Biology subjects

Galgani, T.

Publications and source records attributed to Galgani, T..

3 recordsLinked to original sources

bioRxiv2022

Selective volumetric excitation and imaging for single molecule localization microscopy in multicellular systems

Light sheet fluorescence microscopy (LSFM) has become a leading standard in high-resolution imaging of living samples in 2- and 3-dimensions. Biological samples are however not restricted to a single observation plane and several molecular processes evolve rapidly in 3D. The conventional mechanical scanning required in LSFM limits the range of observable dynamics and are usually restricted in resolution. Here we introduce a new strategy for instantaneous volumetric excitation and volumetric imaging of single-molecules in cell aggregates. The technique combines, for the first time, the use of light sheet microscopy and multifocus microscopy (MFM) and enables a volumetric 4D imaging of biological samples with single-molecule resolution. We engineered the excitation beam to yield a modular and uniform excitation matching the observable detection range of MFM. The strength of the method is highlighted with examples of single-molecule 3D tracking and 3D super-resolution imaging in multicellular samples.

biophysics↗

bioRxiv2022

High-fidelity 3D live-cell nanoscopy through data-driven enhanced super-resolution radial fluctuation

In recent years, the development of new image analysis approaches has highlighted the possibility of recovering superresolution information from short sequences of wide-field images. Our recently developed method, SRRF (Super-Resolution Radial Fluctuations), enables long-term live-cell imaging beyond the resolution limit without specialized hardware. Here, we present eSRRF (enhanced-SRRF), a significant improvement over our initial method, enhancing image fidelity to the underlying structure and resolution. Especially, eSRRF uses automated data-driven parameter optimization, including an estimation of the number of frames necessary for optimal reconstruction. We demonstrate the improved fidelity of the images reconstructed with eSRRF and highlight its versatility and ease of use over a wide range of microscopy techniques and biological systems. We also extend eSRRF to 3D super-resolution microscopy by combining it with multi-focus microscopy (MFM), obtaining volumetric super-resolution imaging of live cells with acquisition speed of ~1 volume/second.

biophysics↗

bioRxiv2021

Probing mechanotransduction in living cells by optical tweezers and FRET-based molecular force microscopy

Cells sense mechanical signals and forces to probe the external environment and adapt to tissue morphogenesis, external mechanical stresses, and a wide range of diverse mechanical cues. Here, we propose a combination of optical tools to manipulate single cells and measure the propagation of mechanical and biochemical signals inside them. Optical tweezers are used to trap microbeads that are used as handles to manipulate the cell plasma membrane; genetically encoded FRET-based force sensors inserted in F-actin and alpha-actinin are used to measure the propagation of mechanical signals to the cell cytoskeleton; while fluorescence microscopy with single molecule sensitivity can be used with a huge array of biochemical and genetic sensors. We describe the details of the setup implementation, the calibration of the basic components and preliminary characterization of actin and alpha-actinin FRET-based force sensors.

biophysics↗