Line scanning system for direct digital chemiluminescence imaging of

A ~ I them. . 1009, 65, 1785-1793

1785

Line Scanning System for Direct Digital Chemiluminescence Imaging of DNA Sequencing Blots Achim E. Karger’,.Robert Weiss, and Raymond F. Gesteland Department of Human Genetics and the Howard Hughes Medical Institute, University of Utah, and Eccles Institute of Human Genetics, Salt Lake City, Utah 84112

Two-dimensionalcharge-coupleddevice (CCD)arrays can be used to substitute film as the recording media in imaging cameras. In the most straightforward operational mode a CCD image is acquired as a snapshot, analogous to the operation of a photographic camera. Due to their high sensitivity, CCD cameras are ideally suited for the digital imaging of electrophoretic gels and blots, and a variety of different detection methods can be applied. High-sensitivity fluorescence CCD imaging has been used to detect DNA,’ proteins,Z and carbohydrates3on snapshots of electrophoretic gels. DNA bands in agarose gels have been visualized on CCD images based on absorption4 and electric birefringence.696 Enzyme-triggeredchemiluminescenceis the most sensitive method for the detection of biopolymers on blots by CCD

imaging. The visualization of DNA covalently bound to a blotting membrane is required in important techniques based on nucleic acid hybridization, such as Southern blotting’ and multiplex DNA sequencing.8 CCD imaging of such blots has been shown for both of today’s most widely used enzymatic chemiluminescence assays, the alkaline phosphatase (AP) triggered chemiluminescence of the AP substrate 3-(2’spiroadamantane)-4-methoxy-4-13’’-(phosphoryloxy)phenyl11,a-dioxetane(AMPPD)SJOandenhanced chemiluminescence (ECL) based on the horseradish peroxidase-luminol system.11-13 The use of AMPPD in a variety of other applications has recently been reviewed.14 The major advantages of digital imaging,in particular, fast visualization, high sensitivity, quantitative imaging, and computer readable data format, are well documented by these studies. When compared to other methods of visualization, however, such as autoradiography using isotope labels and X-ray film,the most obvious limitation of CCD imaging lies in the dimensions of the sensor arrays most commonly used in analytical applications. Their limited size rules out the recording of high-resolution electropherograms on a single frame. The CCD arrays used in the referenced studies offer between 512 and 768 CCD elements along their long axis, depending on the array model. The large number of bands that can be resolved by high-resolution electrophoretic methods by far exceeds the number of bands that can be adequately sampled on such an array. If one requires a minimum of 8-10 data points for the adequate sampling of an electrophoretic band, it becomes evident that none of these imagers can record more than 100 bands per lane on a single frame. Despite this problem, snapshot CCD imaging has been applied to high-resolution electrophoresis, e.g., 2-D protein electrophoresis,2 high-resolution carbohydrate separation? and DNA sequencing.10 One way to obtain a CCD image with adequate sampling over the entire surface of these electropherograms is by manually merging partially overlapping individual frames on a computer screen using image analysis tool.8 This procedure is however time consuming and labor intensive, and the quality of the resulting composite image is compromised by discontinuities. An improvement to the situation can be envisioned by the use of larger CCD arrays, even though no such application has been reported to this day. CCD arrays consisting of 2048

* To whom correspondence should be addressed. Present address: German Cancer Research Center, lm Neuenheimer Feld 506,W-6900 Heidelberg, Germany. (1)Ribeiro, E.A.; Sutherland, J. C. Anol. Biochem. 1991,194,174184. (2)Jackson, P.; Urwin, V.E.; Mackay, C. D. Electrophoresis 1988,9, 330-339. (3)Jackson, P.Biochem. J . 1990,270,705-713. (4)Chan, K. C.;Koutny, L. B.; Yeung, E. S. A d . Chem. 1991,63, 746-750. (5)Lanan, M.;Shick, R.; Morris, M. D. Biopolymers 1991,31,10951104. (6) Lanan, M.; Grossmann, D. W.; Morris, M. D. Anal. Chem. 1992, 64,1967-1972.

(7)Southern, E. M. J. Mol. Biol. 1975,98,503-517. (8)Church, G.; Kieffer-Higgins, S. Science 1988,240,186-188. (9)Karger, A. E.; Ives, J. T.; Weiss, R. B.; Harris, J. M.; Gesteland, R. F.ROC. SPIE 1990,1206,7a-a9. (10)Karger, A. E.;Weiss, R. B.; Gesteland, R. F. Nucleic Acids Res., 1992,40,6657-6665. (11)Misiura, K.; Durran, I.; Evans, M. R.; Gait, M. J. Nucleic Acids Res. 1990,18,4345-4354. (12)Pollard-Knight, D.;Read, C. A,; Domes, M. J.; Howard, L. A.; Leadbetter, M. R.; Pheby, S. A.; McNaughton, E.; Syms, A.; Brady, M. A. W . Anol. Biochem. 1990,185,84-89. (13)Boniszewski, Z.A. M.; Comley, J. S.; Hughes, B.; Read, C. A. Electrophoresis 1990,11,432-440. (14)Beck, S.;Khter, H. Anal. Chem. 1990,62,2258-2270.

A cryogenically cooled charge-coupled device (CCD) camera equipped with an area CCD array is used in a line scanning system for low-lightlevel imaging of chemiluminescent DNA sequencing blots. Operating the CCD camera in timedelayed integration (TDI) mode results in continuous data acquisition independent of the length of the CCD array. Scanning is possible with a resolution of 1.4 line pairs/mm at the 50% level of the modulation transfer function. High-sensitivity, low-light-level scanning of chemiluminescent direct-transfer electrophoresis (DTE) DNA sequencing blots is shown. The detection of DNA fragments on the blot involves DNA-DNA hybridization with oligonucleotide-alkaline phosphatase conjugate and l&dioxetane-based chemiluminescence. The width of the scan allows the recording of up to four sequencing reactions (16 lanes) on one scan. The scan speed of 52 cm/h used for the sequencing blots correspondstoa data acquisition rate of 384 pixels/s. The chemiluminescence detection limit on the scanned images is 3.9 X 10-l8 mol of plasmid DNA. A conditional median filter is described to remove spikes caused by cosmic ray events from the CCD images.

