Anal. Chem. 1994,66, 1424-1431
Simultaneous Monitoring of DNA Fragments Separated by Electrophoresis in a Multiplexed Array of 100 Capillaries Kyoji Uenot and Edward S. Yeung’ Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 500 1 1
Various excitation schemes for distributing a laser beam to a large number of capillaries in an array are evaluated. This led to the construction of a multiplexed system for monitoring the electrophoresis of DNA fragments in 100 capillaries. The laserexcited fluorescence signals from each capillary are simultaneously recorded at the rate of 0.6 frame/s by a CCD camera. The reconstructed electropherograms show excellent reproducibility and minimal cross-talk. The system provides for two simultaneous excitation wavelengths so that it can be adapted for two-color, two-intensity DNA sequencing based on the commercial four-dye chemistry. Only 20 m W per laser line was employed. Further development of this system to accommodate up to 4096 independent sequencing channels at a time is discussed. The worldwide Human Genome Initiative (HGI) is already well under way. There is no need to reiterate the importance of the effort to biomedical science in the 21st century.’s2 It is generally accepted that “because of the high cost and slow rate of D N A sequencing with current technology, sequencing of the entire genome should not be initiated a t present”.2 Rather, new DNA sequencing schemes must first be developed. The magnitude of the problem is that the specific order of the 3 billion bases in the human chromosomes must bedeciphered. Reading the sequence alone a t the rate of four per second would require 24 years. The sequence, if printed a t 60 characters per line and 50 lines per page, would occupy 142 ft of library shelf space. Using current technology, a competent laboratory can sequence about 105-106 bases per year, which means that 1000-10 000 years are needed for the entire human genome. At today’s cost of $ 5 per base sequenced, the $15 billion dollar investment is prohibitive, even if one is willing to wait. There are two main groups of sequencing technologies under investigation as part of the HGI-evolutionary improvements and revolutionary methods. While revolutionary methods show promise for quantum jumps in speed and in reduced cost, their success depends on achieving many technical milestones in diverse fields that have yet to be proven. Evolutionary sequencing improvements build upon existing, proven technology. Each step still requires innovative approaches, but the success rate is likely to be higher and more + Present address: Shionogi & Co., Ltd., Shionogi Research Laboratories, 12-4, Sagisu 5-Chome, Fukushima-ku, Osaka 553, Japan. ( I ) Joint U.S. Department of Energy and US.Department of Health and Human Services Report DOE/ER-O452P. Understanding Our Genetic Inheritance-The U S . Human Genome Project: The First Five Years;Washington, DC, April 1990. (2) National Research Council Report of the Committee on Mapping and Sequencing the Human Genome, Board of Basic Biology, Commission on Life Sciences, National Academy Press, Washington, DC, 1988.
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predictable. One promising evolutionary approach is capillary gel electrophoresis (CGE). It has already been demonstrated that, because of the high applied potential, a sequencing rate per capillary 25 times faster than standard slab-gel instruments is p ~ s s i b l e . ~The key feature is that CGE is inherently compatible with automation and highly multiplexed operation. Automatic loading of the separation medium and the samples, as well as column preconditioning, is already implemented in commercial C E instruments. The small size of the capillaries allows thousands of them to fit in a reasonable amount of space. For example, 4000 capillaries each with 150-pm 0.d. will form an array only 60 cm wide. The small capillary inner diameter means that the running current is 10 pA each, such that a 40-mA power supply will be able to handle 4000 capillaries. The amounts of samples, buffer solution, and separation media required are also low, simplifying the frontend sample preparation procedure, reducing reagent cost, and minimizing the accumulation of hazardous waste in a largescale operation. Especially important is that the separation mechanism and tagging chemistry can be taken directly from the well-established protocols used in commercial instruments. Various approaches have been suggested for highly multiplexed D N A sequencing based on a parallel array in CGE.”’ In one a p p r o a ~ h ,a~confocal .~ fluorescence excitation geometry was employed with one or two phototubes while a 25-capillary array was translated one at a time across the optical region by a mechanical stage. Even though detection is not simultaneous, the scan rate of 1 H z was adequate for the separation speed and the dwell time per capillary was sufficient for sensitive detection. In another arrangement,6the exit ends of the capillaries were placed in a flow cell to couple with matching sheath flows generated by larger capillaries placed a t the opposite end so as to confine and to isolate each of the separation channels. A single laser beam crossed the flow streams in a line for excitation, and an imaging charge-coupled device (CCD) camera was used for simultaneous detection perpendicular to the plane and outside the flow cell. The main advantage is superior stray-light rejection in the sheath flow.8 The main challenges in scaling up to thousands of capillaries are alignment of the individual sheath flows, possible turbulence in the flow paths, improper matching of the laser beam waist over a long distance with the core diameters
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(3) Swerdlow, H.; Zhang, J. Z.; Chen, D. Y.; Harke, H. R.; Grey, R.; Wu, S.;
Dovichi, N . J.; Fuller, C. Anal. Chem. 1991, 63, 2835-2841.
