Biology subjects

Vesga, A. G.

Publications and source records attributed to Vesga, A. G..

3 recordsLinked to original sources

bioRxiv2025

SMOLM-LFM: ratiometric single molecule orientation without polarizers

Single-molecule orientation and localization microscopy (SMOLM) enables the determination of molecular orientation, wobbling, and position. However, most SMOLM implementations rely on complex point spread function (PSF) fitting, which limits analysis throughput and introduces high computational cost. A way to overcome these limitations is to simplify the analysis using a ratiometric intensity estimation, often relying on polarization projections. While effective in 2D, extending these methods to 3D remains challenging. Here, we introduce a new ratiometric strategy for SMOLM in 3D. Building on the principles of Single Molecule Light Field Microscopy, which captures the 3D position information from a single snapshot by segmenting the back focal plane, we extend this strategy to orientation retrieval. Our approach uses the generalized 3D Stokes formalism to linearly decompose the intensity measurements across the light-field channels, allowing computationally-efficient estimations, while avoiding both complex PSF fitting and polarization projections. This framework, called SMOLM-LFM, enables 6D estimation of single molecules (3D position + 3D orientation) with a simplified optical setup and a large depth-of-field. We present the theoretical foundations, experimental implementation, and validation through measurements on calibration beads, single fluorophores, and cells, thereby demonstrating the methods potential and practical limitations.

bioengineering↗

bioRxiv2025

4polar3D : Single molecule 3D orientation imaging of dense actin networks using ratiometric polarization splitting

Single Molecule Orientation and Localization Microscopy (SMOLM) aims at simultaneously measuring the position and orientation of single molecules, generating orientation-encoded super-resolved images by estimating both their 3D mean orientation and the extent of their angular fluctuations (wobble). Most existing SMOLM approaches rely on the engineering of single molecules point spread functions, which requires complex optical setups and long computational times that can be an obstacle in dense cellular environments with high detection density and challenging imaging conditions. In this work, we propose a simpler and effective method named 4polar3D, based on the estimation of single molecule intensities projected onto four polarized channels with controlled numerical apertures. This strategy enables 3D orientation measurements of single molecules in addition to their 2D localization in a fast processing step. We demonstrate that 4polar3D can resolve nanoscale molecular organization in whole cells crowded structures, uncovering 3D-oriented actin filament networks in densely packed lamellipodia and podosomes.

bioengineering↗

bioRxiv2025

VP-CLEM-Kit: An accessible pipeline for visual proteomics using super resolution volume correlative light and electron microscopy (SR-vCLEM)

Visual proteomics (VP) aims to allow researchers to visualise, measure and analyse proteins in the context of cell and tissue structure in health and disease. VP is becoming a reality through technological advances across several domains, including in situ structural biology and correlative light and electron microscopy (CLEM). However, widespread adoption remains limited due to the complexity and cost of the various VP approaches reported to date. Here we present the VP-CLEM-Kit, a disruptive cost-effective pipeline for super resolution volume CLEM (SR-vCLEM) that can be implemented with minimal advanced electron microscopy expertise and equipment, making it accessible to light microscopy facilities and research labs. SR-vCLEM is based on in-resin fluorescence (IRF), where fluorophores are preserved through processing into resin. The easyIRF protocol reported here reduces the requirement for complex costly sample preparation equipment and toxic chemicals compared to standard IRF protocols. easyIRF blocks are cut into ultrathin sections that are imaged using tomoSTORM, a new modular and cost-effective openFrame-based light microscope controlled by the open-source software package Micro-Manager that provides serial single molecule localisation microscopy in array tomography format. Sections are then post-stained and imaged using a tabletop scanning electron microscope controlled by open-source SBEMimage software to run in array tomography format. We demonstrate the potential of the VP-CLEM-Kit by imaging organelle reporters in human cell lines and stem cell derived neurons, fluorescently labelled protein in neurons, and immunolabelled cells in human kidney biopsy tissue from transplant patients. The VP-CLEM-Kit delivers a [~]5-fold improvement in resolution at a [~]7-fold lower cost, and thus provides new technical capability as well as a blueprint for more equitable access to advanced imaging workflows.

cell biology↗