INTRODUCTION

0003-2700/93/0365-1785$04.00/0

0 1993 American Chemical Society

1786

ANALYTICAL CHEMISTRY, VOL. 65, NO. 13, JULY 1, 1993

elements square are commercially available, although at prices that are often prohibitive for analytical applications. Considering that several thousand data points need to be collected when several hundred bands are being separated, we see that even the state-of-the-art, 4-megapixel CCD area array will fall short of the most demanding requirements of highresolution separations. DNA sequencing, for example, requires sampling capability for well above 500 bands on a single lane, translating into much more than 2048 data points. Continuous data acquisition using an area CCD can be achieved by operating the CCD camera in time-delayed integration (TDI) mode. This has been shown for two highspeed, fluorescence DNA sequencing formats: capillary electrophoresis16 and ultrathin slab gels.16 TDI mode has also been used to monitor migrating fluorescent bands in capillary electrophoresis along the length of the column with a CCD camera." The line scanning system for chemiluminescent blots presented here consists of a cryogenically cooled CCD camera equipped with a Thomson TH 7882 CCD area array and a precision translation stage to move the blot. The T H 7882 CCD is 0.5 in. long, making it representative for the category of CCD arrays available for sensitive digital imaging of gels and blots today. The same camera has previously been used to record snapshot images of chemiluminescent DNA sequencing blots and a variety of detection and assay parameters have been investigated.10 As in the on-line fluorescence detector applications mentioned,l"l' TDI operation adds the capability of continuous data acquisition independent of the array length to a CCD camera capable of sensitivity chemiluminescence imaging, thereby meeting a key requirement for digital imaging of high-resolution electrophoretic blots. EXPERIMENTAL SECTION Sequencing of Plasmid DNA. Enzymatic dideoxynucleotide DNA sequencing reactions18 are performed used the pUCderived 2.7-kb double-stranded multiplex sequencing vector K Z P purified by CsCl gradient centrifugation as a template. This vector provides priming and unique recognition sites for oligonucleotide hybridization at both sides of the insert to be sequenced. Two different sequencing protocols are used to generate samples for this study. In one protocol 5 ng of unlabeled primer (18-mer), 1pL of lox reaction buffer (400 mM Tris, pH 7.6,100 mM MgC12,500 mM NaCl), and 2.5 pg of CsCl purified, denatured template DNA are combined to a total volume of 10 pL. The solution is heated to 72 "C and slowly cooled to room temperature over a period of 1 h. One microliter of 0.1 M dithiothreitol, 1.5 pL of 1OX reaction buffer, 1pL of water, and 2 pL of a 1:8 dilution of Sequenase version 2.0 (13 units/pL, United States Biochemical, Cleveland, OH) are added to the templateprimer solution and mixed. For the termination reactions, 2.5 pL of each of the four d/ddNTP mixtures is placed in separate tubes prewarmed to 37 "C, and 3.5 pL of the templateprimer mixture containing the enzyme is added to each tube. All d/ddNTP mixtures are 80 pM dNTP. The d/ddCTP mixture is 4 pM ddCTP, the d/ddATP mixture is 4 pM ddATP, the d/ddGTP mixture is 8 pM ddGTP, and the d/ddTTP mixture is 8 pM ddn'P. The termination reactions are carried out at 37 "C for 10 min before the enzyme is inactivated at 72 OC for 10min. A 4-pL formamide stop solution is added to the samples before heating to 95 OC for 5 min and loading onto the sequencing (15) Karger, A. E.; Harris, J. M.; Gesteland, R. F. Nucleic Acids Res. 1991, 74,4955-4962. (16) Kostichka,A. J.;Marchbanks,M. L.;Brumley,R.L., Jr.;Drossman, H.; Smith, L. M. BiolTechnology 1992,10, 78-81. (17) Sweedler, J. V.; Shear, J. B.; Fishman, H. A.; Zare, R. N.; Scheller, R. H. Anal. Chem. 1991,63,496-502. (18) Sanger,F.;Nicklen,S.;Coulson,A.R.Proc.Natl.Acad.Sci. U.S.A. 1977, 74, 5463-5467. (19) Cawthon, R. M.; Weiss, R.; Xu, G.; Viskochil, D.; Culver, M.;

Stevens, J.: Robertson, M.; Dunn, D.: Gesteland, R. F.; OConnell, P.; White, R. Cell 1990,62, 193-201.

