Direct Observation of Single Native DNA Molecules in a

Strick, T. R.; Allemand, J.-F.; Bensimon, D.; Croquette, V. Biophys. J. 1998, 74, 2016−2028. [Crossref], [PubMed], [CAS]. (17) . Behavior of superco...
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Anal. Chem. 2004, 76, 4459-4464

Direct Observation of Single Native DNA Molecules in a Microchannel by Differential Interference Contrast Microscopy Seong Ho Kang,† Seungah Lee,† and Edward S. Yeung*,‡

Department of Chemistry, Chonbuk National University, Jeonju 561-756, South Korea, and Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011

Direct observation of single native DNA molecules in a microchannel was monitored without fluorescence-dye labeling. At a PDMS/glass microchip, the image of individual λ-DNA molecules appear sharp and distinct in Nomarski differential interference contrast microscopy. Intercalator dyes affected the physical properties and dynamic behavior of individual DNA molecules. From the migration velocities in the microchannel it is evident that native DNA molecules migrated faster than DNA molecules labeled with the intercalator YOYO-1. This is because YOYO-1 increases the molecular weight and size of λ-DNA and decreases the charge. The electric field strength and pH also affected the dynamics of single DNA molecules. We also observed that YOYO-labeled DNA was more stretched out compared to native DNA. Single-molecule detection (SMD) techniques have recently attracted plenty of attention in the field of life science.1 The observation and manipulation of single biomolecules allow their dynamic behaviors to be studied to provide insight into molecular genetics,2-4 biochip assembly,5-8 biosensor design,9-11 DNA biophysics,12-26 and basic separation theories of capillary electro* Corresponding author. Tel: 515-294-8062. Fax: 515-294-0266. E-mail: [email protected]. † Chonbuk National University. ‡ Ames Laboratory-USDOE and Department of Chemistry, Iowa State University. (1) Ishijima, A.; Yanagida, T. Trends Biochem Sci. 2001, 26, 438-444. (2) Herrick, J.; Michalet, X.; Conti, C.; Schurra, C.; Bensimon, A. Proc. Natl. Acad. Sci. (USA) 2000, 97, 222-227. (3) Herrick, J.; Bensimon, A. Chromosome Res. 1999, 7, 409-423. (4) Lyubchenko, Y. L.; Shlyakhtenko, L. S. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 496-501. (5) Hill, E. K.; de Mello, A. J. Analyst 2000, 125, 1033-1036. (6) Turner, S. W. P.; Levene, M.; Korlach, J.; Webb, W. W.; Craighead, H. G. Proc. µTAS 2001 Symp. 2001, 259-261. (7) Yoshinobu, B. Proc. µTAS Symp. 2000, 467-472. (8) Shivashankar, G. V.; Libchaber, A. Curr. Sci. 1999, 76, 813-818. (9) Chan, V.; McKenzie, S. E.; Surrey, S.; Fortina, P.; Graves, D. J. J. Colloid Interface Sci. 1998, 203, 197-207. (10) Chan, V.; Graves, D. J.; Fortina, P.; McKenzie, S. E. Langmuir 1997, 13, 320-329. (11) Jordan, C. E.; Frutos, A. G.; Thiel, A. J.; Corn, R. M. Anal. Chem. 1997, 69, 4939-4947. (12) Bensimon, A.; Simon, A.; Chiffaudel, A.; Croquette, V.; Heslot, F.; Bensimon, D. Science 1994, 265, 2096-2098. (13) Bensimon, D.; Simon, A. J.; Croquette, V.; Bensimon, A. Phys. Rev. Lett. 1995, 74, 4754-4757. (14) Xue, Q.; Yeung, E. S. Nature 1995, 373, 681-683. 10.1021/ac0496143 CCC: $27.50 Published on Web 06/12/2004

