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Fast single-cell biochemistry: theory, open source microscopy and applications

Published version
Peer-reviewed

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Authors

Trinh, Andrew 
Ber, Suzan 
Howitt, Annie 
Oriol Valls, Pablo 
Fries, Maximilian 

Abstract

Fluorescence lifetime sensing enables researchers to probe the physicochemical environment of a fluorophore providing a window through which we can observe the complex molecular make-up of the cell. Fluorescence lifetime imaging microscopy (FLIM) quantifies and maps cell biochemistry, a complex ensemble of dynamic processes. Unfortunately, typical high-resolution FLIM systems exhibit rather limited acquisition speeds, often insufficient to capture the time evolution of biochemical processes in living cells. Here, we describe the theoretical background that justifies the developments of high-speed single photon counting systems. We show that systems with low dead-times not only result in faster acquisition throughputs but also improved dynamic range and spatial resolution. We also share the implementation of hardware and software as an open platform, show applications of fast FLIM biochemical imaging on living cells and discuss strategies to balance precision and accuracy in FLIM. The recent innovations and commercialisation of fast time-domain FLIM systems are likely to popularise FLIM within the biomedical community, to impact biomedical research positively and to foster the adoption of other FLIM techniques as well. While supporting and indeed pursuing these developments, with this work we also aim to warn the community about the possible shortcomings of fast single photon counting techniques and to highlight strategies to acquire data of high quality.

Description

Keywords

HeLa Cells, Humans, Image Processing, Computer-Assisted, Microscopy, Photons, Single-Cell Analysis, Time Factors

Journal Title

Methods and Applications in Fluorescence

Conference Name

Journal ISSN

2050-6120
2050-6120

Volume Title

7

Publisher

IoP Publishing
Sponsorship
MRC (unknown)
MRC (4050551988)
Medical Research Council (G1001522)
Medical Research Council (MC_UU_12022/1)
MRC (MC_UU_12022/8)
We acknowledge funding from the Medical Research Council core grants (MC_UU_12022/1 and MC_UU_12022/8) to ARV.