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.

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: Sep 19, 2018
• 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
• Public URL: https://durham-repository.worktribe.com/output/1319193