gel. The second type of sample is generated using thermostable Vent DNA polymerase (New England BioLabs, Beverly, MA) in a thermal cycle protocol which will be published elsewhere." Both protocols differ from standard sequencing protocols in that no label is incorporated into the fragments. Baaingthe detection on DNA-DNA hybridization on the blot rather than on labeled fragments allows the protocol to be used for multiplexsequencing, a technique in which a blot is sequentially probed with a series of different oligonucleotides.8*21*B The most relevant aspect of the sequencing protocols for the purpose of this study is the amount of oligonucleotide sequencing primer loaded onto the slab gel, because it will obviously have a direct influence on the amount of DNA present in the sequencing bands and therefore the chemiluminescence intensity. The Sequenase sequencing reaction was carried out using 5 ng of primer (18-mer) and 2.5 pg of template DNA, such that the reaction is limited by the 840 fmol of sequencingprimer. The Sequenase sample is not ethanol precipitated after the completion of the reaction, and about 21 fmol of primer in a 1-pL volume is loaded per lane. A 25-cycle Vent sequencing reaction uses 25 ng of primer and 0.4 pg of template vector DNA. The thermal-cycled sample is ethanol precipitated and redissolved in a small volume, such that 420 fmol of primer is loaded per lane. Generation of Direct-Transfer Electrophoresis DNA Sequencing Blots. A direct-transfer electrophoresis (DTE) apparatus constructed in-houseis used for the generation of blots. It is functionallyequivalent to blotters described by Pohlet al.sa The reactions are loaded onto a 4 % Long Ranger (AT BioChem Inc., Malvern, PA) reverse-wedge slab gel, separated at 70 V/cm field strength and blotted onto the positively charged, 0.45-pm pore size nylon membrane Biodyne B (PallBiosupport) moving at a blotting speed of 2.4 mm/min. The DNA sequencing blot is treated with a UV dose of 120 mJ/cmZ in a UV Stratalinker (Stratagene) and dried for 10 min at 60 "C before storage and subsequent membrane development. Membrane Development for Detection. The development protocol is an adaptation of published methods for membranebased DNA-DNA hybridization assays using oligonucleotideAP probes21*22~~ and the AP-triggered chemiluminescence of AMPPD (Tropix Inc., Bedford, MA) as described for chemiluminescent DNA sequencing.n Blots to be probed with the oligonucleotide-AP conjugate are prehybridized in PBS-SB buffer [PBSbuffer: 40 mM potassium phosphate, 130mM NaCl, pH 7.6. PBS-SB buffer: 1% sodium dodecyl sulfate (BioRad, enzyme grade), 0.5% bovine serum albumin (Sigma) in PBS buffer] for 15min at 50 OC. The blots are hybridized in a solution of oligonucleotide-AP in PBS-SB buffer (5 mL/100 cm2) for 15 rnin at 50 OC followed by one wash in PBS-SB buffer for 10 min at 50 OC and three washes in PBS buffer for 10 min each. The blots are then equilibrated in DEA buffer (0.1 M diethanolamine, 1mM MgClz, 0.02% NaNs, pH 9.6) for 10 min and subsequently incubated in 0.40 mM AMPPD in DEA buffer (5 mL/lOO cm2) for 10min in a heat-sealableplastic bag. Excess AMPPD solution is drained off, and the moist membrane is sealed in the bag. To record a CCD scan, the chemiluminescent blot is sandwiched between a 60-cm-longglassplate and transparent plasticwrapping foil (Saran wrap) with the chemiluminescent side of the blot facing the foil. The glass plate is then mounted on the scanner stage, and the blot is scanned thru the thin foil in a light-tight environment. One parameter of the protocol that deserves some attention when AMPPD chemiluminescence is used is the period of time (20) Swerdlow, H.; Dew-Jager, K.; Gesteland, R. F., in preparation. (21) Tizard, R.; Cate, R. L.; Ramachandran, K. L.; Wysk, M.;Voyta, J. C.; Murphy, 0. J.; Bronstein, I. Proc. Natl. Acad. Sci. U S A . 1990,87, 4514-4518. (22) Creaaey,A.; DAngio, L., Jr.; Dunne, T. S.; Kiasinger,C.; O'Keeffe,

T.; Perry-OKeefe, H.; Moran, L. S.; Roskey, M.; Schildkraut, I.; Sears, L. E.; Slatko, B. BioTechniques 1991,11,102-109. (23) Pohl,F. M.; Beck, S. Methods Enzymol. 1987,155, 250-259. (24) Beck, S. Anal. Biochern. 1987,164, 514-520. (25) Richterich, P.; Heller, C.; Wurst, H.; Pohl, F. M. BioTechniques

1989, 7, 52-59. (26) Jablonski, E.; Moomaw, E. W.; Tullis, R. H.; Ruth, J. L. Nucleic Acids Res. 1986, 14, 6115-6127. (27) Martin, C.; Bresnick, L.; Juo, R.-R.; Voyta, J. C.; Bronstein, I. BioTechniques 1991, 11, 11C-113.


for the enzymatic reaction to proceed after the blot is incubated in the substrate solution. Two counteracting effects have to be considered. After AMPPD is added to the blot, the chemilumineacenceintensityemittedfrom the blot increasesover aperiod of 6-10 h before reaching ita maximum.n To establish steady light emission and maximum assay sensitivity per unit time, a dwell time of up to 10 h can be used.1° However, the slight but measurable diffusion broadening of the bands on the blot during prolonged periods of dwell time suggests the image should be recorded soon after the s t a r t of the incubation to obtain optimal band resolution. The right dwell time, generally 1-10 h, will depend on whether the emphasis of the study is on resolution or on sensitivity. Since both aspects are vital to the successful imaging of DNA sequencing blots, a 3-h dwell period is used in this study. Chemiluminescence Imaging. Chemiluminescenceimages are acquired by a CH210 CCD camera (Photometries Ltd., Tucson, AZ) consisting of a side-on, cryogenicallycooled camera head equipped with the full-frame576 X 384 pixel TH 7882 CCD array (Thomson-CSF)and a CC200 external electronic controller unit. A comprehensive evaluation of the characteristic features of the TH 7882 sensor at low light levels is available in the literature." The controller unit is connected to an Arche Rival 286 PC-AT computer via a GPIB-PCIIA board (National Instruments, Austin, TX) providing a fast parallel interface for the downloading of image files from the controller frame buffer to the PC-AT. For TDI scanning, a pulse generator functioning as the TDI clock is connected to the controller unit. During operation theeameraiscooledto-12OoC withliquid Na. Afil.2 50-mm focal-length Pentax-A photographic lens is mounted on the camera head, and chemiluminescent blots are imaged at a 36-em distance from the lens, which is the shortest distance that can be focused. During scanner operation, the blot is moved horizontally in front of the camera by a translationstage consisting of a Model 5062415 rail table, a MD series drive, and a MC3000 controller unit (DAEDAL Inc., Harrison City, PA). The translation stage provides up to 61 em of horizontaltravel at a stepper motor resolution of 25 Fm. For image filtering, analysis, and sequence evaluation, the digital images are transferred from the PC-AT to a HP-Apollo 35M) workstation. Image filtering for the removal of artifacts caused by cosmic ray events is performed using Visualization Workbench (Paragon Imaging, Lowell, MA) general purpose image analysis software. This package isinstalldona Sun4/490 computer for reasons of computational speed. Data analysisand evaluation are performed using Pro-Matlab (The MathworksInc., South Natick, MA) matrix-oriented numerical calculus software. Large-size images are efficiently handled by substituting the Photometries image file header for a file header appropriate for Matlab software, thereby avoiding any data type conversion and increase in file size.

