Biology subjects

Bruggeman, E.

Publications and source records attributed to Bruggeman, E..

5 recordsLinked to original sources

bioRxiv2023

High-density volumetric super-resolution microscopy

Volumetric super-resolution microscopy typically encodes the 3D position of single-molecule fluorescence into a 2D image by changing the shape of the point spread function (PSF) as a function of depth. However, the resulting large and complex PSF spatial footprints reduce temporal resolution by requiring lower labelling densities to avoid overlapping fluorescent signals. We quantitatively compare the density dependence of single-molecule light field microscopy (SMLFM) to other 3D PSFs (astigmatism, double helix and tetrapod) showing that SMFLM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax. We then experimentally demonstrate the high accuracy (>99.2 {+/-} 0.1%, 0.1 locs m-2) and sensitivity (>86.6 {+/-} 0.9%, 0.1 locs m-2) of SMLFM at point detection through whole-cell (scan-free) imaging and tracking of single membrane proteins in live primary B cells. We also exemplify high density volumetric imaging (0.15 locs m-2) in dense cytosolic tubulin datasets.

biophysics↗

bioRxiv2023

Fast and artifact-free excitation multiplexing using synchronized image scanning

We present the Resonator, a simple optical device that provides quasi-simultaneous fluorescence imaging with multiple excitation wavelengths. The device uses a resonant scanning mirror to periodically displace the sample image on a camera sensor at a rate that is much faster than the image acquisition rate. The excitation light is synchronized with the scanner motion to create two laterally shifted copies of the image, each containing the fluorescence excited by a single wavelength. The additional information is then encoded either into the point-spread function of the imaging or as multiple distinct images. Since this multiplexing is performed at very high rates, our design can eliminate or mitigate artifacts caused by temporal aliasing in conventional sequential imaging. We demonstrate the use of our system for the monitoring of fast light-induced dynamics in single quantum dots and for the imaging of Ca2+ signalling in hippocampal neurons.

biophysics↗

bioRxiv2023

POLCAM: Instant molecular orientation microscopy for the life sciences

Current methods for single-molecule orientation localization microscopy (SMOLM) require optical setups and algorithms that can be prohibitively slow and complex, limiting the widespread adoption for biological applications. We present POLCAM, a simplified SMOLM method based on polarized detection using a polarization camera, that can be easily implemented on any wide-field fluorescence microscope. To make polarization cameras compatible with single-molecule detection, we developed theory to minimize field of view errors, used simulations to optimize experimental design, and developed a fast algorithm based on Stokes parameter estimation which can operate over 1000 fold faster than the state of the art, enabling near instant determination of molecular anisotropy. To aid in the adoption of POLCAM, we developed open-source image analysis software, and a website detailing hardware installation and software use. To illustrate the potential of POLCAM in the life sciences, we applied our method to study alpha-synuclein fibrils, the actin cytoskeleton of mammalian cells, fibroblast-like cells and the plasma membrane of live human T cells.

biophysics↗

bioRxiv2022

resPAINT: Accelerating volumetric super-resolution localisation microscopy by active control of probe emission

Points for accumulation in nanoscale topography (PAINT) allows the acquisition of practically unlimited measurements in localisation microscopy. However, PAINT is inherently limited by unwanted background fluorescence at high probe concentrations, especially in large depth-of-field volumetric imaging techniques. Here we present reservoir-PAINT (resPAINT), in which we combine PAINT with active control of probe photophysics. In resPAINT, a reservoir of non-fluorescent activatable probes accumulate on the target, which makes it possible to drastically improve the localisation rate (by up to 50-fold) compared to conventional PAINT, without any compromise in contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate volumetric super-resolution imaging of entire cell surfaces. We then generalise the approach by implementing multiple switching strategies, including photoactivation and spontaneous blinking. We also implement alternative volumetric imaging modalities including the double-helix pointspread function, the tetrapod point-spread function and singlemolecule light field microscopy. Finally, we show that resPAINT can be used with a Fab to image membrane proteins, effectively extending the operating regime of conventional PAINT to encompass a larger range of biological interactions.

biophysics↗

bioRxiv2020

Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis

The scission of lipid membranes is a common biological process, often mediated by ESCRT complexes in concert with VPS4 assembling around the separation point. The functions of the ESCRT-I and ESCRT-III complexes are well established in certain of these cellular processes; however, the role of ESCRT-II remains contentious. Here, we devised a SNAP-tag fluorescent labelling strategy to understand the domain requirements of EAP45, the main component of ESCRT-II, in HIV egress, late endosome recruitment, and cytokinesis. We used TIRF microscopy to measure the spatial co-occurrence of the HIV structural polyprotein Gag with full length EAP45 in both fixed and live cells. Gag colocalises with the full length EAP45 comparably to ALIX, but this is lost on deletion of the EAP45 N terminus. Our findings reveal the H0 domain of the EAP45 protein is essential for linking to ESCRT-I during HIV budding and in anchoring at the late endosomal membrane, however in cytokinesis it is the Glue domain that is critical.

biophysics↗