Xingchi Yan

Intermolecular interactions underlie all cellular functions, yet visualizing these interactions at the single-molecule level remains challenging. Single-molecule localization microscopy (SMLM) offers a potential solution. Given a nanoscale map of two putative interaction partners, it should be possible to assign molecules either to the class of coupled pairs or to the class of noncoupled bystanders. Here, we developed a probabilistic algorithm that allows accurate determination of both the absolute number and the proportion of molecules that form coupled pairs. The algorithm calculates interaction probabilities for all possible pairs of localized molecules, selects the most likely interaction set, and corrects for any spurious colocalizations. Benchmarking this approach across a set of simulated molecular localization maps with varying densities (up to ∼55 molecules μm−2) and localization precisions (1 to 50 nm) showed typical errors in the identification of correct pairs of only a few percent. At molecular densities of ∼5 to 10 molecules μm−2 and localization precisions of 20 to 30 nm, which are typical parameters for SMLM imaging, the recall was ∼90%. The algorithm was effective at differentiating between noninteracting and coupled molecules both in simulations and experiments. Finally, it correctly inferred the number of coupled pairs over time in a simulated reaction–diffusion system, enabling determination of the underlying rate constants. The proposed approach promises to enable direct visualization and quantification of intermolecular interactions using SMLM.

Chromatin has a complex 3D structure and diverse binding proteins that coordinate the genome’s most essential functions. Many microscopy and genomics technologies that map chromatin proteins and modifications rely on the diffusion of antibodies (Abs) to target epitopes within whole nuclei. Here, we reveal a critical flaw in such methods that arises when Abs become trapped at the edge of nuclear structures and fail to reach internally positioned epitopes. This “Ab-trapping” results in artifactual peripheral signal that fundamentally distorts the apparent positions of chromatin features across the genome and nucleus. Using computational modeling and experimental validation, we demonstrate that Ab-trapping is caused by a combination of three compounding factors—high epitope abundance, high Ab affinity, and low Ab diffusion rates. Ab-trapping can thus systematically misrepresent the localization of many prevalent chromatin features like histone modifications, transcription factors, nucleolar proteins, and protein tags. We also show that this artifact manifests in multiple technologies, including immunofluorescence microscopy, more recent CUT&Tag-seq, and likely any method relying on Ab diffusion. Finally, we outline readily implementable strategies to identify and mitigate Ab-trapping. Combined, our work presents a previously unrecognized yet prevalent artifact in Ab-based chromatin mapping methods and the means to resolve it.

Photoacoustic imaging (PAI) uniquely combines the advantages of optical contrast with deep tissue penetration capability of acoustic waves, enabling imaging at depths of several centimeters. Conventional PAI methods have relied on pulsed lasers to induce the photoacoustic effect, coupled with arrays of pressure transducers to detect the resulting ultrasound signals. In this work, we propose an alternative all-optical approach that leverages optical deflection to record photoacoustic waves by an array of detection beams. The measured signal is shown to be the Radon transform of the pressure gradients. An optimization-based inversion procedure is used to reconstruct the initial time pressure gradient field. Subsequently, a Galerkin method is used to reconstruct the pressure field from the pressure gradient field. The new modality offers the potential for enhanced sensitivity and reduced signal distortion, advancing the capabilities of PAI beyond traditional transducer-based systems.

Although the photoacoustic effect is most commonly generated by pulsed or amplitude modulated continuous optical sources, it is possible to generate acoustic waves by moving a constant amplitude, continuous light beam. If the light beam moves at the speed of sound, an amplification effect takes place which can be used in trace gas detection. Here, the properties of the photoacoustic effect are investigated for a continuous optical beam moving in a one-dimensional resonator. The solution shows the additive effects of sweeping the optical beam the length of the cell and back.

The motion of an optically levitated, transparent sphere in an amplitude-modulated, Gaussian laser beam is shown to be described by Mathieu functions. Solutions to the equation of motion for the particle are found with and without the effects of viscosity for sinusoidal optical amplitude modulation. Solutions for step function changes in the amplitude of continuous laser irradiation are found as well. By examination of the Hamiltonian field for the particle, it is shown that the particle motion is not harmonic under sinusoidal modulation of the optical beam intensity. With sinusoidal optical modulation, the particle motion shows a steady ponderomotive force to act on the particle proportional to the second space derivative of the laser intensity.

Photoacoustic excitation of a fluid sphere generates an outgoing ultrasonic wave whose time profile permits determination of the density, sound speed, and diameter of the sphere. Experiments with pulsed laser beams have confirmed the major predictions of existing theory. With regard to acoustic waves generated within spheres, although mathematical expressions for their properties are known, virtually no exploration of the waveforms in theory or experiment has taken place. Here, two cases for photoacoustic excitation of a droplet are discussed: first, absorption of radiation in a region of fluid external to the droplet, and, second, absorption of radiation by the droplet itself. Large amplitude transients, compressions in the former and rarefactions in the latter, are generated as the waves approach the center of the sphere. The high amplitudes of the waves suggest shock wave formation.