Automated smFRET microscope for the quantification of label-free DNA oligos

: Single-molecule FRET (smFRET) spectroscopy is a powerful tool for studying inhomogeneous dynamics in biological systems. However, because of the intrinsic variations that accompany the sample sizes, massive data sets are essential to extract statistically reliable information. In this aspect, a simple motorized stage and autofocusing modification can save time without the expense of a high-end automated microscope. In this report, we describe a simple and economical modification of a commercial inverted microscope with a manual stage to automate the data acquisition and measurement process. We collected 8000 images with a 100 ms exposure time in 1000 fields of view in approximately 13 min, where it would take more than 8 h by manual collection. We demonstrated the method with a DNA oligo quantification experiment. In this experiment, the measurement platform is a FRET signal from a dye-labeled DNA duplex containing unmatched base pairs. The target DNA replaces one of the strands because of the formation of a perfect duplex. This thermodynamically driven exchange reaction causes FRET to disappear, which correlated with the DNA concentration. The data are batch processed with the freeware ImageJ. These modifications are feasible and economical for general smFRET experiments.

DNA strands of surface-bound duplexes, which were dissociated by the target oligo (microRNA let-7g's DNA analog) due to the energy levels. This dissociation decreased the FRET pair numbers, which were proportional to the concentration of the microRNA analog. Because the sensitivity of smFRET is high, the DNA molecules are not amplified or labeled. However, to obtain statistically reliable results, large sample sizes are necessary. While manually changing the microscope stage to collect tens of videos is possible, it becomes a tedious and prolonged procedure to collect thousands of videos. The main time-consuming step is moving the stage and refocusing. Although a time as short as 30 s is enough to change the field of view for a skillful researcher, this step amounts to more than 8 h if 1000 videos are needed. To solve this problem, we implement an automotive stage-scanning setup. For capturing data in 1000 fields of view (100 ms exposure time and 8 frames per field), the acquisition time decreased from more than 8 h to 13 min. If more data are necessary, the time difference will be even larger. Accordingly, the massive amount of data was batch processed with macros developed in the ImageJ program. Our method provides an alternative assay to directly quantify nucleic acids.

TIRF microscope optics
An objective-based TIRF (total internal reflection fluorescence) microscope was built on a Nikon Eclipse Ti inverted microscope (Fig. 1). Two solid-state lasers (532 nm -75 mW, 640 nm -20 mW, Crystalaser, Reno, NV) were used. The original laser spots (approximately Ø 0.5 mm) were expanded with a pinhole spatial filter (KT110, Thorlabs) to a size of Ø 1.5 cm and then directed to the edge of a 60 x 1.49 NA oil immersion objective (Nikon Instruments). The light was focused at the back focal plane of the objective and then hit the coverslip of the flow cell at a glancing angle to be totally reflected at the glass-liquid interface. Therefore, only surface-bound molecules can be excited by the 100-nm-thin evanescent wave radiation. The emitted fluorescent light was collected via the same objective, passed through dichroic mirrors, and after separation by a dual view apparatus (Optical Insights) formed mirrored images on a back-thinned CCD camera (532B Cascade II; Photometrics). The sample cell was made of a cover slip (ThermoFisher rectangular cover slips, 24 x 40 mm, thickness #1 (0.17 μm)) and a glass slide (Fisherbrand, 25x75 mm) via double-faced tape. Six channels (3x13 mm) were made in one glass with two holes in each channel. One hole was connected a pipette tips for solution delivery, and the other hole was connected to a syringe or syringe pump for suction ( Fig. 1). A few studies have described details on building a TIRF microscope for smFRET experiments [15][16][17]. The physical setup from our lab is shown in Fig. 8 in the Appendix. Step motors were connected to the stage and the microscope focus knob for the automation modification.