(4) Huang, X. C.; Quesada, M. A,; Mathies, R . A. Anal. Chem. 1992, 64,967-
972.
( 5 ) Huang, X. C.; Quesada, M . A.; Mathies, R. A. Anal. Chem. 1992,64,21492154. (6) Kambara, H.; Takahashi, S. Nature 1993, 361, 565-566. (7) Taylor, J. A.; Yeung, E. S . Anal. Chem. 1993, 65, 956-960. (8) Zarrin. F.; Dovichi, N. J . Anal. Chem. 1985. 57, 269&2692
0003-2700/94/0366- 1424$04.50/0
0 1994 American Chemlcal Society
containing the eluted fragments, attenuation of the laser beam in subsequent channels due to cumulative absorption, anti the necessity of having an extra space between the capillaries to accommodate the sheath flow. Recent work in our laboratory7v9centered around excitation via optical fibers inserted into the ends of the separation capillaries (axial-beam mode) and detection perpendicular to the capillary array with a CCD camera. Advantages include easy and efficient coupling of the laser beam to each capillary, the complete lack of moving parts, and a mechanical alignment system that is rugged and reproducible. The main disadvantage is the intrusion of the optical fiber into the separation capillary, affecting electroosmotic flow and increasing the likelihood for contamination and column blockage. In this article, we evaluate the various design parameters that are important for parallel laser-excited fluorescence detection in C E in large arrays. A functional system with an array of 100 capillaries is successfully assembled and tested. The possibility of scaling up to 4096 capillaries for DNA sequencing with straightforward modifications of this system will be discussed.
EXPERI MENTAL SECTION Comparison of Excitation Configurations. The configurations compared are shown in Figure 1. In each case, the CCD camera system is placed vertically above the plane of the capillary array: (a) the laser beam was focused using a 1OX microscope objective into an optical fiber and coupled at (1) Oo (axial beam), the incident light beam was directed along the capillary axis through the optical fiber which was inserted into the ~ a p i l l a r y (2) ; ~ 45O, the incident optical fiber was directed perpendicular to the capillary wall at 45O relative to the vertical; and (3) 90°, the incident optical fiber was directed perpendicular to the capillary wall at 90° relative to the vertical; (b) the laser beam was directed through two mirrors and then focused into a line by a plano-convex cylindrical lens and incident in the (4) opposite direction, i.e., the horizontal direction of the beam was opposite to that of the flow of fluorophores; and ( 5 ) same direction, Le., the horizontal direction of the beam was the same as that of the flow of fluorophores. The binning sizes were varied to obtain the lowest detection limits for each scheme. Thus the CCD camera was operated in a 20 to 1 row and a 10 to 1 column binning mode for the axial beam, 45O, and 90' schemes, and in a 20 to 1 row and a 20 to 1 column binning mode for both of the cylindrical-lens schemes. A 488-nmargonionlaser (Uniphase, Model 2201-10SLSC, San Jose, CA, maximum power 10 mW) was used as the light source. For the studies with an optical fiber, the laser beam was partially focused to one end of a glass optical fiber (Edmund Scientific, Barrington, N J , Model P31735, 0.002-in.0.d.) using a microscopeobjective(Bausch and Lomb, Rochester, NY, Model BM2888, 1OX). The free end of the illuminated fiber was either inserted into a capillary or aligned with the outer wall of a capillary at the proper angle. For the studies with a plano-convex cylindrical lens, the laser beam was directed through three mirrors on adjustable mounts and focused onto a capillary placed on an aluminum block. ( 9 ) Taylor, J. A.; Yeung, E. S.Anal. Chem. 1992, 64, 1741-1744.
