arXiv subjects

W. E. Moerner

Publications and source records attributed to W. E. Moerner.

9 recordsLinked to original sources

arXiv2024

Stimulated emission does not radiate in a pure dipole pattern

Stimulated Emission (StE) remains relatively unused as an image-forming signal despite having potential advantages over fluorescence in speed, coherence, and ultimately resolution. Several ideas for the radiation pattern and directionality of StE remain prevalent, namely whether a single molecule would radiate StE itself in a pure dipole pattern, or whether its emission direction depends on the driving field. Previous StE imaging has been carried out in transmission, which would collect signal either way. Here, we introduce the StE driving field (the probe) at an angle, using total internal reflection to avoid incident probe light and its specular reflections in our detection path. In this non-collinear detection configuration which also collects some fluorescence from the sample, we observe fluorescence depletion even in the spectral window where an increase in detected signal from StE would be expected if StE radiated like a simple classical dipole. Because simultaneous direct measurement of the fluorescence represents a calibration of the potential size of StE were it spatially patterned like a classical dipole emitter, our study clarifies a critical characteristic of StE for optimal microscope design, optical cooling, and applications using small arrays of emitters.

physics.optics↗

arXiv2020

Addressing systematic errors in axial distance measurements in single-emitter localization microscopy

Nanoscale localization of point emitters is critical to several methods in optical fluorescence microscopy, including single-molecule super-resolution imaging and tracking. While the precision of the localization procedure has been the topic of extensive study, localization accuracy has been less emphasized, in part due to the challenge of producing an experimental sample containing unperturbed point emitters at known three-dimensional positions in a relevant geometry. We report a new experimental system which reproduces a widely-adopted geometry in high-numerical aperture localization microscopy, in which molecules are situated in an aqueous medium above a glass coverslip imaged with an oil-immersion objective. We demonstrate a calibration procedure that enables measurement of the depth-dependent point spread function (PSF) for open aperture imaging as well as imaging with engineered PSFs with index mismatch. We reveal the complicated, depth-varying behavior of the focal plane position in this system and discuss the axial localization biases incurred by common approximations of this behavior. We compare our results to theoretical calculations.

physics.optics↗

arXiv2019

BGnet: Accurate and rapid background estimation in single-molecule localization microscopy with deep neural nets

Background fluorescence, especially when it exhibits undesired spatial features, is a primary factor for reduced image quality in optical microscopy. Structured background is particularly detrimental when analyzing single-molecule images for 3D localization microscopy or single-molecule tracking. Here, we introduce BGnet, a deep neural network with a U-net-type architecture, as a general method to rapidly estimate the background underlying the image of a point source with excellent accuracy, even when point spread function (PSF) engineering is in use to create complex PSF shapes. We trained BGnet to extract the background from images of various PSFs and show that the identification is accurate for a wide range of different interfering background structures constructed from many spatial frequencies. Furthermore, we demonstrate that the obtained background-corrected PSF images, both for simulated and experimental data, lead to a substantial improvement in localization precision. Finally, we verify that structured background estimation with BGnet results in higher quality of super-resolution reconstructions of biological structures.

physics.optics↗

arXiv2019

Accurate phase retrieval of complex point spread functions with deep residual neural networks

Phase retrieval, i.e. the reconstruction of phase information from intensity information, is a central problem in many optical systems. Here, we demonstrate that a deep residual neural net is able to quickly and accurately perform this task for arbitrary point spread functions (PSFs) formed by Zernike-type phase modulations. Five slices of the 3D PSF at different focal positions within a two micron range around the focus are sufficient to retrieve the first six orders of Zernike coefficients.

eess.IV↗

arXiv2019

Interferometric scattering enables fluorescence-free electrokinetic trapping of single nanoparticles in free solution

Anti-Brownian traps confine single particles in free solution by closed-loop feedback forces that directly counteract Brownian motion. The extended-duration measurement of trapped objects allows detailed characterization of photophysical and transport properties, as well as observation of infrequent or rare dynamics. However, this approach has been generally limited to particles that can be tracked by fluorescent emission. Here we present the Interferometric Scattering Anti-Brownian ELectrokinetic trap (ISABEL trap), which uses interferometric scattering rather than fluorescence to monitor particle position. By decoupling the ability to track (and therefore trap) a particle from collection of its spectroscopic data, the ISABEL trap enables confinement and extended study of single particles that do not fluoresce, that only weakly fluoresce, or which exhibit intermittent fluorescence or photobleaching. This new technique significantly expands the range of nanoscale objects that may be investigated at the single-particle level in free solution.

