Single-DNA analysis combines fluorescence microscopy with a method of stretching a single DNA molecule from its random coil shape to a linear shape. Although super-resolution imaging methods can be used for analyzing the DNA with a higher lateral resolution, these methods require several seconds to minutes to obtain a super-resolution image of the entire DNA molecule. Therefore, immobilizing the stretched DNA molecules on the substrate surface is essential for preventing the decrease in the lateral resolution caused by thermal fluctuations of the molecule. Previous studies utilized a method to use super-resolution imaging methods, in which a DNA molecule can be stretched by the surface tension of the air–liquid interface and immobilized on a glass surface treated with a silane coupling agent. However, achieving control over the stretch ratio of the DNA molecule poses challenges because of the difficulty in accurately adjusting the surface tension. In this study, we used the combination of stretching DNA molecules using pressure flow in a microchannel and immobilizing them on a glass surface treated with a silane coupling agent. Our results indicated that this method enabled the control of the stretch ratio of the molecule by adjusting the flow velocity and the super-resolution imaging while reducing thermal fluctuation by immobilizing the molecule on the surface. Combining the method with the super-resolution imaging method enables the analysis of single DNA molecules with higher accuracy.

Single DNA analysis typically uses the method of stretching a single DNA molecule from its random coil shape to a linear shape in combination with direct observation by fluorescence microscopy. The DNA analysis includes size, protein–DNA interactions, and sequence-specific analyses.1–10 Super-resolution imaging methods such as stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), binding-activated localization microscopy (BALM), stimulated emission depletion (STED) microscopy, and structured illumination microscopy (SIM) have been proposed to overcome the limit of the lateral resolution of microscopy, which is several hundred nanometers because of the diffraction limit. Utilizing these super-resolution imaging methods enhances the lateral resolution of single-DNA analysis. Previous studies have demonstrated that STED microscopy obtains the super-resolution imaging of single DNA molecules with a lateral resolution of 45 nm, which is five times smaller than that of confocal imaging.11 Utilizing this method enabled accurate and precise analysis of the lengths of single DNA molecules ranging from a few hundred base pairs (bp) to tens of kilobase pairs (kbp).12 In addition, by using BALM with a vortex point spread function, 3D localization microscopy of single DNA molecules was achieved with a lateral resolution of 20 nm. This enabled the orientation estimation of 48 kbp DNA and the analysis of intertwined structures on supercoiled DNA at the super-resolution level.13 

However, these methods require several seconds to minutes to obtain the image of an entire DNA molecule. Given that the thermal fluctuation of the DNA molecule is several hundred nanometers,14 it is essential to immobilize the molecules stretched with a high stretch ratio, which is defined as the stretch length divided by the contour length, to prevent the decrease in the lateral resolution. The DNA combing method has been used as a general method to stretch and immobilize DNA molecules.15–19 In this method, a substrate surface treated with a silane coupling agent such as 3-aminopropyltriethoxysilane (APTES) is immersed in a DNA solution to immobilize one end of the molecule onto the surface. The substrate is then lifted from the solution, creating an air–liquid interface that stretches the DNA molecules due to surface tension. Immobilization on the substrate surface occurs as the air–liquid interface traverses over the DNA molecule. The super-resolution imaging of DNA molecules stretched and immobilized using this method has been achieved using STED microscopy and direct STORM.11,12,20 Although surface tension can be adjusted by changing the surface treatment, controlling the stretch ratio of the DNA molecule is challenging.

Conversely, methods for stretching a single DNA molecule using a flow field in a microchannel have been proposed.21–24 After one end of the molecule is immobilized on a microbead using a biotin–streptavidin interaction, the bead is trapped in the microchannel using an optical tweezer and the DNA molecule is stretched by pressure flow. In other methods, the ends of the molecule are immobilized on the surface using the biotin–avidin interaction or microfabricated structures, and the molecule is stretched by a flow field.25,26 In these methods, the stretch ratio can be controlled precisely by adjusting the velocity of the flow field because the force required for stretching the DNA molecule is the shear force generated by the flow. However, the position of the stretched DNA molecule changes over time because of thermal fluctuations caused by only ends of the DNA molecule being immobilized on the beads or the structures. Methods for stretching a single DNA molecule in a nanochannel with the depth and width smaller than the diameter of randomly coiled DNA have also been proposed;27–30 however, in these methods, the DNA molecule was not immobilized in the nanochannel.

