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.
Highlights
Emission imaging microscopy[1,2,3,4] has revolutionized our understanding of living systems by allowing their structure to be vizualised with sub-micron resolution and biochemical function to be monitored at the molecular level
The new method expands the available imaging timescales 1000-fold whilst maintaining the essential spatial resolution, and allows complete background rejection
We illustrate the power of TP-time-resolved emission imaging microscopy (TREM) through its application to a range of problems in biological imaging on the example of diverse live cells and histological tissues
Summary
Emission imaging microscopy[1,2,3,4] has revolutionized our understanding of living systems by allowing their structure to be vizualised with sub-micron resolution and biochemical function to be monitored at the molecular level. High-resolution visualization with complete background rejection, could be achieved by employing molecular probes that emit on a substantially longer, microsecond, timescale.[15,16,17,18,19,20] at the same time, an orders-of-magnitude increase in sensitivity of lifetime imaging to bio-relevant analytes including oxygen[12,13,14] should be achievable through such an extension in lifetime This concept of time-resolved emission imaging microscopy (TREM) would allow gating out of auto uorescence at early times and selective imaging of the probe’s emission at later times. The general method overcomes the limitations given above and combines for the rst time microsecond imaging with enhanced multiphoton resolution and diffraction-limited pointscanning in a fast correlated individual-photon tagging implementation It uses phosphorescent rather than uorescent probes and achieves time-resolved detection on a timescale several orders of magnitude longer than that available in FLIM yet maintaining the essential sub-micron spatial resolution afforded by two-photon excitation, thereby offering dramatic improvements in both contrast and sensitivity in emission imaging. Exploiting, amongst others, the rst water-soluble member of a family of platinum-based oxygen-sensitive probes with microsecond timescale emission, we demonstrate how TPTREM can provide background-free multiphoton microsecond depth-imaging of diverse live cells and histological tissues, and variation in the probe lifetime in vivo orders of magnitude higher than that available with current techniques or probes
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