Diredionaftranslation stage movement

1787

-

A G T C Chemibminescasnl blot

i

T

-

Direnion of CCD charge transfer

F@wel. Sch+maticviewoftheTDIscanner: CCDamy,photograpMc lens. and moving chemiluminescent DNA sequencing blot. Light originatlng from a vettical line on the moving membrane contributes to the charge on a synchronously moving scan line of the CCD. Synchronization isobtained by wntrollingthetransferof chargeacross the CCD in the direction of the output register (on the left side of the CCO array) using the CCDs TDI trigger line. Light collectton from a Scan line is terminated when the charge reaches the CCD output

register.

register. Having arrived in the output register, the charge packets are shifted one by one in the perpendicular direction toward theon-chip amplifier andsubsequent AID conversion. When a CCD snapshot is acquired, all photogenerated charge packets are read out in an uninterrupted series of parallel transfers followed hy serial transfers and digitization after the shutter has closed. No charge transfer takes place during the exposure. In TDI mode, in contrast, the parallel shifting of the charge toward the output register is under the control of an external trigger, the TDI clock. The parallel transfer is delayed until the arrival of a pulse on the TDI trigger line. The controlled transfer of charge is used while the photoactive area is exposed to light and the accumulation of photogenerated charge takes place. A line in the resulting TDI image represents light from a line in the object plane that is scanned across the field of view of the camera, the scan speed controlled by the frequency of the TDI clock. The TDI mode can be used to image a moving object, provided the movement is synchronized with the TDI clock. The major advantage of using the TDI mode is that the number of scan lines isnotlimited to thenumber oflinesofthe pardel register as in standard full-frame operation. With the equipment described in the Experimental Section, the limitations to the RESULTS AND DISCUSSION length of the TDI scan is given either by the 24-in. maximum travel of the translation stage or by the 6630 scan lines that Advantages of TDI Scanning for Low-Light-Level can be acquired with the 4.8-Mbyte frame buffer memory Imaging. The abilityto operate in time-delayed integration installed in the controller of our system. Both limitations mode greatly enhances the imaging capabilities of the CH210 are purely technical in nature and could easily be overcome camera. Figure 1 gives an overview of the TDI line scanning if necessary. To obtain the highest resolution possible in the system designed to scan DNA sequencing blots. To underdirection of the scan, each of the 576 lines of the T H 7882 stand TDI operation mode, we recall that an area CCD array imager, also called TDI stages in this context, is digitized is composed of two major functional components, the parallel individually. The dimensions of the TDI stages can be register consisting of a two-dimensional array of light-sensitive adapted to applications requiring less spatial resolution than CCD elements, organized in 576 lines of 384 elements each DNA sequencing by charge binning. in the case of the T H 7882, and a single-line linear output In addition to continuous data acquisition and flexibility register, positioned along one side of the parallel register and in its resolution, a line scanner consisting of an area array in connected to theon-chipamplifier. Photons striking the CCD TDI mode offers increased sensitivity per unit time when surface generate photoelectrons, which are trapped in the compared to a line scanner equipped with a linear array, CCD element nearest to the location of photon incidence. because light is collected simultaneously on the entire After the array has been exposed to light, readout of the photoactive surface of the CCD. Because the sensitivity of photogenerated charge is performed by simultaneously shifta cryogenically cooled CCD in low-light-level measurements ingthechargepacketsofalllinesinparalleltowardtheoutput is approximately proportional to the size of the photoactive area, the sensitivity of an area CCD operated in TDI mode (28) Beal,G.:Boueharlst,G.:Chabbal,J.;Dupin,J.P.;Fort,B.;Mellier, is increased over the sensitivity of a linear array consisting Y.Opt. Ent. 1981,26,902-910.

1788


which predicts that the resulting synchronization error U , ~ C = 0.60q is limited to a fraction of the width of a pixel. The overall effect of the synchronization error and other sources of band broadening on the optical resolution is reflected in the modulation transfer functions (MTFs) of the camera in the snapshot imaging and in the TDI line scanning mode. The MTF quantitatively describes the overall performance of an imaging system by measuring modulation, or contrast, in the image as a function of the spatial frequency of a test object. A standard object consists of a series of black and white lines of equal width and spacing, characterized by ita spatial frequency in line pairs (lp) per millimeter. Modulation M is defined as where ,I and Imhstand for the maximum and minimum signal intensity of the white and black bars. One way to establish the MTF of an imaging system is by measuring the modulation in the image for several bar patterns with different spatial frequencies and recording the image modulation, as a function of the spatial frequency of the object. The USAF-1951resolution target with clear l i e s on opaque chrome (Newport Corp., Fountain Valley, CA) provides bar (29) Waehkurak, W,D.; Chamberlain,5.G.; Jenkins,P.T.CCD Image Sensors; DALSA Inc.: Waterloo, ON, Canada, 1988; pp 198-201. (30) Kelly,P. C.; Horlick, G. Anal. Chem. 1973,45,518-527. (31) Horlick, G. Anal. Chem. 1975,47,352-354.

