Wednesday, September, 29th, 2021: 12:00pm – 1:30pm
Protein-protein interactions at the cellular interface: Biophotonics approaches to quantitative FRET measurements
School of Cancer and Pharmaceutical Science, King’s College London, Guy’s Campus, London SE11UL
The current COVID19 pandemic caused by SARS-CoV2 has led to over 185 million cases and more than 4 million deaths worldwide (as of 08/07/2021, source WHO). While there has been a significant effort to understand how the virus infects individuals and why some people are
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.
I will discuss 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 live cell imaging in iPSC derived cultures. Beyond this demonstration, I will discuss application of fluorescence lifetime imaging to imaging flow cytometry and single molecule spectroscopy.
Simon M. Ameer-Beg is Professor of Optical Oncology (BioImaging and Biophysics) in the School of Cancer and Pharmaceutical Sciences 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 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. Simon developed high-content screening methodologies based on fluorescence anisotropy/lifetime for protein interaction perturbation using siRNA and biologics. As technology development lead for the CRUK KCL Imaging Centre, he developed high-resolution multiphoton FLIM instrumentation for in vivo applications and the world’s first TCSPC based lifetime flow cytometer. In collaboration with Prof Robert Henderson (Edinburgh), Simon has developed some of the world’s most advanced fluorescence lifetime imaging microscopes. He heads several projects including development of an analysis pipeline for patient derived biopsy and exosomes using novel microfluidics and fluorescence lifetime spectroscopy techniques; ISO-FLIM, a novel isotropic resolution massively parallel fluorescence lifetime imaging platform; ASTORM, a project to apply Förster resonance energy transfer (FRET) by fluorescence anisotropy to single-molecule localisation microscopy. SAB is co-PI for the UK Interdisciplinary Biological Imaging Network, co-founder of the King’s Imaging Network (KIN), Biophotonics Interest Group and Microscopy Innovation Centre at KCL. In May 2020, SAB founded NanoClinical Ltd, a biomedical start-up developing FRET based clinical diagnostics, with long term collaborator Prof Tony Ng.
Quantitative real-time imaging of intracellular FRET biosensor dynamics using rapid multi-beam confocal FLIM
A High Speed Multifocal Multiphoton Fluorescence Lifetime Imaging Microscope For Live-cell FRET Imaging
Time-domain microfluidic fluorescence lifetime flow cytometry for high-throughput FRET screening