Biology subjects

Gardini, L.

Publications and source records attributed to Gardini, L..

5 recordsLinked to original sources

bioRxiv2026

Extracellular Matrix Stiffness Controls Actin Cytoskeletal Tension

Mechanical properties of the extracellular matrix (ECM) regulate cell behavior and strongly impact the cytoskeleton, yet direct measurements of actin tension across different ECM stiffnesses are lacking. Using a genetically encoded FRET-based actin tension sensor, we show that cells cultured on stiffer substrates exhibit increased spreading, reduced multicellular aggregation, and higher molecular tension within filamentous actin. Complementary measurements using an -actinin tension sensor support increased cytoskeletal mechanical loading. These results provide direct evidence that ECM stiffness modulates intracellular actin tension during mechanotransduction.

biophysics↗

bioRxiv2024

A compact prism-based microscope for high sensitive measurements in fluid biopsy

The increasing demand for sensitive, portable, and cost-effective disease detection methods has raised significant interest in the development of biosensors for rapid and early-stage diagnosis, population mass-screening, and bedside monitoring. Although a certain number of sensing devices with high sensitivity has experienced considerable progress, their practical application has been hindered by challenges in replicating instrumental systems, obtaining rapid and multiplexed signals, and the high cost of manufacturing. In response, we present a streamlined prism-based Total Internal Reflection system which, in combination with surface functionalization techniques on gold nanoparticles, is capable of facilitating Evanescent Wave scattering for the highly sensitive and rapid detection of specific analytes in synthetic liquids and real human samples. The system achieves a remarkable limit of detection of 1 fg/mL concentration for the targeted pathological biomarker. Our innovative design addresses the limitations of existing technologies by reducing costs, minimising overall size, and ensuring swift biofluid analysis with remarkable sensitivity. To validate its efficacy, we conducted scattering experiments in synthetic and human serum samples, exploiting functionalized AuNPs to recognize bacterial lipopolysaccharides as biomarkers for sepsis disease. The cohesive integration of these techniques hopefully makes this biosensing setup a promising candidate for potential clinical deployment, meeting the pressing requirements for rapid personalised diagnosis, large-scale population screening and bed-monitoring.

biophysics↗

bioRxiv2023

Optimization of highly inclined Illumination for diffraction-limited and super-resolution microscopy

In HILO microscopy, a highly inclined and laminated light sheet is used to illuminate the sample, thus drastically reducing background fluorescence in wide-field microscopy, but maintaining the simplicity of the use of a single objective for both illumination and detection. Although the technique has become widely popular, particularly in single-molecule and super-resolution microscopy, a limited understanding of how to finely shape the illumination beam and how this impact on the image quality complicates setting HILO to fit the experimental needs. In this work, we build up a simple and comprehensive guide to optimize the beam shape and alignment in HILO and to predict its performance in conventional fluorescence and super-resolution microscopy. We model the beam propagation through Gaussian optics and validate the model through far- and near-field experiments, thus characterizing the main geometrical features of the beam. Further, we fully quantify the impact of a progressive reduction of the inclined beam thickness on the image quality of both diffraction-limited and super-resolution images and we show that the most relevant impact is obtained by reducing the beam thickness to sub-cellular dimensions (< 3 m). Based on this, we present a simple optical solution to reduce the inclined beam thickness down to 2.6 m while keeping a field-of-view dimension suited for cell imaging and allowing an increase in the number of localizations in super-resolution imaging of up to 2.6 folds.

biophysics↗

bioRxiv2021

Particle localization using local gradients and its application to nanometer stabilization of a microscope

Accurate localization of single particles plays an increasingly important role in a range of biological techniques, including single molecule tracking and localization-based superresolution microscopy. Such techniques require fast and accurate particle localization algorithms as well as nanometer-scale stability of the microscope. Here, we present a universal method for three-dimensional localization of single labeled and unlabeled particles based on local gradient calculation of microscopy images. The method outperforms current techniques in high noise conditions, and it is capable of nanometer accuracy localization of nano- and micro-particles with sub-ms calculation time. By localizing a fixed particle as fiducial mark and running a feedback loop, we demonstrate its applicability for active drift correction in sensitive nanomechanical measurements such as optical trapping and superresolution imaging. A multiplatform open software package comprising a set of tools for local gradient calculation in brightfield and fluorescence microscopy is shared to the scientific community.

biophysics↗

bioRxiv2020

α-catenin regulates cell junction fluidity by cooperative mechanosensing

-catenin is a crucial protein at cell junctions that provides connection between the actin cytoskeleton and the cell membrane. At adherens junctions (AJs), -catenin forms heterodimers with {beta}-catenin that are believed to resist force on F-actin. Outside AJs, -catenin forms homodimers that directly connect the cell membrane to the actin cytoskeleton, but their mechanosensitive properties are inherently unknown. Surprisingly, by using ultra-fast laser tweezers we found that a single -{beta}-catenin heterodimer does not resist force but instead slips along F-actin in the direction of force. Conversely, the action of 5 to 10 -{beta}-catenin heterodimers together with force applied toward F-actin pointed end engaged a molecular switch in -catenin, which unfolded and strongly bound F-actin as a cooperative catch bond. Similarly, an -catenin homodimer formed an asymmetric catch bond with F-actin triggered by protein unfolding under force. Our data suggest that -catenin clustering together with intracellular tension engage a fluid-to-solid phase transition at the membrane-cytoskeleton interface.

biophysics↗