© 2004 American Chemical Society

phoresis (CE) and liquid chromatography (LC).27-33 Generally, the detection of fluorescence from individual molecules of interest is used for these ultrasensitive analytical and biophysical applications. However, although studies at the single-molecule level have been reported extensively, the results did not come directly from the native molecules of interest but indirectly from the derivatized molecules. Therefore, a new detection method for native biomolecules (without fluorescence-dye labeling) in solution are needed to facilitate real-time monitoring and for dynamic studies of individual molecules. The basic principle of differential interference contrast (DIC) microscopy as developed by Nomarski et al. and its variants is similar to conventional interferometers in that the measuring and comparison beam systems are made to interfere with each other, the difference being that the comparison beam also passes through the object.34 The distance between the two beams is always very small (the order of 1 µm) and is sometimes deliberately chosen to be below the minimum resolvable distance defined by the diffraction limit.35 The optical path differences make the objective visible. The plasticity of the image obtained with (15) Houseal, T. W.; Bustamante, C.; Stump, R. F.; Maestre, M. F. Biophys. J. 1989, 56, 507-516. (16) Auzanneau, I.; Barreau, C.; Salome, L. C. R. Acad. Sci., Ser. III 1993, 316, 459-462. (17) Strick, T. R.; Allemand, J.-F.; Bensimon, D.; Croquette, V. Biophys. J. 1998, 74, 2016-2028. (18) Fan, F.-R. F.; Bard, A. J. Science 1995, 267, 871-874. (19) Funatsu, T.; Harada, Y.; Tokunaga, M.; Saito, K.; Yanagida, T. Nature 1995, 374, 555-559. (20) Chiu, D. T.; Zare, R. N. J. Am. Chem. Soc. 1996, 118, 6512-6513. (21) Nie, S.; Chiu, D. T.; Zare, R. N. Science 1994, 266, 1018-1021. (22) Yokota, H.; Saito, K.; Yanagida, T. Phys. Rev. Lett. 1998, 80, 4606-4609. (23) Enderlein, J. Biophys. J. 2000, 78, 2151-2158. (24) Xu, X.-H.; Yeung, E. S. Science 1997, 276, 1106-1109. (25) Dickson, R. M.; Norris, D. J.; Tzeng, Y.-L.; Moerner, W. E. Science 1996, 274, 966-969. (26) Ma, Y.; Shortreed, M. R.; Yeung, E. S. Anal. Chem. 2000, 72, 4640-4645. (27) Xu, X.-H. N.; Yeung, E. S. Science 1998, 281, 1650-1653. (28) Shorteed, M. R.; Li, H.; Huang, W.-H.; Yeung, E. S. Anal. Chem. 2000, 72, 2879-2885. (29) Smith, S. B.; Aldridge, P. K.; Callis, J. B. Science 1989, 243, 203-206. (30) Ueda, M. J. Biochem. Biophys. Methods 1999, 41, 153-165. (31) Kang, S. H.; Shortreed, M. R.; Yeung, E. S. Anal. Chem. 2001, 73, 10911099. (32) Zheng, J.; Yeung, E. S. Anal. Chem. 2002, 74, 4536-4547. (33) Kang, S. H.; Yeung, E. S. Anal. Chem. 2002, 74, 6334-6339. (34) Allen, R. D.; David, G. B.; Nomarski, G. Z. Wiss. Mikr. 1969, 69, 193-221. (35) James, J. Light Microscopic Techniques in Biology and Medicine; Martinus Nijhoff Medical Division: Netherlands, 1976; pp 185-192.

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Figure 1. Experimental setup, (A), for single-DNA molecules monitored by DIC at a PDMS/glass microchannel, (B) and (C).