1) Axial-beam
2) 45"
3) 90"
kwr
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4) Plano-convex cylindrical lens opposite direction Laser
3
CL
cL&
4
+
Flow
C
-
5) Plano-convex Cylindrical lens same direction
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.-++
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C
Flow
+
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Flgurr 1. Various configurations for directing a laser beam to a capillary: upper, sMe view: lower, top view. C, caplllary; OF, optical fiber: CL, plano-convex cyllndrlcal lens.
Translational stages were used for fine alignment of the focal point of the laser beam. The detection region of the capillary was imaged onto a CCD imaging system (Spectra Source Instruments, Westlake Village, CA, Model MCDlOOO with CCD02-06 CCD sensor) through the camera extension of a binocular microscope (Bausch and Lomb, Model Stereozoom 7). Two cutoff filters (Melles Griot, Irvine, CA, Model OG5 15) were used in front of the CCD camera to reduce background noise caused by scatter and stray light. The inethods for data acquisition and extraction were similar to those described below except for the binning size and the image size. A fused-silica capillary (Polymicro Technologies, Phoenix, AZ, Model TSP075150,75-pm i.d., 150-pm o.d., total length 40 cm, effective length 38 cm) was prepared by removing 2 cm of polyimide coating with boiling sulfuric acid at 37 cm from the injection end. The capillary was filled with running buffer (pH 9.0, 10 mM carbonate buffer). Electrokinetic injection was used throughout at 5 s at 500 V/cm, and the electrophoretic separation was driven at 500 V/cm using a high-voltage power supply (Spellman, Plainview, NY, Model UHR50PN50) with a platinum electrode at the positive highvoltage end and a chrome1 electrode at ground. Relative laser intensities were measured by a photodiode (Hamamatsu Corp., Middlesex, NJ, Model S1227-66BQ). AnaMical Chemistry, Vol. 66,No. 9, May I , 1994
$425
m
48 8
514
I
I I
A
Flgure 2. Schematic diagram of the fluorescence detection system for multicapHlaryelectrophoresis: CCD, chargecoupleddevice camera; F, cutoff and long-pass filters; WL, wide-angle lens with extension tube; BE, beam expander; C, capillary; M, mirror; CL, plano-convex cylindrical lens; AB, aluminum block; P, prism; R, buffer reservoir; GND, ground potential; NEG, negative high voltage.
The output of the photodiode was monitored by a digital multimeter (Keithley Instruments, Cleveland, OH, Model 197 autoranging DMM). The laser intensities for both of the cylindrical lens schemes were measured through a 1-mm slit and then converted to that for a 75-gm capillary by multiplication with the factor of 0.075. Fluorescence Detection System for Multiple Capillaries. A schematic diagram of our fluorescence detection system for multi-capillary electrophoresis is shown in Figure 2. The light source for excitation was an air-cooled argon ion laser (Uniphase, San Jose, CA, Model 221 3- 150ML) operating simultaneously at several visible lines that were separated externally using a glass prism. The diameters of all output lines were expanded with a laser beam expander (Oriel Corp., Stratford, CT, Model 15610output assembly and Model 15630 20X diverging input lens). Each of the 5 14- and 488-nm lines was directed through two mirrors on adjustable mounts and focused onto the capillaries held by an aluminum block using a plano-convex cylindrical lens (Melles Griot, Model OlLCP155, focallength 100 mm). The beams from theother lines were blocked with paper painted with ultraflat black paint. Several translational stages wereused for finealignment of the focal points of the laser beams. From a total laser power of 50 mW, each of the laser lines at 514 nm and at 488 nm was estimated to be -20 mW. There are 100 triangleshaped grooves machined on the surface of the aluminum block for setting the 100 capillaries, as shown in Figure 3. The block was painted black to reduce stray light. The injection ends of the capillaries were bundled, inserted, and then glued through a hole in a screwed cap to fit a common sample vial. A stainless-steel tube, which supplied high-pressure nitrogen gas, was also inserted and glued through another hole. Both cleaning of the capillaries with solvent (water and methanol) and refilling them with polymer solution can be easily accomplished with this pressure-driven flushing system. The capillary array in the detection region was imaged onto the CCD sensor through a 24-mm wide-angle lens (Canon, Tokyo, Model FD24mm F1.4L) and a 10-mm extension tube (Canon). The front of the camera lens was placed 50 mm from the capillaries. Two cutoff filters (Melles Griot, Model OGSSO) and two long-pass filters (Corion, Hollison, MA, Model LL-550-R) were placed in front of the lens to reduce background noise caused by scatter and stray light. When 100 capillaries were used for the separation of DNA fragments, the CCD camera was operated in a 25 to 1 row 1426
Analfllcal Chemistry, Vol. 66, No. 9,May 1, 1994
;z/T:-=F# 488nm
514nm
- _ _
. -- _. _ - _
, _
I ' 1 Figure 9. Closeup figures of the aluminum block for mounting multiple caplllarles.