In this study, we used the combination of the stretch of a single DNA molecule using pressure flow in a microchannel and its immobilization on a substrate whose surface was treated with APTES. A single DNA molecule in the microchannel was observed by fluorescence microscopy for clarifying the stretch and immobilization processes. In addition, the dependence of the stretch ratio of the DNA on the flow velocity was investigated. Finally, the effectiveness of this method for super-resolution imaging was investigated by performing the direct STORM imaging of stretched and immobilized DNA molecules in a microchannel. These results indicated that this method enables the control of the stretch ratio of the molecule by adjusting the flow velocity and super-resolution imaging while reducing thermal fluctuation by the immobilization of the molecule.

The straight microchannel was fabricated using PDMS. A photoresist (SU-8-2, Kayaku Advanced Materials, Inc.) was coated on the Si substrate using a spin coater. The substrate was exposed to a mercury lamp via a photomask after prebaking it at 65 °C for 1 min and soft baking it at 95 °C for 3 min. The substrate was post-baked at 65 °C for 1 min and 95 °C for 1 min; it was developed with the SU-8 developer for 1 min. Then, it was rinsed with isopropyl alcohol for 1 min and dried with N2. Thereafter, the substrate underwent hard-baking at 200 °C for 30 min. A PDMS prepolymer (SILPOT 184 W/C, Dow Corning Toray Co. Ltd.) mixed with a curing agent at a ratio of 10:1 w/w was poured onto the Si substrate, and the pattern on the Si substrate was transferred onto the PDMS at 120 °C for 1 h. The glass substrate was treated with air plasma for 1 min and then immersed in an APTES solution diluted to 0.01% using pure water for 30 min. The glass and PDMS substrates were bonded at 100 °C for 30 min. The width, depth, and length of the microchannels were 50, 3 μm, and 9 mm, respectively.

Lambda DNA (48.5 kbp, Takara Bio) was used as the DNA sample, 6 μl of 500 ng/μl was used as the DNA solution, and 10 ml of 0.1 μM YOYO-1 (Molecular Probes®; excitation wavelength: 491 nm and fluorescence wavelength: 509 nm) was introduced as a fluorescent dye. The length Lc of the DNA molecule with YOYO-1 is 24.5 μm,31 and the final concentration of the molecules in YOYO-1 was 0.03 ng/μl. An inverted fluorescence microscope (Olympus, IX73) with an oil immersion objective (Olympus, Apochromat, 60×, NA 1.42) was used for fluorescence observations. The pressure pump was connected to the reservoir of the fabricated device using a pressure controller. An LED light source with a wavelength of 460 nm was used as the excitation light source for the fluorescence observations in the microchannel. The excitation power of the sample was 0.1 kW/cm2, and the fluorescent images were acquired using a high-sensitivity camera (Andor iXon Ultra 897).

The DNA molecules were introduced into the microchannel by applying pressure after 5 μl of DNA molecules were introduced into the reservoir of the device. Stretch and immobilization were observed using a fluorescence microscope. The exposure time and EM gain were set to 50 ms and 500, respectively. The frame interval for acquiring images was 83 ms. Pressure in the range of 15–80 kPa was applied to verify the dependence of the stretch ratio of the DNA molecules on the flow velocity. The velocity v of the DNA molecules migrating at each pressure was measured to be 98–3100 μm/s based on the time variation of the fluorescent images. Direct STORM imaging was performed for the super-resolution imaging of DNA molecules, which uses the spontaneous photoblinking of fluorescent molecules. Direct STORM imaging of DNA molecules using YOYO-1 was performed according to the procedure described in our previous study.20 After the DNA molecule was stretched and immobilized in the microchannel, the switching buffer (phosphate buffered saline with oxygen scavenger [0.5 mg/ml glucose oxidase (Sigma-Aldrich), 40 μg/ml catalase (Sigma-Aldrich), 10% w/v glucose (Thermo Fisher Scientific), and 50 mM β-mercaptoethylamine (MEA, Fluka)]) was introduced into the microchannel via the reservoir. The photoblinking of the fluorescent molecules was captured using an EMCCD camera. The exposure time and EM gain were set to 100 ms and 500, respectively. The frame number and interval for image acquisition were 4600 and 125 ms, respectively. The center position was identified with an accuracy beyond the diffraction limit by the Gaussian fitting of the fluorescence distribution of the emitted YOYO-1 dye using the ImageJ plugin ThunderSTORM. A super-resolution image was obtained by reconstructing center positions.