I

I,

of the same type of elements approximately by the number of scan lines that are simultaneously monitored.% Effect of TDI Operation on the Image Resolution. The operational price for continuous TDI data acquisition is the requirement to synchronize the translational motion of the imaged object with the velocity of the scanned lines. A brief discussion will show how accurately these two velocities can be matched with the equipment used and estimates the resulting synchronization error. The synchronization error, Cup&, is given by the length to which the image of a point in the object plane is drawn out in the direction of the scan due to lack of synchronization between the CCD scan speed and the motion of the translation stage. Three independent contributors to the synchronization error in our system are considered: the stage velocity setting, the stage velocity, and the TDI clock. The velocity to which the translation stage is set is determined by measuring the field of view of the camera in the direction of the scan. This measurement is done by acquiring snapshot images of a metric ruler that is repositioned between exposures and determining the CCD line numbers that fall onto two widely spaced (>95% of the total field of view in the scan dimension) ruler marks. The length of the field of view of the CCD array is then calculated from the difference. Each measured CCD line number is subject to quantization error, the variance of which can be approximated by q2/12, where q is the magnitude of the quantization level.mJ1 The quantization level in this case is given by the size of a pixel. Because two measurements are required from each calibration image, the error contribution of determining the length of the field of view to the overall synchronization error is Ufield = 4/61/2 = 0.41q. This error is comparable to the 0.055% error to which the frequency of the TDI clock is calibrated, correspondingto u d d = 0.329 (0.055%of 576pixels). Equally, the stage velocity is calibrated to deviate from a set value by no more than 0.050%, corresponding to u8-e = 0.29q. The contribution of the three sourcesto the horizontal broadening of the image of a point source is given by,

0.8

1

\\

\ i

i

Splcirl Frequency (line pain / mm)

Figure 2. Vertical modulation transfer functions (MTFs) of the CH210 camera equipped with the a f/1.2 50-mm focaHength photographic lens and the TH 7882 CCD array in snapshot imaging (X) and TDI The MTF in the directionof the scanner motion scanning operation (0). is derived from images of the USAF-1951 resolution target. CondC tions: 38cm focusing distance,52 cm/h scanner speed, 7 pixeis/mm sampling rate (Nyquist frequency 3.5 line paks/mm), 1-Hz TDI clock frequency.

~

charts for the purpose of focusing and establishing the MTF. Spatial frequencies in the range of 1-3.17 lp/mm on that target are used to establish the MTF of the CCD camera in the direction of the scan in standard imaging as well as in the scanning TDI mode. Using CCD terminology, in which the output register and the lines of the parallel register are defined as being horizontal, the MTF in scan direction is correctly addressed as the vertial MTF of the CCD array. This term is therefore used, despite the fact that due to the orientation of the array in our camera the movement of the imaged object on the translation stage is actually horizontal during scanner operation. At the closest focusing distance, the sampling frequency in the object plane is 7 samples/mm, corresponding to a Nyquist frequencyof 3.5 lp/mm. The vertical MTF values measured on the resolution target are shown in Figure 2 as well as their approximation by Gaussian curves. Specifying an MTF value of 50% as a criterion for minimum contrast, the camera can resolve 1.6 lp/mm in the snapshot mode and has slightly less resolution of 1.4 lp/mm in the TDI scanning system. Equally spaced (0.30-mm-wide bars (2.1 pixels) are resolved on snapshot images and 0.36-mm-wide bars (2.6 pixels) on TDI scans at the MTF = 50% level. The DTE procedure gives the operator control over the spacing of the sequencing bands on the blot by selecting an appropriate blotting speed. For subsequent CCD imaging at a sampling rate of 7 pixels/mm, the blotter speed is set to result in at least 1-mm interband spacing. As can be seen from the MTFs in Figure 2, the CCD camera is able to image spatial frequencies in the range of 1 lp/mm without major loss in contrast in the snapshot as well as in the scanning mode. In establishingthe MTFs in Figure 2, the same lens aperture setting of f/1.2 and focusing procedure was used as during chemiluminescencescans. Because of the very narrow depth of field under these conditions,focusingerror will occur easily, with negative consequences for the image resolution. It is therefore important to note that Figure 2 represents the MTFs under operating conditions, which are not necessarily the best MTFs achievable with this camera and scanning equipment. Scanning of Chemiluminescent DNA Sequencing Blots. A DNA sequencing blot is produced according to the sequencing,blotting, and membrane developmentprocedures given in the Experimental Section. The blot is scanned at