and a 2 to 1 column binning mode. Binning provided compression of 175 rows and 400 columns of image data into a 7 X 200 array. Digitized data were transferred from the MCDl 000 to an IBM-PC-compatible 80486-basedcomputer. Each frame of data, corresponding to a 1-0-s CCD exposure taken every 1.6 s, was stored in an individual file. Using a simple BASIC algorithm, all of the data from one selected spatial region representing each separation capillary was extracted. These time-dependent intensity data were plotted as electropherograms and analyzed by standard chromatographic software (ChromPerfect, Justice Innovations, Palo Alto, CA). Separation. DB-1 coated G C capillaries (J& W Scientific, Folsom, CA, 75-pm id., 150-pm o.d., total length 50 cm, effective length 35 cm) were prepared by removing 3 cm of polyimidecoating with boiling sulfuric acid at 33 cm from the injection end. The capillaries were filled with 0.5% methyl cellulose solution in 100 mM tris-borate buffer (pH 8.2, containing 2 mM ethylenediaminetetraacetic acid). Electrokinetic injection was used throughout at 10 s a t 100 V/cm and the electrophoretic separation was driven at 50 V/cm using a high-voltage power supply with a platinum electrode at the negative high-voltage end and a chrome1 electrode at ground. Materials. DNA restriction fragments, Hue111 4 x 1 74, were obtained from United States Biochemical (Cleveland, OH). TOTO-1 iodide [ 1,1-(4,4,7,7-tetramethy1-4,7-diazaundecamethy1ene)-bis-4- [3-methyl-2,3-dihydro(benzo-1,3thiazole)-2-methylidene]quinolinium tetraiodide] was obtained from Molecular Probes (Eugene, OR). The buffer components, tris(hydroxymethyl)aminomethane,boric acid, ethylenediaminetetraacetic acid, sodium hydrogen carbonate, and sodium hydroxide were all from Sigma Chemical (St. Louis, MO). Methyl cellulose (4000 CPat 25 OC for a 2% solution) was also obtained from Sigma Chemical. Fluorescein disodium salt was obtained from Aldrich (Milwaukee, WI).
The 0.5% methyl cellulose buffer solution was prepared according to the method described in our previous paper.1° Procedure. To 8 pL of TOTO-1 aqueous solution (concentration 1 X 10-6 M) in a 100-pL centrifuging tube was added 2 pL of Hue111 6 x 1 7 4 buffer solution (concentration 480 pg/mL) (final concentration of DNA 96 pg/mL). The mixed solution was ultrasonicated for 30 s and stood in the dark at room temperature for at least 1 h. The sample solution was stored in the dark prior to injection.
RESULTS AND DISCUSSION Comparison of Excitation Configurations. In order to determine which configuration is the best one for directing the incident laser beam onto the capillaries, five schemes were compared on the basis of the estimation of the detection limit for fluorescein and the measurement of the excitation intensity in each case. Naturally, these excitation schemeswere selected because each is capable of conveniently dividing the output from a single laser for highly multiplexed operation. Distribution of the output from a laser to thousands of capillaries can be conveniently accomplished by optical fibers, as discussed earlier.7 An optical-fiber bundle with 500 50pm fibers costs around $50. One can readily expand the laser beam to couple with the 1-cm-diameter bundle without critical alignment. Given the close-pack arrangement, -50% of the light can be effectively used. In scheme 1 of Figure 1, insertion of the 50-pm-0.d. fibers into the 75-pm4.d. capillaries can be accomplished mechanically by first mounting each of the arrays on metal blocks which have prealigned parallel grooves for each channel. Once fitted to the capillaries, there is no need for optical adjustments. Since the intrusion of the fiber into the electrophoretic capillary may disturb the separation, we also investigated coupling schemes based on direct contact of the optical fiber to the capillary walls. Because of the cylindrical shape of the capillary, light from the 50-pm fiber (in a 60° cone) is partially collimated by the l5O-pm outer wall so that most of the photons pass through the liquid core for efficient excitation. A 45' arrangement (scheme 2) projects the transmitted light away from the camera lens used to collect fluorescence. For highly multiplexed operation, one can again have an aluminum block with grooves to mount the capillaries but with 45O channels drilled