Enhanced DNA imaging using super-resolution microscopy and simultaneous single-molecule orientation measurements

Abstract

Single-molecule orientation measurements provide unparalleled insight into a multitude of biological and polymeric systems. We report a simple, high-throughput technique for measuring the azimuthal orientation and rotational dynamics of single fluorescent molecules, which is compatible with localization microscopy. Our method involves modulating the polarization of an excitation laser, and analyzing the corresponding intensities emitted by single dye molecules and their modulation amplitudes. To demonstrate our approach, we use intercalating and groove-binding dyes to obtain super-resolved images of stretched DNA strands through binding-induced turn-on of fluorescence. By combining our image data with thousands of dye molecule orientation measurements, we develop a means of probing the structure of individual DNA strands, while also characterizing dye-DNA interactions. This approach may hold promise as a method for monitoring DNA conformation changes resulting from DNA-binding proteins.

Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking

“…The molecular orientation of labels can be used to infer information about local environments,(202, 276, 277) and such measurements can be combined with 3D measurements to powerful effect. Additional well-known local single-molecule variables such as lifetime, energy transfer, spectra, etc.…”

“…In some experiments, it is quite useful to measure orientations of single molecules along with positions,(202) but this extra step requires more detected photons. Alternatively, there is a direct, purely optical solution to the mislocalization problem, proposed recently by Lew et al (201) and illustrated in Figure 14C.…”

Fundamental Limits on Measuring the Rotational Constraint of Single Molecules Using Fluorescence Microscopy

“…The prefactor s and image b ∈ R n×1 represent the integrated signal photons and background photons, respectively, for each measurement g. The second moment vector ζ = [ζ x , ζ y ] † = [µ x , µ y ] † / µ 2 x + µ 2 y is the normalized projection of molecular orientation µ into the xy plane, and the angle brackets · denote the temporal average taken over one camera frame or the equivalent average over the orientation domain. The basis images can be computed for any excitation-modulation method by computing the SM's response to varying excitation polarization [3] and for any emission-modulation technique using vectorial diffraction models [24][25][26][27][28].…”

Chromatographic Behaviour of Antibiotics on Thin Layers of Zeolite

“…Thin-layer chromatography is one of the most important analytical techniques used to separate components of mixtures. TLC is often used as a quick, easy and simple method [10][11][12][13][14][15][16]. The effective separation by the depends on the properties of the sample, and those of the mobile and stationary phases [17][18].…”