Stage motorization and stepping size calibration
To gain statistical significance, it is essential to automate the data acquisition. For the purpose of automating a random scanning of the fields of view of the sample, it is not necessary to implement high-precision expensive automated stages. Thus, two economical SilverPak17C step motors (Lin Engineering) were coupled to the 2D-manual stage actuators. The stepping size of the motor was calibrated with a patterned grid as shown in Fig. 2. Three different motor stepping sizes were measured: P50, P100 and P150 (these are the motor parameters for stepping). For example, at the P50 setting, 7 images were taken at 7 consecutive steps of the stage movement. The images can be aligned with their succeeding image via the same grid in the different images because every grid line has a unique thickness. Therefore, the stage movement step can be measured in pixels by comparing the pixel differences of the same grid in consecutive images. To quantify the displacement more precisely, the grid pattern in each image was obtained via the "vertical profile plot" tool in ImageJ (plots (a-c) in Fig. 2). Each colored profile represents one grid pattern at one stage position. Six offsets with the best overlay of the consecutive profiles were obtained and averaged. This was done similarly for P100 and P150. By visual inspection of Fig. 2, the step size increased from P50 to P150. In Fig. 2(d), the stepping sizes are plotted against the P-parameter, and a linear relationship is obvious. The slope is approximately 2.7 pixels/P. Therefore, the P300 setting moves the stage 800 pixels, which is 58% more than the 512 pixels of the camera area. On the other hand, the standard deviations in Fig. 2(d) indicate that random errors occurred during the stepping. However, because returning back to the exact same field of view is not required in most smFRET experiments, the small uncertainty in the stage movement is not a concern for these experiments, as long as the new field of view does not overlap with the previous field of view. The P300 setting achieved this goal without moving the stage at an excessively large distance. Consequently, even though a more precise estimate of the error can be obtained by hundreds of repeated measurements, it is trivial in the current study. Instead, 6 movements and measurements at each P-setting were sufficient for calibration purposes.   Figure 3 )-(20 steps up pixels, which c a 60x objectiv e total channel ore steps, as w g the data size or keeping the e set of 40 field ell ensemble. A p ich is shown as th annels is the cover ) Illustration of the ngs, P50, 100 and e distinguishable g grid in different im igning the vertical and averaged to g movement (Y-) shows the de )), and one rou p). Based on th corresponded t ve

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smFRET platform
All of the DNA oligos were purchased from IDTdna. The duplex formed between S1 and S2 was prepared by mixing 10 μM S1 and S2 in an exchange buffer (10 mM Tris (pH 8.0), 1 M NaCl, 1 mM EDTA), heated at 95 °C for 5 min and then cooled to room temperature very slowly. The duplex was then stored at −20 °C in aliquots. Before loading into the imaging chamber, the duplex was diluted 100,000 times to a final concentration of 0.1 nM. FRET images of the imaging chamber were first collected as a reference; then, S3 solutions with various concentrations were injected into the sample chamber and incubated at 37 °C for 30 min under water vapor conditions to prevent drying. Afterwards, the sample chamber was washed with buffer extensively to remove free floating DNA strands, and FRET images were taken again. The imaging solution was the same as the exchange buffer with the addition of an oxygen scavenger cocktail containing (3 mg/mL glucose, 100 mg/mL glucose oxidase, 48 mg/mL catalase, and 2 mM trolox). The sequences of S1-3 are tabulated in Table 1. Table 1. DNA sequences (S1-S2 forms the platform, and S3 is the analyte), the shaded part formed a stem loop to specifically recognize short DNAs.
Two smFRET platforms were tested (Figs. 5(b) and 10(b)). In Fig. 5(a), the DNA duplex was tethered to the glass surface via the biotin-streptavidin-biotin interaction via S1 (red strand), which was labeled with biotin at its 3′-end. The S2 (green strand) was complementary to S1 except at its 5′-terminal (an A-A pair), while S3 was completely complementary to S1 (an A-T pair). A set of FRET-paired dyes Cy3-Cy5 were labeled at S1 and S2, respectively, which yielded an average FRET state centered at approximately 0.6 ( Fig. 5(c)). Before loading S3, a FRET image is shown in Fig. 5(b) (left panel). In the presence of S3 (canonical let-7g analog), S2 was removed from the duplex and washed away because of the formation of the more stable S1-S3 duplex. This replacement caused dissociation of the FRET-paired dyes and was proportional to the S3 concentration. Consequently, a lower density of Cy5 fluorescence was observed after incubation with S3 ( Fig. 5(b), right panel). The number of FRET pairs was counted by the simultaneous emissions from both channels (boxed particles in Fig. 5(b)) and normalized with respect to the Cy3 particles among different fields of view. As reported before, one-step bleaching processes of both Cy3 and Cy5 were observed to verify that the signals were from single dyes only [18]. Figure 5(d) shows the FRET-parried fluorescence emissions from the Cy3 and Cy5 channels superposed with each other. The offset of the alignment between the paired spots is exaggerated for illustration.
In Fig. 10(a), the duplex was the same as in Fig. 5(a), except that the biotin was labeled on . Therefore, after exchange, the Cy5-labeled S2 remained on the surface, while the S1-S3 duplex diffused away in the solution. The exchange efficiency was calculated from the ratio of the Cy5 emission via FRET to the Cy5 emission via direct excitation. Figure 9(b) shows an example of a data set before and after the exchange. The left two images (before exchange) are the Cy5 channel emission (binary data are shown for presentation) via direct excitation and via FRET. The right two images (after exchange) are in the same arrangement. The images were obtained via the abovementioned alternating laser illumination procedure. The advantage of this setting compared to that of Fig. 5 is the removal of nonspecific emission in the Cy3 channel, which is more common than that in the Cy5 channel. Nevertheless, similar results were obtained with either platform.

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