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 IMAGING SYSTEM 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. ELECTRONIC COLUMN IMAGING SYSTEM + 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 449 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 SUTHERLAND 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- ELECTRONIC 0 0 I...,..........1 150 300 COLUMN 450 NUMBER IMAGING 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 451 SYSTEM FOR GEL FLUORESCENCE 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 k : SUTHERLAND 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. ELECTRONIC IMAGING ii X 64 _ 0 I v. 0 2 INTEGRATION 4 6 TIME 453 SYSTEM FOR GEL FLUORESCENCE 8 <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 IMAGING 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). FOR GEL 455 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. REFERENCES 1. Freeman, S. E., Blackeet, A. D., Monteleone, D. C., Setlow, R. B., Sutherland, B. M., and Sutherland, J. C. (1986) Anal. 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