescence. Electronic imaging with a CCD
camera offers three advantages compared to
photographic
film in the measurement
of
single-strand breaks using agarose gel electrophoresis: the response of a CCD detector is a
linear function of the intensity of the fluorescent light, the dynamic range of the detector
is greater than that of photographic film, and
the time required to obtain image data in
digital form is reduced dramatically.
Net
costs favor electronic imaging if many gels
are processed.
The optical density of photographic film is
not a linear function of the intensity of light
to which it was exposed. Several methods
have been reported to convert film density to
a parameter proportional to incident intensity (8,10-l 3) but some are tedious and all
require extra processing of experimental data
which may result in less accurate results. In
contrast, CCD detectors respond linearly to
incident light over a wide range of intensities.
The dynamic range of a detector is the
ratio of incident intensity that starts to saturate the output to the equivalent intensity of
the dark signal. For the Polaroid type 55 film
used previously in the measurement of single-strand breaks by agarose gel electrophoresis (1,2,8) the dynamic range is about 506
while the Fairchild CCD 222 sensor in our
camera has a dynamic range greater than
1000.7 However, all of the detector’s dynamic range cannot be used in a single expo6 Value estimated from the characteristic curve for
type 55 negatives supplied by Polaroid Corp. (Cambridge, MA).
7 From specifications for the type CCD 222 area image
sensor, Fairchild Weston Systems, Inc. (Sunnyvale, CA).
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FLUORESCENCE
sure because the 8-bit ADC of the frame
grabber limits the precision of measurement
to 1 part in 256. Multiple exposures for different periods of integration can result in a
higher effective dynamic range.
The electronic imaging system requires
much less time between completion of the
exposure and storage of the photometric data
in computer memory ready for analysis. The
time required to develop, rinse, dry, and scan
a photographic negative containing 15 lanes
is typically 2-3 h using our present gel scanner (8); the electronic imaging system requires only 33 ms. The scanner acquires a
few hundred points for each of the 15 lanes
while the imaging system generates a 480 by
768 pixel image thus facilitating background
subtraction and averaging across the width of
a lane (8).
Rapid acquisition of complete two-dimensional images also makes possible more sophisticated methods of background subtraction and image normalization.
Suppose that
Z,, (i, j) is the value of the pixel in row i and
column j of the image of some sample and
that I&,(& i) is the value of this same pixel
after background subtraction and normalization for system response. The corrected
image, Zz,, is calculated from the experimental image using the equation
~~,(i,i) =
km-@,
j)
-
zbkg(i,
-
~“d~i)
i)
t11
Lzf(i,A
A background image, Zbkg, can either be determined experimentally using a gel containing no DNA that has been processed like the
sample gel or be calculated from the sample
image using points outside of the lanes. A
reference image, Zref, is generated by recording an image of a fluorescent object such as
our orange glass plate that converts incident
uv light into fluorescence with constant efficiency. Finally, a null image, InUll, is obtained
by performing a timed exposure while blocking all incident light from reaching the detector. The exposure time for the sample
image must equal that of the background
image and the exposure time for the refer-
456
SUTHERLAND
ence image must equal that for the null
image. The denominator of Eq. [l] corrects
for spatial variations in both the intensity of
the excitation source and in the sensitivity of
the CCD detector. For short exposure times,
the amplitude of this null image is usually
insignificant.
The initial cost of an electronic imaging
system is greater than a photographic system,
but we believe that the improvements
in
photometric performance and the more sophisticated data analysis that the complete
images make possible justify the difference.
If many gels are analyzed, incremental operating costs are much lower for the electronic
imaging system. The greatest savings are in
personnel costs. Each 15-lane gel requires
several hours longer to process if photography is used. Media costs also favor electronic
imaging. The cost of the portion of an optical
disk required to store the image of a gel is
about only 10% of the cost of one exposure of
type 55 Polaroid positive-negative film.
We find that background levels are more
uniform across an image generated with the
CCD camera system compared to photographic film. This improvement
in uniformity appears due to the inherently more uniform response of the CCD detector as well as
the more homogeneous output of our new
excitation source. The combined effects of
the improved signal to noise ratio of lane
profiles demonstrated in Fig. 6 compare favorably with results obtained with photographic detection (1,7). The more sophisticated procedures for background subtraction
and image normalization
made practicable
by the rapid digitization of the entire image
plus the improved dispersion of long DNA
molecules obtained with pulsed-field electrophoresis (2) yield a significant improvement
in the sensitivity of measuring DNA damage.
For example, the difference in number average molecular lengths for the two DNA profiles shown in Fig. 6 corresponds to 0.45 pyrimidine dimer per million bases, and the
quality of the data in Fig. 6 suggests that even
higher sensitivity is possible. In contrast, the
ET AL.
sensitivity limit using our previous system
was a one dimer per million bases (1).
Alternate designs for electronic imaging
systems.The imaging system we assembled
was based in part on components already
available in our laboratory. Many other configurations based on TV cameras are possible
with presently available equipment.
Since
electronic imaging is a rapidly evolving technology, we anticipate continued improvements in performance and decreases in cost.
Slow-readout CCD cameras designed for scientific applications can also be used to measure fluorescence from electrophoretic gels.
We tested a cryogenically cooled CCD camera supplied by Princeton Scientific Instruments (Princeton,
NJ), easily obtaining
images of ethidium bromide-stained DNA in
an agarose gel. While we have not had the
opportunity to evaluate the performance of a
slow-readout CCD camera in detail, certain
differences are clear. The analog signal from
a slow-readout camera has less noise and can
be digitized to 14 or even 16 bits giving resolutions of one part in 16,384 or 65,536, respectively. The inherent dynamic range of
available scientific CCD detectors are greater
than 70,000.8 Thus fluorescence can be measured more precisely and over a wider range
of intensities in a single exposure. Scientific
CCD detectors can be cooled to below
-80°C
resulting in integration
times of
hours without significant buildup of thermally induced background. Longer exposure
times can be used to lower the limits of DNA
detection provided a means can be found to
reduce background fluorescence from fluorophore not bound to DNA. Some scientific
CCD detectors have many more pixels (up to
2048 by 2048) than the CCDs used in TV
cameras. The major disadvantages of slowreadout CCD cameras are their higher initial
cost and the lack of a VIEW mode to facilitate focusing of the optics and alignment of
the gels.
*From preliminary
specifications for the type
TK5 12M imager, Tektronix, Inc. (Beaverton, OR).
ELECTRONIC
IMAGING
SYSTEM
ACKNOWLEDGMENTS
We thank Drs. J. M. Preses and Jon Hanson, Brookhaven National Laboratory for numerous helpful discussions. We also thank John Lowrance, Princeton Scientific Instruments, for letting us use a slow-readout
CCD camera system to record fluorescence from a gel.
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