Imaging ONE WORLD - "Protein-protein interactions at the cellular interface: Biophotonics approaches to quantitative FRET measurements"

8 March 2021

Online

RMS Hosted Event

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Overview

This week will feature Simon Ameer-Beg from King's College London.

Scientific Organisers: Stefanie Reichelt, Alex Sossick, Nick Barry, Alessandro Esposito and Kirti Prakash

The meeting will begin at 13:00GMT.

As part of the 'Imaging ONE WORLD' series, the focus of these lectures is on microscopy and image analysis methods and how to apply these to your research. Almost all aspects of imaging such as sample preparation, labelling strategies, experimental workflows, ‘how-to’ image and analyse, as well as facilitating collaborations and inspiring new scientific ideas will be covered. Speakers will be available for questions and answers. The organisers, CRUK CI core facility staff, Gurdon Institute, MRC-LMB, MRC Cancer Unit and NPL will be able to continue the discussion and provide advice on your imaging projects.


Speaker

  • Dr Simon Ameer-Beg

    King's College London
    Simon M. Ameer-Beg is a Professor in the school of cancer at King’s College London (KCL). He studied Physics with Laser Physics at University of Essex before undertaking a PhD in ultrafast spectroscopy in industry with British Nuclear Fuels Ltd. He has since developed an interdisciplinary research portfolio principally in the field of time-resolved multiphoton fluorescence lifetime imaging (FLIM) for application to quantitative analysis of protein-protein interactions. Much of his research relates to high-content screening methodologies based on fluorescence anisotropy/lifetime for protein interaction perturbation using siRNA and biologics.

Speaker Abstract

For high precision FLIM, time-correlated single photon counting (TCSPC) is unparalleled in its measurement accuracy particularly for multi-exponential decays. Until recently, high speed FLIM could only be performed using modulated or time-gated image intensifier systems] as TCSPC was fundamentally limited with respect to photon counting rate in implementations of laser scanning microscopy. This has restricted its use in a number of time-critical applications including live cell imaging.

We have previously demonstrated multifocal multiphoton fluorescence lifetime imaging microscopy (MM-FLIM) for applications utilizing TCSPC which increases the acquisition rate of high-resolution fluorescence lifetime imaging by a factor of 64 by parallelizing excitation and detection. The system consists of a two dimensional array of ultrafast beams which are then optically conjugated with a Megaframe camera consisting of 32×32 individual 10-bit time-to-digital convertor (TDC) array with integrated single-photon avalanche diodes (SPADs), each of which operates in TCSPC mode and provides FLIM capability.

We report the development of a novel massively parallelised multifocal multiphoton FLIM laser scanning high speed microscope that we term SWept Array Microscopy (SWARM) with the ability to acquire ~250 million photon arrival events per second. This allows us to operate in full frame mode (32×32 beamlets) unlocking more potential from the Megaframe camera for FLIM imaging. Utilising a diffractive optical element to generate the beamlet array and an innovative scanning approach we have been able to simplify and reduce the optical footprint of the system. We demonstrate the applicability of the system to HCS tissue sections and to live cell imaging. I will conclude with some future perspectives and novel applications which showcase the potential of FRET to answer real world biological questions. 



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