DNA molecules within composites were imaged using a home-built light sheet microscope with a 10× 0.25 numerical aperture (NA) excitation objective, a 20× 0.5 NA imaging objective, and an Andor Zyla 4.2 CMOS (complementary metal-oxide semiconductor) camera.

Single-molecule conformational tracking. For each sample, 45 videos displaying ~10 DNA molecules per frame were recorded at 10 frames/s (fps) for 500 frames. All data presented are for an ensemble of ~1000 molecules from two different samples, each tracked for a minimum of 2.5 s. Custom-written software (Python) was used to track the COM positions (x, y) as well as the lengths of the major axis (Rmax) and minor axis (Rmin) of each molecule in each frame. From COM positions, we computed the MSD = ½(<(Δx)2> + <(Δy)2>) and corresponding transport coefficients and scaling exponents via MSD = Ktα (Figs. 1B and 2 and fig. S1). From the major and minor axis length measurements, we calculated an effective coil size Rcoil = [½(Rmax2 + Rmin2)]½ (Figs. 1B and 3) (19). Lastly, we characterized the time-dependent conformational fluctuations of single molecules by calculating the fractional fluctuation length Lf(t) = <|Rmax(t) − Rmax(0)|>/<Rmax> for all lag times t. Lf(t) quantifies the time scale and fractional length scale over which single molecules fluctuate between different conformational states. These analysis methods, depicted in Fig. 1, have been described and validated previously (1921, 35).

Differential dynamic microscopy. For each sample, eight videos with a 256 pixel × 1280 pixel (49.6 μm × 248.3 μm) field of view were recorded at 18 fps for 5000 frames at different regions within the sample. For DDM analysis [described in (62)], videos were then split into 256 pixel × 256 pixel (49.6 μm × 49.6 μm) ROIs (19, 6264). Each ROI was analyzed individually and averaged together after analysis (Fig. 1C). A two-dimensional (2D) Fourier transform was taken from the difference between images separated by time lags of 0.05 to 166.55 s (Figs. 1C and 2). Because of dynamics are isotropic, the 2D Fourier transform was radially averaged for all lag times t, resulting in the DDM matrix D(q,t), where q is the magnitude of the wave vector. The DDM matrix can be fit to D(q,t) = A(q)[1 − f(q,t)] + B(q), where f(q,t) is the ISF. We used a stretched exponential for the ISF (fig. S2). While these fits for each q do not follow the data over all t for ring DNA, they did allow us to extract the parameters A(q) and B(q). With the measured D(q,t) and extracted A(q) and B(q), we plotted the ISF, f(q,t), for a particular wave vector to compare the rate at which the ISF decays (Figs. 1C and 3).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.