DIC may be rather impressive, even though the observed heights and depths only represent local differences in the gradient of the optical path through the sample.36 However, despite the known properties, DIC is relatively untapped as an imaging method compared to conventional fluorescence microscopy. DIC microscopy has only been applied to obtaining images of fixed samples by using a conventional cover slip and a glass slide.37 However, we are not aware of any papers related to single biomolecule detection and manipulation in flow streams using DIC microscopy. Recently, microchip techniques such as micro total analysis system (µ-TAS)38 or lab-on-a-chip39 have been developed, especially in the field of microfluidics. Microfluidics has enabled many developments in chemical analysis of biomolecules including highspeed separation, high-throughput, parallel assays, and microscale sample preparation.40 In this work, we demonstrate singlemolecule detection and manipulation by DIC optical microscopy without any modification or fluorescence-dye labeling. The technique was successfully applied to follow real-time dynamic behavior of individual λ-DNA molecules in a microchannel. EXPERIMENTAL SECTION Buffer Solutions. Gly-Gly, CHES, and sodium hydroxide were A.C.S. grade and purchased from Sigma Chemical Co (St. Louis, MO). The various buffer systems used are as follows: sodium acetate/acetic acid (pH 6.0-7.5), Gly-Gly/NaOH (pH 8.2), and CHES/NaOH (pH 9.0-10.0). A.C.S. grade or higher glacial acetic acid, sodium acetate, and sodium chloride (all from Fisher (36) van Munster, E. B.; van Vliet, L. J.; Aten, J. A. Quantitative interferometric imaging using a conventional differential interference contrast microscope. In Optical Diagnostics of Biological Fluids and Advanced Techniques in Analytical Chemistry; Priezzhev, A. V., Asakura, T., Leif, R. C., Eds.; Proceedings SPIE, 1997; Vol. 2982, pp 458-467. (37) Visscher, K.; Gross, S. P.; Block, S. M. IEEE J. Quantum Electron. 1996, 2, 1066-1076. (38) Harrison, D. J.; van den Berg, A. Micro Total Analysis Systems ’98; Kluwer Academic Publishers: Dordrecht, 1998. (39) Graves, D. J. Trends Biotechnol. 1999, 17, 127-134. (40) Ropper, M. G.; Shackman, J. G.; Dahlgren, G. M.; Kennedy, R. T. Anal. Chem. 2003, 75, 4711-4717.

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Scientific, Fair Lawn, NJ) were dissolved in ultrapure (18 MΩ) water using the same procedure as described by Kang et al.31 Sodium acetate buffer solutions were prepared at various pHs using 1 M solutions of acetic acid, sodium acetate, and sodium chloride. All other chemicals were A.C.S. grade. All solutions were filtered through a 0.2-µm filter prior to use. DNA Sample Preparation. λ-DNA (48 502 bp) was obtained from Life Technologies (Grand Island, NY). A 1-kb fragment of DNA was purchased from Seegene Co. (Seoul, Korea). All DNA samples were prepared in 10 mM Gly-Gly buffer, pH 8.2. DNA samples at a concentration of 200 pM were labeled with an intercalating dye YOYO-1 (Molecular Probes, Eugene, OR) at a ratio of one dye molecule per five base pairs according to the manufacturer’s instructions. DNA samples labeled with YOYO-1 were allowed to incubate for 5 min before further dilution and use. However, in DIC detection, some DNA samples were simply prepared in 10 mM Gly-Gly buffer without intercalating dye. For single-molecule imaging experiments, these DNA samples were further diluted to 10-20 pM immediately prior to the start of the experiment in the appropriate buffer. It was verified that the addition of the Gly-Gly buffer did not noticeably change the pH of the final sample solution. DIC Microscope and CCD Camera. An upright Olympus BX51 microscope (Olympus Optical Co., Ltd., Shinjuku-ku, Tokyo, Japan) equipped with a DIC slider (U-DICT, Olympus) was used for most investigations (Figure 1A). A 20× objective lens (Olympus UPLFL20×/0.5 N.A., W.D. 1.6) was used. A CCD (Cool SNAP fx, Photometrics, Tucson, AZ) camera was mounted on top of the microscope with a halogen lamp and a Hg lamp. The CCD exposure time was 5-500 ms at 20 MHz digitization speed. Electrophoresis and manipulation of single-DNA molecules were performed with a high-voltage power supply (DBHV-100, Digital Bio Technology Co., Ltd., Seoul, South Korea) in the range of 0-5 kV. DNA samples were driven at 0.41-81.6 V/cm. Then 0.2and 0.5-µm microbeads (FluoSpheres carboxylate-modified microspheres, Molecular Probes) were used to calibrate the deter-

Figure 2. Fluorescence image of λ-DNA molecules stained with the intercalator YOYO-1 at a PDMS/glass microchannel from an epi-fluorescence microscope. Electric field, 20.4 V/cm; objective lens, UPLFL20×/0.5 W.D. 1.6; CCD exposure time, 300 ms; sample, 10 pM λ-DNA labeled with YOYO-1.