a rate of 1line/s, corresponding to a 576-8 exposure time for each point of the image. This scan rate and the demagnification as described in the Experimental Section result in a scan speed of 52 cm/h. A total of 4320 scan lines are acquired. Figure 3 shows the TDI scan of the chemiluminescent membrane with two reactions each of the two different sequencing protocols separated side by side. The two reactions on the left are generated by the thermal cycle sequencing protocol and are characterized by a strong chemiluminescence emission,while the two reactions on the right are generated by the standard protocol. Readable sequence is obtained up to fragments 420 nucleotidesin length from the cycle sequencing reactions. The first detectable band is the fragment 57 nucleotides in length, reflecting the fact that the DNA bands carry no label,but are rather detected by DNA-DNA hybridization at the probing site downstream of the primer (see Figure 4B). Data processing starts by extracting lanes from the digital image. The center of a lane on the image is manually assigned at 10-20 points along the length of the lane, and a third-order polynomial is used to interpolate the pixel coordinates of the lane center. A third-order polynomial is required for many lanes to model the deviation of the lane centers of long lanes from a straight line sufficiently well. Using the interpolated centers, the lanes are then averaged using an appropriate bandwidth. Increasing the averaging bandwidth improves the S/N ratio but finds its limits when cross-talk from the adjacent lanes occurs. The lanes extracted from a cycle sequencing sample (second from left in Figure 3) are shown in Figure 4A. An averaging bandwidth of 11 pixels corresponding to 1.5mm is used to avoid interlane cross-talk, even though the lane centers are spaced 3 mm apart, given by the width of the gel loading wells. The relevant sequence of the vector and the priming and the probing sites are given in Figure 4B. Figure 5 shows the peak heights of a representative lane (T-lane) of the thermal cycling reaction as a function of the length of the fragments. As in other DNA sequencing formats, multiple factors contribute to the decrease in signal with increasingfragment 1engthinDTE. The most intense T-band is 153 nucleotides in length (TIM)with 132.4 CCD counts while the least intense band in the readable part is T a with 8.6 counts. This corresponds to signal-to-noise ratios ranging from 208 to 13. The bands of the standard samples are about 3-fold less intense than those generated by the thermal cycling protocol. One has to consider, however, that this increase in signal results from a 20-fold increase in the amount of sequencing primer. The sensitivity of the development and scanningprocedure was determined by processing and scanning two slot blots in parallel with the DNA sequencing blot shown in Figure 3. The slot blots carry two dilution series each of denatured plasmid template DNA spotted on Biodyne B nylon membrane. The scan of the slob blots is quantitated with the same software used to extract lanes from scans of the DNA sequencing blots. Peak heights on the scanned slot blots are used to establish a log-log calibration curve. The detection limit (S/N = 3) under these conditions is 3.9 X 10-18 mol of plasmid DNA. It is important to note that the detection sensitivity for denatured plasmid DNA spotted onto a membrane can only approximate the sensitivity for singlestranded fragments bound to the DNA sequencing blot. Neglecting the possible difference in sensitivity by using the standard curve for plasmid DNA to estimate the amount of DNA in the bands of the T-reaction, we find 1.0 fmol to 28 am01 of DNA per band. Constant interband spacing is an important feature of DTE blots, and the conditions used to generate this blot result in

1789

Figure 9. Chemiluminescence image of a DTE DNA sequencing blot acquired with the scanner. Four sequencing samples of the PUG derived muitlplex vector KZ2 were separated. The two samples to the left are generated by thermal cycle sequencing; the two samples to the right orlginate from a standard protocol. Separatlon and assay conditions: 4 % Long Ranger gel, 8 0 t m length, 100-200-pm reverse wedge, 70 V/cm electric field, 3-mm sharks tooth comb, 2.4 mmlmln blottlng speed, Biodyne B nylon membrane, DNA-DNA hybrldlzatlon assay using 6 nM ollgonucleotlde (20-mer)-alkaline phosphatase conjugate; imaging and display CCD scan speed 52 cm/h, TDI clock frequency 1 Hr (1 scan linels), line exposure time 576 s. The area displayed Is 46 X 5.5 cm in sire, representing3200 TDI scan Ilnes, photonegative display.

bands spaced 1.4 mm on average. The spacial resolution of the bands of the T-reaction expressed as R = As/4u, ranges from 0.59 to 0.93 in the range of the informative sequence. Only a slight decrease in resolution is observed in going from

1790

ANALYTICAL CHEMISTRY, VOL. 65, NO. 13, JULY 1, 1993 4

140

I'

T

A

E

-i

U

Y

1

l ao-

I

Y

R

IJ I

03 I

0

2

0

4

6

1

10

12

I4

50

16

100

150

200

250

300

350

4w

450

Nucleotide Number

Peak height as a function of fragment length, shown for the bands in the T-reaction lane in Figure 4. Figure 5.

1R a

1

..

u a A

2 1J 1

20 -G

30 40 50 60 C T G G A T C C U CCATCCT ACC CCACACCTTT T M A C C A A C T

90 100 110 120 70 80 AACCTCACTC CCTCGAATTC ACTGGCCGTC GTTTTACAAC GTCGTGACTG GGAAAACCCT

130 14 0 150 160 17 0 180 GGCGTTACCC M C T T A A T C G CCPTGCAGCA CATCCCCCPT TCGCCAGCTG GCGTAATAGC

190 200 210 220 230 240 GAAGAGGCCC GCACCGATCG CCCPTCCCAA CAGTTGCGCA GCCTGAATGG CGAATGGAAA 280 290 300 260 270 250 TTGTAAGCGT TAA'ITCGACC TGCAGGGGGG GGGGGGCGCT GAGGTCTGCC TCGTGAAGAA 310 320 330 340 350 3 60 GGTGTTGCTG ACTCATACCA GGCCTGAATC GCCCCATCAT CCAGCCAGAA AGTGAGGGAG 370 380 390 400 410 420 CCACGGTTGA TGAGAGCTTT GTTGTAGGTG GACCAGTTGG T G A T m G A A ClTITGCITT

Figuro 4. (A) Lanes derived from a sequencing sample generated by thermal cycllng (lanes 5-8 on the D E blot in Figure 3). The 3-mm wide lanes on the digital TDI image are averaged to the right and to the left of theircenter uslnga 1.5mm bandwidth;the chemlkminescence signal Is normalized. (B) Sequence of the vector KZ2: the priming site (l&mer) and the annealing site for the ollgonucleotide-akaiine phosphatsee direct conjugate probe (20-mer) used for DNA-DNA hybrklization are underlined.

