IIT Projects Search

Augmented fluorescence correlation spectroscopy with a novel SPAD array detector to observe complex biological processes in living cells

Biomolecular dynamics and interactions in living cells are poorly understood despite their importance in several cellular processes, such as RNA transcription or synaptic transmission. In particular, the connection between biomolecular dynamic processes and cellular function is still unclear. Here, we propose a new class of microscopy techniques to observe complex biological processes in living cells, with a major focus on synaptic protein dynamics. The proposed methods are based on fluorescence correlation spectroscopy (FCS), a technique often used in biophysics to study the movement of molecules, in which the fluorescence fluctuations generated by a population of moving molecules are recorder by a singlephoton detector and correlated retrieving information about sample dynamics. Recently, a new single-photon avalanche-diode (SPAD) array detector was introduced by Vicidomini’s group showing how to further enhance conventional FCS with lower architecture complexity and reduced sample photodamage. These detectors can record single photons with a series of spatiotemporal tags, which cover both a large spatial and temporal range (from nanoseconds to seconds, from tens of nanometres to micrometres), creating an enriched dataset. We will take advance of the fast timescales granted by the SPAD array detector to (i) perform pulsed-interleaved fluorescence cross-correlation spectroscopy, which will provide us information about molecule interactions (O1) and, (ii) “functional” SPAD-FCS, which will provide us the tool to link cell function with molecule movements (O2). We are going to apply these results to study of the interaction between GABAA receptors and scaffolding proteins during synaptic plasticity in inhibitory neurons (O3). We will be able to reveal how the molecular organization affects synaptic function in GABAergic neurons, establishing a novel biophysical framework, where the enriched SPAD array detector dataset is employed to study protein dynamics.

Project information
TRAPEZIO 2022 - Perego
Start date
End date
People involved
Eleonora Perego
Molecular Microscopy and Spectroscopy
Total budget: 172.750,08€
Total contribution: 172.750,08€