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Long-lived metal complexes open up microsecond lifetime imaging microscopy under multiphoton excitation: from FLIM to PLIM and beyond

Baggaley, Elizabeth; Botchway, Stanley W.; Haycock, John W.; Morris, Hayley; Sazanovich, Igor V.; Williams, J.A. Gareth; Weinstein, Julia A.

Long-lived metal complexes open up microsecond lifetime imaging microscopy under multiphoton excitation: from FLIM to PLIM and beyond Thumbnail


Authors

Elizabeth Baggaley

Stanley W. Botchway

John W. Haycock

Hayley Morris

Igor V. Sazanovich

Julia A. Weinstein



Abstract

Lifetime imaging microscopy with sub-micron resolution provides essential understanding of living systems by allowing both the visualisation of their structure, and the sensing of bio-relevant analytes in vivo using external probes. Chemistry is pivotal for the development of the next generation of bio-tools, where contrast, sensitivity, and molecular specificity facilitate observation of processes fundamental to life. A fundamental limitation at present is the nanosecond lifetime of conventional fluorescent probes which typically confines the sensitivity to sub-nanosecond changes, whilst nanosecond background autofluorescence compromises the contrast. High-resolution visualization with complete background rejection and simultaneous mapping of bio-relevant analytes including oxygen – with sensitivity orders of magnitude higher than that currently attainable – can be achieved using time-resolved emission imaging microscopy (TREM) in conjunction with probes with microsecond (or longer) lifetimes. Yet the microsecond timescale has so far been incompatible with available multiphoton excitation/detection technologies. Here we realize for the first time microsecond-imaging with multiphoton excitation whilst maintaining the essential sub-micron spatial resolution. The new method is background-free and expands available imaging and sensing timescales 1000-fold. Exploiting the first engineered water-soluble member of a family of remarkably emissive platinum-based, microsecond-lived probes amongst others, we demonstrate (i) the first instance of background-free multiphoton-excited microsecond depth imaging of live cells and histological tissues, (ii) over an order-of-magnitude variation in the probe lifetime in vivo in response to the local microenvironment. The concept of two-photon TREM can be seen as “FLIM + PLIM” as it can be used on any timescale, from ultrafast fluorescence of organic molecules to slower emission of transition metal complexes or lanthanides/actinides, and combinations thereof. It brings together transition metal complexes as versatile emissive probes with the new multiphoton-excitation/microsecond-detection approach to create a transformative framework for multiphoton imaging and sensing across biological, medicinal and material sciences.

Citation

Baggaley, E., Botchway, S. W., Haycock, J. W., Morris, H., Sazanovich, I. V., Williams, J. G., & Weinstein, J. A. (2013). Long-lived metal complexes open up microsecond lifetime imaging microscopy under multiphoton excitation: from FLIM to PLIM and beyond. Chemical Science, 5(3), 879-886. https://doi.org/10.1039/c3sc51875b

Journal Article Type Article
Acceptance Date Oct 15, 2013
Online Publication Date Oct 16, 2013
Publication Date Oct 16, 2013
Deposit Date Sep 18, 2018
Publicly Available Date Mar 28, 2024
Journal Chemical Science
Print ISSN 2041-6520
Electronic ISSN 2041-6539
Publisher Royal Society of Chemistry
Peer Reviewed Peer Reviewed
Volume 5
Issue 3
Pages 879-886
DOI https://doi.org/10.1039/c3sc51875b

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