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Biochemical resolving power of fluorescence lifetime imaging: untangling the roles of the instrument response function and photon-statistics.

Published version
Peer-reviewed

Type

Article

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Authors

Abstract

A deeper understanding of spatial resolution has led to innovations in microscopy and the disruption of biomedical research, as with super-resolution microscopy. To foster similar advances in time-resolved and spectral imaging, we have previously introduced the concept of 'biochemical resolving power' in fluorescence microscopy. Here, we apply those concepts to investigate how the instrument response function (IRF), sampling conditions, and photon-statistics limit the biochemical resolution of fluorescence lifetime microscopy. Using Fisher information analysis and Monte Carlo simulations, we reveal the complex dependencies between photon-statistics and the IRF, permitting us to quantify resolution limits that have been poorly understood (e.g., the minimum resolvable decay time for a given width of the IRF and photon-statistics) or previously underappreciated (e.g., optimization of the IRF for biochemical detection). With this work, we unravel common misunderstandings on the role of the IRF and provide theoretical insights with significant practical implications on the design and use of time-resolved instrumentation.

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Keywords

Journal Title

Biomedical optics express

Conference Name

Journal ISSN

2156-7085

Volume Title

12

Publisher

Sponsorship
Medical Research Council (MC_UU_12022/1, MC_UU_12022/8)
Cancer Research UK (OncoLive, C54674/A27487)