smaller to larger fragments, and the limit of the informative portion of the scan is defined by lack of sensitivity rather than by deterioration of the band resolution. The bandwidth of the most narrow peak found in the T-lane is a, = 2.8 pixels, assuming a Gaussian peak profile. The bandwidth corresponds to a data sampling rate of so = 0.36u8,indicating that the image demagnification used is sufficient for the adequate sampling of the narrow DTE bands. Figure 6 shows 576 lines of the scan in detail to further illustrate the quality of the data acquired. A segment of this size corresponds to the field of view of the CCD camera and is acquired during 9.6-min scan time. Figure 6 shows raw CCD image data to also illustrate the presence of artifacts caused by cosmic rays on long CCD exposures. About 16 such artifacts can be identified on this portion of the scan. They are limited in size to only a few pixels and are identifiable as intense black dots on the photonegative display. The digital filter used to remove these artifacts from the CCD images is described in the following section. The lanes extracted frim the scan detail shown in Figure 6, after correction for cosmic ray events, are shown in Figure 7. A total of 55 sequencing bands can be recorded along the field of view of the imager. The sensitivity is clearly sufficient to detect all peaks in this portion of the electropherogram. The dominating noise that can potentially interfere with the correct assignment of peaks is small false stop bands originating from the sequencing chemistry or possibly from cross-talk from adjacent lanes. The extent of lateral diffusion of bands leading to some cross-talk between the lanes and the minor bands caused by false stops can also be seen on the original image in Figure 6. While the noise associated with the sample preparation for DNA sequencing cannot be improved by detector instrumentation, the artifacts caused by the cosmic ray events clearly are a CCD-specific instrumentation problem that requires attention. Digital Filter for Removal of Artifacts Caused by Cosmic R a y Events. Like other CCD arrays, the TH 7882 is sensitive to environmental radiation, in particular y rays, summarily referred to as cosmicradiation in this context.*~32~33 Cosmic rays cause a large number of photoelectrons to be generated a t the location of incidence on the CCD surface, contaminating the image with spikes. Particularly in applications requiring long exposure times, like CCD-basedRaman spectroscopy and low-light-levelimaging, cosmicray artifacts can become the dominant source of noise. For the removal of cosmic ray spikes from CCD-derived Raman spectra, Phillips and Harris have developed a one-dimensional digital (32) Murray, C. A.;Dierker, S. B. J. Opt. SOC.Am. A 1986,3, 21512159. (33) Phillips, G.R.;Harris, J. M. Anal. Chem. 1990, 62, 2351-2357.


TCGATCGATCGATCGA

17Sl

G272

A220 Figure 8. Detail of a 8.3 X 5.5 cm portion of Figure 3. The 576 scan lines shown correspond to the length of the field of view of the camera and are acquired in 9.6-min scan time. Raw CCD Image data are shown to Illustrate the presence of about 16 cosmic ray artifacts, recognizable as intense black dots in the image.

Figwe 7. Lanes derived from the second sequencing sample (Vent polymerase, thermal cycle protocol) from the left of the D E blot In Figure 6. The lanes were extracted from an image corrected for cosmic ray artifacts by conditional median fiitering.

The filter parameters are chosen after the cosmic ray noise in our system is characterized. Figure 8 presents a typical intensity distribution of cosmic ray events on a long-term exposure of the TH 7882 installed in the CH210 camera. The statistic was established from a subarea comprising 86 % of the CCD surface. A total of 333 events are recorded over a period of 4 h, corresponding to a rate of 96 events/h over the entire surface. The most frequent type of event generates about 1X 109 photoelectrons, while the most intense spikes consist of more than 15 X 109 photoelectrons. The frequency of events on the 4-h dark exposure is in close agreement with the rate seen on the TDI scan in Figure 6. On a portion of the scan corresponding to the full size of the array and an exposure time of 9.6 min we expect 15 events. Thick, frontside-illuminated CCD arrays like the TH 7882 have a tendency to spill charge generated by a single event into several pixels.% Charge spilling from the spike center into adjacent pixels results in 4.2 pixels/ event being affected on the 5, signal level with our camera.

filter." Here we describe a two-dimensional filtering procedure for the CCD images which is based on median filtering.

(34) Janesick, J. R.; Elliot, T.;Collins,S.; Blouke, M. M.; Freeman, J. Opt. Eng. 1987,26,692-714.

.o

I

'5

G

230

1s -

i'!

C

20

21

22

23

24

U

16

27

28

Posidon on DTE blot (cm)


1792

50 i

1

-1

50

35 L? c

:

30

e

$

i

I00 Signal (CCDcounrs)

Figure 8. Intensity dlstributlon of cosmic ray events seen on the TH 7882 installed In the CH210 camera. In a subarea comprlslng 86% of the total surface, 333 events were recorded In 4 h. System galn, 16.78 photoelectrons per CCD count.

200

300

500

400

CCD lme n u m k r

Figure 9. Effect of condltknal median fllterlng. Lanes extracted at the same location from an unfiltered and a flltered 4-h dark exposure using a 1l-plxel averaglng bandwldth. 'Lanes offset for illustration. Filter parameters: 5 X 5 plxel median fllter mask, 25CCD count

condltlon threshold.

