In:
The Journal of Neuroscience, Society for Neuroscience, Vol. 42, No. 4 ( 2022-01-26), p. 552-566
Abstract:
Fluorescence imaging is an indispensable method for analysis of diverse cellular and molecular processes, enabling, for example, detection of ions, second messengers, or metabolites. Intensity-based approaches, however, are prone to artifacts introduced by changes in fluorophore concentrations. This drawback can be overcome by fluorescence lifetime imaging (FLIM) based on time-correlated single-photon counting. FLIM often necessitates long photon collection times, resulting in strong temporal binning of dynamic processes. Recently, rapidFLIM was introduced, exploiting ultra-low dead-time photodetectors together with rapid electronics. Here, we demonstrate the applicability of rapidFLIM, combined with new and improved correction schemes, for spatiotemporal fluorescence lifetime imaging of low-emission fluorophores in a biological system. Using tissue slices of hippocampi of mice of either sex, loaded with the Na + indicator ING2, we show that improved rapidFLIM enables quantitative, dynamic imaging of neuronal Na + signals at a full-frame temporal resolution of 0.5 Hz. Induction of transient chemical ischemia resulted in unexpectedly large Na + influx, accompanied by considerable cell swelling. Both Na + loading and cell swelling were dampened on inhibition of TRPV4 channels. Together, rapidFLIM enabled the spatiotemporal visualization and quantification of neuronal Na + transients at unprecedented speed and independent from changes in cell volume. Moreover, our experiments identified TRPV4 channels as hitherto unappreciated contributors to neuronal Na + loading on metabolic failure, suggesting this pathway as a possible target to ameliorate excitotoxic damage. Finally, rapidFLIM will allow faster and more sensitive detection of a wide range of dynamic signals with other FLIM probes, most notably those with intrinsic low-photon emission. SIGNIFICANCE STATEMENT FLIM is an indispensable method for analysis of cellular processes. FLIM often necessitates long photon collection periods, requiring the sacrifice of temporal resolution at the expense of spatial information. Here, we demonstrate the applicability of the recently introduced rapidFLIM for quantitative, dynamic imaging with low-emission fluorophores in brain slices. RapidFLIM, combined with improved correction schemes, enabled intensity-independent recording of neuronal Na + transients at unprecedented full-frame rates of 0.5 Hz. It also allowed quantitative imaging independent from changes in cell volume, revealing a surprisingly strong and hitherto uncovered contribution of TRPV4 channels to Na + loading on energy failure. Collectively, our study thus provides a novel, unexpected insight into the mechanisms that are responsible for Na + changes on energy depletion.
Type of Medium:
Online Resource
ISSN:
0270-6474
,
1529-2401
DOI:
10.1523/JNEUROSCI.0819-21.2021
DOI:
10.1523/JNEUROSCI.0819-21.2021.video.1
DOI:
10.1523/JNEUROSCI.0819-21.2021.video.2
DOI:
10.1523/JNEUROSCI.0819-21.2021.video.3
Language:
English
Publisher:
Society for Neuroscience
Publication Date:
2022
detail.hit.zdb_id:
1475274-8
SSG:
12
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