The left side of Fig. 1 represents a typical fluorescent image of a single DNA molecule at a flow velocity of 105 μm/s, which indicates that the DNA molecule was stretched and immobilized using pressure flow in the microchannel. The right side of Fig. 1 shows a schematic of the process based on fluorescent images. First, one of the random-coil-shaped DNA molecules migrating in the microchannel was trapped to the APTES surface of the glass substrate. This trapping occurred as the hydrophobic end of the DNA molecule was specifically adhered to the hydrophobic surface modified with APTES, following a method similar to the DNA combing method in previous studies.12 The DNA molecule was stretched by the shear force of the pressure flow. Finally, the stretched DNA molecule was immobilized on the surface after the DNA vibrated perpendicular to the stretch direction because of its thermal fluctuation. This is because the DNA molecule had several hydrophobic parts, except for the ends, because of defects in the double helix strand.15 These parts were immobilized on the surface when the stretched DNA molecule approached the surface. The DNA molecule remained stationary, unaffected by pressure flow or thermal fluctuations, as observed through fluorescence imaging.

FIG. 1.

Fluorescence observation of a single DNA molecule migrating in the microchannel and schematic of the stretch and immobilization processes. The DNA molecule was stained with YOYO-1. After one DNA molecule was trapped on the surface treated by APTES, the DNA molecule was stretched by pressure flow and immobilized.

FIG. 1.

Fluorescence observation of a single DNA molecule migrating in the microchannel and schematic of the stretch and immobilization processes. The DNA molecule was stained with YOYO-1. After one DNA molecule was trapped on the surface treated by APTES, the DNA molecule was stretched by pressure flow and immobilized.

Close modal

Figure 2 shows a typical fluorescence image of the microchannel after applying pressure. Although many DNA molecules were stretched and immobilized, there were more than a few DNA molecules with a low degree of stretching. There are two possible reasons for this issue:

  1. The order of the stretch and immobilization processes

    As shown in Fig. 1, a single DNA molecule can be stretched and immobilized in a linear shape in the following sequence: trapping one end of the DNA molecule to the surface, stretching it by pressure flow, and immobilizing the other parts. However, the initial trapping of the DNA molecules’ hydrophobic parts on the surface occurred randomly. Therefore, stretched DNA molecules, whose parts except for their ends were first trapped on the surface, had a partially random-coil shape, resulting in a reduced degree of stretch. Using a surface modification method that interacts more strongly with the ends of the DNA molecule and less strongly with the rest of the molecule is important to overcome this issue. The biotin–streptavidin interaction can strongly trap the ends of a DNA molecule on the surface.32 Therefore, it may be possible to accurately determine the order of the stretch and immobilization processes and improve the degree of stretch of the molecule using biotin–streptavidin interaction in addition to APTES.

  2. The cleavage of the DNA molecule

    This results in DNA molecules with lengths of several micrometers, which is clearly a low degree of stretching. The stretch force required to cleave a single DNA molecule is ∼10 nN.15 In contrast, the stretch force caused by the shear force of the pressure flow can be calculated to be of pN order, which is considerably smaller than the force required for cleavage. Therefore, DNA molecules are not cleaved during the stretching process. DNA molecules may have been cleaved by shear forces in the bulk state during the preparation of the DNA sample. However, a clear conclusion regarding this issue is yet to be reached.

FIG. 2.

Fluorescence image of DNA molecules with YOYO-1 in the microchannel after applying the pressure flow. Although many DNA molecules were stretched and immobilized, there were many DNA molecules with a low degree of stretch.