mination of the size of individual single-DNA molecules in the microchannel. MetaVue software (Version 5.0, Downingtown, PA) was used for DNA image collection and data processing. Microchip Preparation for DIC. The PDMS/glass microchip for DIC is illustrated in Figure 1B,C. The microchips were produced at the Digital Bio Technology Microfabrication Facility (Seoul, Korea). A 4.9-cm-long straight channel was fabricated from poly(dimethylsiloxane) (PDMS) made with Sylgard 184A and B (Dow Corning, Corning, NY). The master composed of positive relief structure of a channel was made in a Corning silicon wafer by using standard microphotolithographic technology. The PDMS was prepared, degassed in a vacuum for 30 min, and poured over the channel master. After curing at 80 °C for 3 h and cooling at room temperature for 3 h, the PDMS was peeled off from the silicon mold, producing a pattern of negative relief channels. Then it was cut to a suitable size by a blade and holes (2-mm diameter) were punched into the cured polymer to create access reservoirs by a punch machine (Bollmanncrip). The cover-glass plate (No. 1 Corning cover glass; 60-mm length × 24-mm width, 0.15-mm thick) and the PDMS with negative channel was attached by using the PDC-32G-2 Basic Plasma Cleaner (Harrick Scientific Co., Broadway Ossing, NY) to enclose the microchannel and create stability for the flexible PDMS chip. A small PDMS plate with a 2-mm-diameter hole was left above the PDMS/glass microchip to increase the volume of solution in the reservoirs. The microchip was composed of two PDMS plates (well for reservoir, 1.5 mm thick, and cover for microchannel, 0.26 mm thick) and one glass plate (No. 1 Corning cover glass). The channel dimension was 49 mm long, 50 µm wide, and 50 µm deep. The reservoirs were 2.0 mm in diameter and 1.76 mm in total depth. RESULTS AND DISCUSSION According to our previous report,31 individual λ-DNA molecules at a fused-silica/water interface formed random coils and their conformations fluctuated rapidly above pH 5.5. As the pH further decreased to 4.5, DNA molecules were gradually dragged onto the fused-silica surface, similar to the process of molecular combing.3,11,41 However, at the PDMS/glass microchip, both the λ-DNA molecules stained with the intercalator dye YOYO-1 (YOYO-DNA) and the native λ-DNA molecules were not eluted

below pH 6.5 because of adsorption on the PDMS surface of the microchannel. Above the pH 7.0 and the 300-ms exposure time, the fluorescence images of YOYO-DNA exhibit a linear shape (Figure 2 and movie M1 in Supporting Information). This is due to the motion of DNA during the long exposure, resulting in an integrated trajectory in the form of a streak.28 The streaks show different thicknesses depending on the vertical location of the molecule relative to the focal plane. Real-time monitoring of the motion of individual native λ-DNA in the microchannel was facilitated by the Nomarski differential interference contrast (DIC) microscopy without labeling with YOYO-1. The trajectory of native λ-DNA molecules resemble an earthworm at an exposure time of 300 ms (Figure 3A,B). However, the intercalator changed the detailed dynamic behavior of λ-DNA molecules (movie M2A, YOYO-DNA and M2B, native λ-DNA molecules in Supporting Information). The fact that the streak lengths are primarily the result of DNA motion (electrophoresis) is confirmed in Figure 3C,D. Therefore, the short exposure times essentially eliminated the contributions of motion to the streak lengths. The migration velocities of λ-DNA molecules as a function of the electric field in the microchannel are shown in Figure 4. The velocity of λ-DNA molecule increased with the applied electric field and the native DNA molecules (black circles in Figure 4) migrated faster than the YOYO-DNA molecules (open triangles in Figure 4). At pH 8.2, both the native λ-DNA and YOYO-DNA have the net negative charge. However, YOYO-1 intercalates one out of five DNA bases. YOYO-1 (C49H58I4N6O2; M.W. 1270.65) adds molecular weight and size to the DNA. Furthermore, because YOYO-1 is positively charged, it decreases the electrophoretic mobility of the DNA-YOYO complex by reducing the ionic charge similar to ethidium bromide.42 So the YOYO-DNA showed a slower velocity than the native DNA molecules. In general, because the molecules are moving relative to the camera during exposure, the trajectories show up as streaks in the images. In our previous work, the streak length was used to determine the electrophoretic mobilities in the molecular iden(41) Allemand, J.-F.; Bensimon, D.; Jullien, L.; Bensimon, A.; Croquette, V. Biophys. J. 1997, 73, 2064-2070. (42) Guttman, A.; Cooke, N. Anal. Chem. 1991, 63, 2038-2042.