through the block and prealigned with thegrooves. This way, alignment is rugged and reproducible. To test whether stray light is a limiting factor in this external-fiber coupling scheme, we can study coupling at 90° (relative to the fluorescence collection direction) (scheme 3). Excitation efficiency should beidentical to scheme 2, but refraction in the direction of the camera lens will be altered. A totally different method for distributing laser light to a large number of capillaries is to focus the laser to a thin line which intersects the axes of the capillaries. For 150-pm-0.d. and 75-pm-i.d. capillaries packed next to each other, half of the total laser light will be irradiating the liquid core to produce fluorescence. There is still refraction at the cylindrical capillary walls. However, grooves can be constructed to optically isolate each capillary from the others. So, schemes 4 and 5 were tested to evaluate the arrangement in Figure 3. The results are summarized in Table 1. The detection limits listed facilitate comparison among the various excitation
-
Table 1. Detection UmHr for Fluorascoln Udng Sovoral Excltatbn contlgwationr a$ Shown In FWn 1
configuration optical fiber (1) axial beam (2) 4 5 O (3) 9oo
cylindrical lens (4) opposite direction (5)same direction (5) same direction
laser output (mW)
detection limit (M, S/N = 3)
10 9 8
6.2 X 1o-B 5.0 X 10-10 2.1 x 10-8
3 3 6
7.9 x 10-10 P 1.1 x 1 4.0 X 1&lo
geometries but have not been optimized for detection in an array of capillaries. For example, picomolar detection limits have been demonstrated in the axial-beam arrangement.9Since in each case here detection was limited by the detector noise and not shot noise in the CCD, the potential detection power, which is critical for actual D N A sequencing applications, is dependent on the total laser power, fluorescence collection efficiency, cooling of the CCD element, exposure time, etc. Although the highest laser intensity was applied for the axialbeam scheme, the poorest detection limit was obtained. It seems that this result was caused by photochemical bleaching of the fluorophores. It is one of the advantages of laserinduced fluorescence detection that higher excitation intensity provides larger analyte signals. The result suggests that the axial-beam scheme could not benefit from this completely. The lowest detection limit was obtained with the plano-convex cylindrical-lens configurations even though much lower laser intensities (per capillary) were used. There is no significant difference between the two cylindrical-lens schemes. This indicates that photochemical bleaching is not important, given the small intersection region and the characteristic flow rates of the fluorophores through this region. So, the arrangement in Figure 2 was selected in subsequent experiments for multiplexed runs. Alignment (focusing) is more critical, but the lack of contact with the liquid and the more uniform distribution of excitation intensities (compared to transmission through fibers) are major advantages. Monitoring of Multiple Capillaries. The optical design leading to the arrangement shown in Figure 2 is based on several important principles. First, the laser is a broad-band continuous-wave Ar ion laser identical to those used in the commercial fluorescence-based DNA sequencer. Eventual adaptation to DNA sequencingbased on proven dye technology is straightforward. The 488- and 514-nm lines are available simultaneously and are the most prominent ones in these standard laser systems. A simple glass prism, beam-folding mirrors, and cylindrical lenses readily focus these onto the capillary as two spatially separate lines to achieve two-color excitation. The weaker laser lines are blocked in our studies, but these can conceivably be used advantageously in a fourcolor excitation coding scheme, especially if mixed Ar-Kr gas is used. Second, a beam expander is used to allow focusing into a fine line, as is predicted by Gaussian optics. The enlarged beam diameter also lengthens the focused line to cut across a large number of capillaries. In our case, a 5-cm line is
*
(10) McGregor, D. A.; Yeung, E. S. J . Chromatogr. A 1993,652, 67-73. (11) Smith, L. M.; Sanders, J. Z.; Kaiser, R. J.; Hughes, P.; Dodd, C.; Connell, C. R.;Heiner, C.; Kent, S. B.;Hood, L. E. Nature 1986, 321, 674-679.