FIG. 2.

Fluorescence image of DNA molecules with YOYO-1 in the microchannel after applying the pressure flow. Although many DNA molecules were stretched and immobilized, there were many DNA molecules with a low degree of stretch.

Close modal

Figure 3 shows the dependence of the average and maximum stretch ratios on flow velocity v. The stretch lengths of several dozen DNA molecules were measured under the same conditions, and the stretch ratio was calculated by dividing the average stretch length Lave and maximum stretch length Lmax by the length Lc. Furthermore, Lave/Lc and Lmax/Lc increased with an increase in the flow velocity v. However, the ratio of Lave/Lc was ∼40%, with a considerable deviation due to the presence of several DNA molecules exhibiting low stretch lengths, as shown in Fig. 2. Lmax/Lc was larger than 90% when v was larger than 1000 μm/s.

FIG. 3.

Results of the average and maximum stretch ratio of DNA molecules with the flow velocity. The solid line is the theoretical result calculated by Eq. (3), and the experimental maximum stretch ratio agreed well with the theoretical result.

FIG. 3.

Results of the average and maximum stretch ratio of DNA molecules with the flow velocity. The solid line is the theoretical result calculated by Eq. (3), and the experimental maximum stretch ratio agreed well with the theoretical result.

Close modal
Based on the observation of the stretch and immobilization processes, the stretch ratio of the DNA molecule was determined by the stretch process using the shear force after one end was trapped. We assumed that the dependence of the stretch ratio on v was based on the model of stretching the tethered DNA proposed in a previous study.33 When the stretch length is L, the relationship between the stretch ratio L/Lc and shear rate γ can be expressed as
(1)
where τ represents the relaxation time of the molecule. If z is the depth direction of the microchannel and z = 0 is the center of the microchannel, the velocity distribution v(z) can be expressed as
(2)
where vave and h represent the average flow velocity and depth of the microchannel, respectively. Therefore, the shear rate γ on the glass surface in the microchannel can be written as 6vave/h. From Eq. (2), the relationship between L/Lc and vave can be expressed as
(3)
The solid line in Fig. 3 represents the theoretical results calculated using Eq. (3), where τ and h are 0.3 s and 3 μm, respectively. The experimental Lmax/Lc values agreed well with the theoretical results, indicating that Lmax/Lc can be adjusted by controlling the velocity of the pressure flow in the microchannel.
Figure 4(a) shows an epifluorescence image of the DNA molecule stretched and immobilized in the microchannel. The fluorescent molecules were photoblinked after photobleaching the fluorescent molecule combined with the DNA molecule, as shown in Fig. 4(b). Figure 4(c) shows a super-resolution image obtained by reconstructing the center positions of the fluorescent molecules using ThunderSTORM. Figure 4(d) shows the cross-sectional fluorescence distribution at five points in a super-resolution image of the DNA molecule. The full width at half maximum (FWHM) was measured to be 80 nm by fitting each fluorescence distribution to a Gaussian distribution. We compared the theoretical FWHM calculated from the identification accuracy of the center positions of the emitted fluorescent molecules with the experimental FWHM. The identification accuracy can be expressed as
(4)
where N, s, a, and b represent the sum of the fluorescence intensity, standard deviation of the fluorescence distribution, pixel size, and standard deviation of the background noise, respectively.34 The theoretical FWHM was calculated as 70 nm by substituting each average value obtained from the fluorescence distributions of the fluorescent molecules emitted during image acquisition into Eq. (4). The center position of the same fluorescent molecule would change with time because of the thermal fluctuation of the DNA molecule if the DNA molecule was not immobilized on the surface of the substrate; this is several hundred nanometers, and the experimental FWHM would be considerably larger than the theoretical FWHM. Therefore, this result indicated that the immobilization of the stretched DNA molecule on the surface reduced the thermal fluctuation, and super-resolution imaging was obtained.
FIG. 4.