The procedure used for the removal of cosmic ray artifacts from the TDI scans can be described as conditional median filtering. Two-dimensional median filtering is provided as a tool in general purpose image analysis software packages. Fast algorithms have been developed for this filter,%@which is conceptually similar to two-dimensional convolution. It acts on an image by substituting the center value of a N X N mask of pixels, where N is an odd integer, with the median of the pixel values of the mask. Median filtering has a smoothing effect, eliminating objects smaller than half the size of the mask from the image. The unconditional application of a median filter to the images of DNA sequencing blots results in unacceptable degradation of the band resolution. In the conditional median filter suggested here, the filter is therefore only applied to pixels that are flagged as spikes. Pixels are flagged when a threshold condition is met that asks for the pixel value to exceed the median by more than the threshold value. Because the average cosmic ray event adds significant charge to more than 1pixel on the TH 7882 array, a 5 X 5 median filter mask is considered to be more effective than the minimum mask size of 3 X 3 pixels. The choice of a suitable filter threshold value takes into account the spike intensity distribution as well as the height and width of the bands in the image. Given the intensity distribution in Figure 8 and sequencing bands as characterized in the previous section, a threshold value of 26 CCD counts was found to discriminate well between spikes and the broader signal modulation of the sequencing bands. Figure 9 illustrates the effect of the conditional median filter on lanes extracted from the original and the filtered 4-h CCD dark exposure. The lanes are generated by averaging across an 11-pixel bandwidth, the same bandwidth that is applied to the images of the sequencingblots. A lane combines data from 2.8% (11/384) of the total image area, such that a typical lane derived from the unfiltered image is contaminated with 10-11 events. Figure 9 shows the lane extracted from the filtered image from the same location and indicates the CCD lines containing flagged pixels. As the lanes are generated by averaging across an 11-pixel bandwidth, the largest spike-related noise that is not flagged will contribute 2.3 CCD counts (26/11) to the lane. Some noise of that magnitude persists in the filtered data in the immediate vicinity of the spike centers.

This study describes a low-light-levelline scanning system based on an area CCD array that meets the size and resolution requirements for the imaging of high-resolution electrophoretic gels and blots. Direct imaging of DNA sequencing blots exemplifies the need for digital data acquisition capability that exceeds the frame size of even the largest CCD area sensors available today. The described line scanning system provides continuous data acquisition, with the length of the scanned area not limited by the length of the sensor as in snapshot operation. The sensitivity of the system is reflected in the chemiluminescence detection limit of 3.9 X lo-'* mol of plasmid DNA in a 9.6-min exposure time (S/N = 3). This exposure time was used for the acquisition of data

(35) Huang,T.5.; Yang,G.J.;Tang, G.Y .IEEE T M ~ Acoust.,Speech, s. Signul Process. 1979,27, 13-18. (36) Narenda, P. M. ZEEE Tram. Pat. Anal. Mach. Intell. 1981, 3, 20-29.

(37) Campion, A.; Perry, S. S. Laser FOCUSWorld 1990,8, 113-124. (38) Pemberton, J. E.; Sobwinski,R.L.J. Am. Chem. SOC.1989,111, 432-434. (39) Epperson, P. M.;Denton,M. B. Anal. Chem. 1989,61,1513-1619.

One of the analytical fields that has benefited the most from the advent of CCDs is Raman spectroscopy.32~~~3'~~ Because CCD-based Raman spectroscopy is similar to lowlight-level imaging in that long exposure times are often required, the two-dimensional filter described here should be effective in that application as well. Two-dimensional digital filtering requires the sacrifice of the reduction of measurement noise that can be gained by binning, the onchip combination of charge from multiple adjacent CCD elements. The advantage of binning over digital summation when Raman spectra are generated from readout- and shotnoise-dominated CCD images has been assessed.39 The same reasoning does not, however, necessarily apply to the treatment of CCD data predominantly contaminated with cosmic ray noise. Phillips and Harris described pixels affected by cosmic ray events as outlying data points,33 concluding that their removal should be performed by identifying the erroneous points and replacing them with estimates derived from adjacent unaffected data points rather than by smoothing. As a consequence, they should also be removed prior to summing with unaffected data points. This is true regardless of whether the summation is done digitally, as when electrophoretic lanes are extracted from a CCD image, or by binning, as commonly done in CCD Raman spectroscopy. In both cases, the removal of cosmic ray spikes is therefore best done as a first step in the processing of the CCD data, using the two-dimensional data set.

CONCLUSIONS

~

~

~~

ANALYTICAL CHEMISTAY, VOL. 65, NO. 13,JULY 1, lQQ3 l7QS

from DNA sequencingblots, resulting in fragments up to 420 nucleotides in length to be readable on the scanned image. The use of a larger area CCD could bring about further improvementsin the high-sensitivity digital recording of DNA sequencingblots. The width of the scanned area in our system is limited to 384 picture elements, relatively modest considering that only a 5.5-cm-wide area can be scanned at the necessary resolution. This scan width corresponds to about 16 lanes, each 3 mm wide, on a DNA sequencingblot. Future work has to be aimed at scanning the full width of a highthroughput, possibly multiplexed DNA sequencing blot carrying up to 96 such lanes, which would require a 28.8-cm scan width. The use of a large area sensor with 2048 elements per scan line could accomplish this, if good imaging optics withacceptable lens aberrationcan be found. As an additional benefit, the increase in the number of TDI stages could be traded into increased scan speed or sensitivity.

ACKNOWLEDGMENT The authors gratefully acknowledge the help of Diane Dunn and Leonard DiSera in operating the DTE apparatus. We also thank Kerry Dew-Jager for preparing the thermal cycle DNA sequencing sample and Andy Marks for writing some of the image f i e conversion routines. This work was supported by the U.S. Department of Energy Grant DE-FGO288ER60700 and the Utah Center for Human Genome Research Grant 5-P30-HG00199 from the National Institutes of Health.

RECEIVEDfor review October February 8, 1993.

13, 1992.

Accepted

Line scanning system for direct digital chemiluminescence imaging of

Recommend Documents