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Figure 3. DIC images of (A) λ-DNA molecules labeled with YOYO-1 at 300-ms exposure; (B) native λ-DNA molecules at 300-ms exposure; (C) λ-DNA molecules labeled with YOYO-1 at 10-ms exposure; and (D) native λ-DNA molecules at 10-ms exposure. Electric field, 10.2 V/cm; sample, 20 pM λ-DNA.

Figure 4. Migration velocity of individual λ-DNA molecules at the PDMS/glass microchannel as a function of the applied electric field monitored by DIC microscopy. CCD exposure time, 10 ms; sample, 20 pM λ-DNA molecule. Solid circles: native λ-DNA. Open triangles: λ-DNA labeled with YOYO-1. Error bars are the standard deviations of five measurements.

tity.28 That is, the faster molecules leave a longer streak and the slower molecules leave a shorter streak. Here, the measured length of both types of λ-DNA molecules varied roughly linearly like the exposure time (Figure 5). The native DNA molecules showed a ball shape at the shortest exposure time of 10 ms (movie M3 in Supporting Information), showing they are not distorted by the flow. However, Figure 5 shows that the YOYO-labeled DNA molecules are more extended than the native DNA molecules. This can be explained by the fact that intercalation stiffens the chain and creates a more rodlike molecule. Figure 6 shows the effect of the pH on the length of native λ-DNA molecules at exposure times of 10 ms (open circle) and 300 ms (solid circle). Above pH 7.0, the surface of glass has 4462 Analytical Chemistry, Vol. 76, No. 15, August 1, 2004

Figure 5. Streak lengths of individual DNA molecules under an electric field of 10.2 V/cm. Sample, 20 pM λ-DNA molecule. Solid circles: native λ-DNA molecules. Open triangles: λ-DNA molecules labeled with YOYO-1. Other conditions were the same as those in Figure 3.

negatively charged silanoate (SiO-) groups. The electrostatic repulsive force dominates the interaction between the surface of the microchannel and DNA molecules. The DNA molecules can move freely above the surface and create streaks at the longer exposure time. Below pH 6.9, hydrophobic interaction between the microchannel and the DNA molecule become stronger than the electrostatic force. The effect of pH is different from that reported before31 because the PDMS surface has a different chemical composition (Si(CH3)2-O-Si(CH3)2-O-Si(CH3)2-OSi(CH3)2-O) compared to the glass surface (SiO-). Thus, the DNA molecules were adsorbed onto the PDMS surface of the microchannel by hydrophobic interaction. As the pH further decreased to 6.5, λ-DNA molecules were immobilized at the PDMS surface, so the motion-related streak length of DNA molecules decreased even at the long exposure time (solid circles in Figure 6). Below pH 6.5, the DNA molecules were strongly adsorbed on

Figure 6. Streak length of native λ-DNA molecules (10 pM) at different pH. Electric field, 10.2 V/cm; run buffer, 25 mM sodium acetate/acetic acid (pH 6.5-7.5), 10 mM Gly-Gly/0.1 M sodium hydroxide (pH 8.2), 25 mM CHES/0.1 M sodium hydroxide (pH 9.010.0). Open circles: 10-ms exposure time. Solid circles: 300-ms exposure time.

the PDMS surface, and the DNA molecules did not elute out through the PDMS/glass microchannel. These results indicate that hydrophobic interaction rather than electrostatic interaction at the liquid-solid interface of the microchip is the major driving force for DNA adsorption, even though both interactions have been implicated in adsorption.3,4,31,41 The individual λ-DNA molecules did not show the same motion and shape in the direction of fluidic flow at pH 8.2. While the solution containing λ-DNA molecule was electrokinetically transported through the microchannel, some DNA molecules started to adsorb onto the surface. We have already shown that adsorption of one end of a DNA molecule under bulk flow causes a