Super-resolution imaging of the DNA molecule stretched and immobilized in the microchannel using direct STORM with YOYO-1. (a) Epifluorescence image of the DNA molecule, (b) photoblinking image of fluorescent molecules, (c) super-resolution image of the DNA molecule, and (d) cross-sectional fluorescence distribution of the super-resolution image. The experimental FWHM was measured to be 80 nm.

FIG. 4.

Super-resolution imaging of the DNA molecule stretched and immobilized in the microchannel using direct STORM with YOYO-1. (a) Epifluorescence image of the DNA molecule, (b) photoblinking image of fluorescent molecules, (c) super-resolution image of the DNA molecule, and (d) cross-sectional fluorescence distribution of the super-resolution image. The experimental FWHM was measured to be 80 nm.

Close modal

These results indicate that the stretch and immobilization method used in this study can be combined with the super-resolution imaging method, although the method still faces challenges regarding significant deviation in the stretch ratio, as shown in Fig. 2. Therefore, this method can be used to analyze DNA molecules of known lengths. However, addressing the significant deviation in the stretch ratio is crucial when analyzing DNA molecules of unknown length. Therefore, it is essential to optimize the surface modification in the microchannel and the sample preparation, as we had described. In addition, the experimental FWHM of the super-resolution image was 80 nm, which was slightly larger than that of the theoretical FWHM, which can be attributed to the mechanical drift of the microscope stage during imaging and the drift caused by thermal fluctuations of the fluorescent molecule bound to the DNA molecule. Based on Eq. (4), an increase in the fluorescence intensity N and a decrease in the background noise b can decrease the FWHM, which means the improvement of the lateral resolution of direct STORM imaging. The excitation power of the light source used in this study was 0.1 kW/cm2, which was 5 to 10 times smaller than that used in previous studies for direct STORM imaging using YOYO-1.35 Therefore, in this study, it is expected that the lateral resolution can be improved by using a light source with a higher power. In contrast, as the lateral resolution improves, the effects of the mechanical drift and thermal fluctuations of the fluorescent molecule on the experimental FWHM become more significant. We can use the methods proposed in a previous study to correct the mechanical drift in super-resolution imaging, in which the amount of mechanical drift can be identified and corrected to a precision of several nanometers based on the change in the position of the images.36,37 In contrast, it is difficult to reduce the drift caused by the thermal fluctuations of the fluorescent molecules. Previous studies have demonstrated the achievement of super-resolution imaging at cryogenic temperatures with an accuracy of several nanometers.38 This capability can be applied for high-resolution single DNA analysis by integrating this method with the stretch and immobilization technique proposed in this study.

In summary, we used the combination of stretching DNA molecules using pressure flow in a microchannel and immobilizing it on a surface treated with APTES. The fluorescence observation of a single DNA molecule in the microchannel clarified the stretch and immobilization processes. We investigated the dependence of the stretch ratio on the flow velocity, and the stretch ratio increased with increasing flow velocity, thereby indicating that the stretch ratio could be adjusted by controlling the velocity. Super-resolution imaging using direct STORM of the stretched and immobilized DNA molecules indicated that the method can be used effectively for super-resolution imaging when the thermal fluctuation of the DNA molecule is reduced. Despite the large deviations in the stretch ratio observed with this method, it is expected that combining it with the super-resolution imaging method will enable the analysis of single DNA molecules with higher accuracy.

This work was supported in part by JST FOREST Program Grant No. JPMJFR2257, JST ACT-X Grant No. JPMJAX21B2, and Tokai Pathways to Global Excellence (T-GEx), a part of the MEXT Strategic Professional Development Program for Young Researchers, Japan.

The authors have no conflicts to disclose.

Naoki Azuma: Conceptualization (lead); Data curation (equal); Formal analysis (equal); Methodology (equal); Writing – original draft (lead); Writing – review & editing (equal). Ryotaro Suzuki: Data curation (equal); Formal analysis (equal); Methodology (equal); Writing – review & editing (equal). Kenji Fukuzawa: Methodology (supporting); Writing – review & editing (equal). Shintaro Itoh: Methodology (supporting); Writing – review & editing (equal). Hedong Zhang: Methodology (supporting); Writing – review & editing (equal).

The data that support the findings of this study are available within the article.

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