physics.ins-det↗

arXiv2017

Measurement-based estimation of global pupil functions in 3D localization microscopy

We report the use of a phase retrieval procedure based on maximum likelihood estimation (MLE) to produce an improved, experimentally calibrated model of a point spread function (PSF) for use in three-dimensional (3D) localization microscopy experiments. The method estimates a global pupil phase function (which includes both the PSF and system aberrations) over the full axial range from a simple calibration scan. The pupil function is used to refine the PSF model and hence enable superior localizations from experimental data. To demonstrate the utility of the procedure, we apply it to experimental data acquired with a microscope employing a tetrapod PSF with a 6 micron axial range. The phase-retrieved model demonstrates significant improvements in both accuracy and precision of 3D localizations relative to the model based on scalar diffraction theory. The localization precision of the phase-retrieved model is shown to be near the limits imposed by estimation theory, and the reproducibility of the procedure is characterized and discussed. Code which performs the phase retrieval algorithm is provided.

physics.optics↗

arXiv2014

The regulatory switch of F1-ATPase studied by single-molecule FRET in the ABEL Trap

F1-ATPase is the soluble portion of the membrane-embedded enzyme FoF1-ATP synthase that catalyzes the production of adenosine triphosphate in eukaryotic and eubacterial cells. In reverse, the F1 part can also hydrolyze ATP quickly at three catalytic binding sites. Therefore, catalysis of 'non-productive' ATP hydrolysis by F1 (or FoF1) must be minimized in the cell. In bacteria, the epsilon subunit is thought to control and block ATP hydrolysis by mechanically inserting its C-terminus into the rotary motor region of F1. We investigate this proposed mechanism by labeling F1 specifically with two fluorophores to monitor the C-terminus of the epsilon subunit by Förster resonance energy transfer. Single F1 molecules are trapped in solution by an Anti-Brownian electrokinetic trap which keeps the FRET-labeled F1 in place for extended observation times of several hundreds of milliseconds, limited by photobleaching. FRET changes in single F1 and FRET histograms for different biochemical conditions are compared to evaluate the proposed regulatory mechanism.

q-bio.BM↗

arXiv2013

Robust Hypothesis Tests for Detecting Statistical Evidence of 2D and 3D Interactions in Single-Molecule Measurements

A variety of experimental techniques have improved the 2D and 3D spatial resolution that can be extracted from \emph{in vivo} single-molecule measurements. This enables researchers to quantitatively infer the magnitude and directionality of forces experienced by biomolecules in their native cellular environments. Situations where such forces are biologically relevant range from mitosis to directed transport of protein cargo along cytoskeletal structures. Models commonly applied to quantify single-molecule dynamics assume that effective forces and velocity in the $x,y$ (or $x,y,z$) directions are statistically independent, but this assumption is physically unrealistic in many situations. We present a hypothesis testing approach capable of determining if there is evidence of statistical dependence between positional coordinates in experimentally measured trajectories; if the hypothesis of independence between spatial coordinates is rejected, then a new model accounting for 2D (3D) interactions should be considered to more faithfully represent the underlying experimental kinetics. The technique is robust in the sense that 2D (3D) interactions can be detected via statistical hypothesis testing even if there is substantial inconsistency between the physical particle's actual noise sources and the simplified model's assumed noise structure. For example, 2D (3D) interactions can be reliably detected even if the researcher assumes normal diffusion, but the experimental data experiences "anomalous diffusion" and/or is subjected to a measurement noise characterized by a distribution differing from that assumed by the fitted model. The approach is demonstrated on control simulations and on experimental data (IFT88 directed transport in the primary cilium).

q-bio.QM↗

arXiv2007

Interferometry of a Single Nanoparticle Using the Gouy Phase of a Focused Laser Beam

We provide a quantitative explanation of the mechanism of the far-field intensity modulation induced by a nanoparticle in a focused Gaussian laser beam, as was demonstrated in several recent direct detection studies. Most approaches take advantage of interference between the incident light and the scattered light from a nanoparticle to facilitate a linear dependence of the signal on the nanoparticle volume. The phase relation between the incoming field and the scattered field by the nanoparticle is elucidated by the concept of Gouy phase. This phase relation is used to analyze the far-field signal-to-noise ratio as a function of exact nanoparticle position with respect to the beam focus. The calculation suggests that a purely dispersive nanoparticle should be displaced from the Gaussian beam focus to generate a far-field intensity change.

physics.optics↗