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formed even though the array width is only 3 cm. This allows the use of only the center part of the Gaussian intensity profile to achieve more uniform excitation intensities. Third, sharpcutoff rather than band-pass filters were used in front of the CCD camera to reject laser light while passing fluorescence. For the fluorophore used here (TOTO-DNA), this is clearly adequate. However, the same filters should be applicable to DNA sequencing based on the well-established four-dye system. This is because each of the two laser lines excites a pair of the dyes." At the same time, each dye in a pair emits at different wavelengths and will produce different intensities a t the detector after the sharp-cutoff filters. So the system is already suitable for two-color, two-intensity coding based on the standard four-dye chemistry. Fourth, a camera lens is used for light collection and imaging. This offers a larger observation field compared to a microscope objective7to image the larger number of capillaries. The choice of a largediameter lens is obvious for good light-collection efficiency. Here, a 50-mm-diameter lens is used. An internal aperture limits the f-number to f/1.4. Camera lenses easily provide good, flat-field imaging without spherical or chromatic aberrations in the visible region. A wide-angle lens provides a lower magnification ratio to match the 75-pm capillaries to the 2 5 - ~ mpixels in the C C D element. An extension tube is used to allow close focusing and thus a better effectivef-number without additional image distortion. Actually, even a t the 4000 capillary level, the image resolution required is substantially below that found in high-quality photographic applications (1 Fm in a 35-mm-wide field). Multiplexed Capillary Electrophoresis. To test the performance of the system, simultaneous capillary electrophoresis was attempted in 102 capillaries. The test mixture consisted of the 11 Hue111 4 x 1 7 4 fragments that have been used extensively in the development of size-based DNA separat i o n ~The . ~11 ~fragments ~ ~ ~are ~ a ~t 72,~ 118, 194, 234, 271, 281,310,603,872,1078,and 1353 bp. Theseparationmedium selected is in the class of replaceable linear polymers that has already been successfully used for D N A sequencing.14 Identical capillaries, separation medium, buffer solutions, and samples wereused to evaluate the parallel runs. A rudimentary injection and column flushing system was all that was required. To avoid irreproducibility in coating the capillary columns with a hydrophobic material, we used standard G C columns that are already bonded with a nonpolar phase. In the CCD image produced by our particular optical system, 385 active pixels (columns) map out the direction along the focused laser line. Of these, 366 span the 102 capillaries. To reduce the number of data points and to improve the signal-to-noise ratio (S/N), 2: 1 binning was implemented. So, each capillary spans 1.83 data channels. One essentially has every other binned pixel tracking a different capillary. In the axial direction of the capillaries, fluorescence does spread out beyond the immediate focused laser line because of consecutive reflections along the capillary walls as a result of the 45' entrance angle. Binning in this direction (12) Chan, K. C . ; Whang, C. W.; Yeung, E. S . J . Liq. Chromafogr. 1993, 16. 1941-1962. (13) Cohen, A. S.; Najarian, D. R.; Paulus, A,; Guttman, A,; Smith, J. A,; Karger, B. L. Proc. Nail. Acad. Sci. U.S.A.1988, 85, 9660-9663. (14) Ruiz-Martinez, M. C.; Berka, J.;Belenkii,A.; Foret, F.;Miller, A. W.; Karger, B. L. Ana/. Chem. 1993, 65, 2851-2858.
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31
32
33
34
35
36
37
a
TIME (min) Flgure 4. Electropherogams(partial)from consecutivebinned channels 97, 98, and 99 in a mukiplexed run. Capillaries are separated by 1.83 binned channels each. Excitation A = 514 nm.
therefore increases the S/N. In the end 200 X 2 binned data points were analyzed per frame of the CCD image to accommodate both laser lines; 400 reconstructed electropherograms were thus obtained in one run. Selected sets of these electropherograms are presented below to illustrate the performance of the system. Cross-Talk. While separation is performed in 102 discrete capillaries, the optical signals can interfere with each other. This is particularly problematic in the cylindrical-shaped optical regions. It is known that diffraction patterns can be set up in the plane of the incident laser beam perpendicular to the axes of the ~y1inders.I~ Spread of the excitation beam over to adjacent capillaries is not a problem, since each capillary is excited along the same focused line by design. The reverse of this is of concern, i.e., fluorescence from adjacent capillaries being refracted by the cylindrical walls to reach the CCD camera. We have already observed such cross-talk in capillary array^,^ but the contributions are small when the capillaries are shielded from each other. Figure 4 shows plots of three consecutive data channels on an expanded scale to highlight the early-eluting fragments. The top and the bottom electropherograms clearly represent two adjacent capillaries with distinctly different migration times. This is confirmed by the nearly identical plots obtained from the corresponding channels a t 488 nm. The middle plot is the isolation region in-between capillaries. We can see that signals from the adjacent capillaries are present there. However, cross-talk between the top and the bottom plots is small (
