DNA phase transitions drive life processes and are key to the development of DNA-based biotechnologies. Accordingly, quantifying the physical properties of DNA is an essential endeavor. However, the narrow width (2 nm) of the DNA molecule prohibits direct visualization of its structural dynamics using optical microscopy. To address this challenge, we employ concurrent polarization imaging and DNA manipulation to probe the orientations and rotational dynamics of DNA-intercalated dyes—small fluorescent molecules that bind between adjacent DNA base pairs. The method uses optical tweezers to precisely extend, align and (re)orient a single DNA molecule within the image plane of a fluorescence microscope. Our data shows that at extensions beyond the so-called “overstretching transition” intercalators adopt a dramatically tilted orientation relative to the DNA-axis (approx. 54 degrees), distinct from the perpendicular orientation (approx. 90 degrees) normally observed at lower extensions. Strikingly, by imaging single intercalated dye molecules with polarized illumination, we also demonstrate that intercalators rapidly rotate (i. e. “twirl”) about the DNA-axis, revealing underlying Brownian twisting dynamics of the DNA substrate. Taken together, these results shed new insight on S-DNA: a DNA phase that forms under tension that, at present, is not well understood.
We extend the information content of the microscope’s point-spread-function (PSF) by adding a new degree of freedom: spectral information. We demonstrate controllable encoding of a microscopic emitter’s spectral information (color) and 3D position in the shape of the microscope’s PSF. The design scheme works by exploiting the chromatic dispersion of an optical element placed in the optical path. By using numerical optimization we design a single physical pattern that yields different desired phase delay patterns for different wavelengths. To demonstrate the method’s applicability experimentally, we apply it to super-resolution imaging and to multiple particle tracking.
We present a means of measuring the dipole orientation of a fluorescent, rotationally fixed single molecule (SM), using a specially designed phase mask, termed a “quadrated pupil,” conjugate to the back focal plane of a conventional widefield microscope. In comparison to image-fitting techniques that infer orientation by matching simulations to defocused or excessively magnified images, the quadrated pupil approach is more robust to minor modeling discrepancies, defocus, and optical aberrations. Precision on the order of 1°-5° is achieved in proofof- concept experiments for both azimuthal (φ) and polar (θ) angles. Since the phase mask is implemented on a liquid-crystal spatial light modulator (SLM) that may be deactivated without any mechanical perturbation of the sample or imaging system, the technique may be readily integrated into conventional imaging studies.
Single-molecule-based super-resolution fluorescence microscopy has recently been developed to surpass the diffraction
limit by roughly an order of magnitude. These methods depend on the ability to precisely and accurately measure the
position of a single-molecule emitter, typically by fitting its emission pattern to a symmetric estimator (e.g. centroid or
2D Gaussian). However, single-molecule emission patterns are not isotropic, and depend highly on the orientation of the
molecule’s transition dipole moment, as well as its z-position. Failure to account for this fact can result in localization
errors on the order of tens of nm for in-focus images, and ~50-200 nm for molecules at modest defocus. The latter range
becomes especially important for three-dimensional (3D) single-molecule super-resolution techniques, which typically
employ depths-of-field of up to ~2 μm. To address this issue we report the simultaneous measurement of precise and
accurate 3D single-molecule position and 3D dipole orientation using the Double-Helix Point Spread Function (DH-PSF)
microscope. We are thus able to significantly improve dipole-induced position errors, reducing standard deviations
in lateral localization from ~2x worse than photon-limited precision (48 nm vs. 25 nm) to within 5 nm of photon-limited
precision. Furthermore, by averaging many estimations of orientation we are able to improve from a lateral standard
deviation of 116 nm (~4x worse than the precision, 28 nm) to 34 nm (within 6 nm).