lengthening of the DNA molecule.31 However, permanent immobilization on the channel surface was not observed. This adsorption/desorption behavior of DNA molecules at the basic pH is likely caused by the strong hydrophobic interaction and weak electrostatic repulsion between the PDMS surface and the DNA molecules. We found that λ-DNA molecules in 25 mM sodium acetate buffer (pH 6.0) were strongly adsorbed on the surface of PDMS and did not elute through the microchannel. Thus, we could not observe any DNA molecules at the end of the microchannel (Figure 7A). This confirms that we are seeing DNA molecules and not other types of molecules. However, in the same pH buffer with 0.3% poly(vinylpyrrolidone) (PVP, Mr 1 000 000), the polymer dynamically coated the surface of the microchannel and the individual DNA molecules could be electrokinetically driven (Figure 7B). This result is consistent with the fact that PVP can suppress electroosmotic flow and prevent the adsorption of DNA onto the PDMS as well as the fused-silica surfaces in CE43 and at liquid-solid interfaces.31 The minimum size detectable in the PDMS/glass microchip by this DIC method was a 1-kb DNA molecule (about 85 nm, Figure 8A). We could also easily observe 500-nm microspheres without any pretreatment (Figure 8B). CONCLUSION Direct observation of the dynamic behavior of native singleDNA molecules was achieved by the DIC microscope technique at a microchannel without fluorescence-dye labeling. The basic principle of DIC is that only local differences in optical thickness contribute so that gradients of optical path differences of singleDNA molecules were made visible in the channel. However, images of λ-DNA molecules could only be obtained by using 2 mm or thinner microchannels (Figure 1B,C) because of the working distance of our DIC objective. The real-time kinetics of

Figure 7. Real-time DIC images showing the effect of PVP on the adsorption of λ-DNA molecules at a PDMS/glass microchannel. Electric field, 10.2 V/cm; CCD exposure time, 10 ms; sample, 10 pM native λ-DNA molecules. Run buffer: (A) 0% PVP and (B) 0.3% PVP (Mr 1 000 000) in 25 mM sodium acetate/acetic acid buffer (pH 6.0). Molecules can be seen moving along the channel in (B) but not in (A) because of adsorption.

Figure 8. DIC images of (A) native 1-kb DNA molecule and (B) 500-nm carboxylate-modified microspheres in a PDMS/glass microchip. Electric field, 10.2 V/cm; CCD exposure time, 10 ms; run buffer, 10 mM Gly-Gly buffer (pH 8.2 with 0.1 M NaOH). *Arrow indicates an intact 1-kb DNA molecule.

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individual λ-DNA molecules at the PDMS/glass surfaces were affected by the balance of adsorption/desorption between the DNA molecules and the surface of the microchannel and translation due to the applied electric field. We found that the intercalator dye YOYO-1 affects the behavior of native single-DNA molecules such that native λ-DNA molecules always migrated faster than YOYO-DNA. This new SMD technique (DIC microscopy) should be applicable to a wide variety of biochip studies and to the investigation of DNA biophysics in liquids at room temperature. ACKNOWLEDGMENT We thank Digital Bio Technology for supplying the PDMS/ glass microchip. This study was partially supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (02-PJ10-PG4-PT02-0042). E.S.Y. thanks the Robert Allen Wright Endowment for Excellence for support. The Ames Laboratory is operated for the U.S. Department of Energy (43) Gao, Q.; Yeung, E. S. Anal. Chem. 1998, 70, 1382-1388.

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by Iowa State University under Contract No. W-7405-Eng-82. This work was supported by the Director of Science, Office of Basic Energy Sciences, Division of Chemical Sciences. SUPPORTING INFORMATION AVAILABLE Four AVI movie files: movie M1 (fluorescence), dynamics of YOYO-labeled λ-DNA molecules at the PDMS/glass microchannel at a CCD exposure time of 300 ms; movie M2A (DIC), dynamics of λ-DNA molecules labeled with YOYO-1 (YOYO-DNA) and M2B (DIC), dynamics of native λ-DNA molecules at a CCD exposure time of 300 ms; and movie M3, dynamics of native λ-DNA molecules at the PDMS/glass microchannel at a CCD exposure time of 10 ms. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review March 11, 2004. Accepted May 10, 2004. AC0496143