Wide-field fluorescence lifetime imaging of neuron spiking and subthreshold activity in vivo

Abstract

The development of voltage-sensitive fluorescent probes suggests fluorescence lifetime as a promising readout for electrical activity in biological systems. Existing approaches fail to achieve the speed and sensitivity required for voltage imaging in neuroscience applications. We demonstrated that wide-field electro-optic fluorescence lifetime imaging microscopy (EO-FLIM) allows lifetime imaging at kilohertz frame-acquisition rates, spatially resolving action potential propagation and subthreshold neural activity in live adult Drosophila . Lifetime resolutions of <5 picoseconds at 1 kilohertz were achieved for single-cell voltage recordings. Lifetime readout is limited by photon shot noise, and the method provides strong rejection of motion artifacts and technical noise sources. Recordings revealed local transmembrane depolarizations, two types of spikes with distinct fluorescence lifetimes, and phase locking of spikes to an external mechanical stimulus.

Fast volumetric fluorescence lifetime imaging of multicellular systems using single-objective light-sheet microscopy

“…Previously employed detectors for light-sheet FLIM suffer from high dark currents in the case of intensified or modulated cameras 7,8 , from limited throughput in the case of multichannel plate-based detectors 9 or from limited fill factor and sensor size in the case of a novel SPADarray 10,11 . A promising approach for high-speed FLIM of live samples is time-gating via fast Pockels cells 12,13 , but this specialized technology is not readily available. Therefore, lightsheet FLIM has not yet realized its potential to enable faster lifetime imaging than state-ofthe-art laser scanning microscopes.…”

Nanocrystals for Deep-Tissue In Vivo Luminescence Imaging in the Near-Infrared Region

“…Time-resolved imaging is a crucial technique for discerning fluorescence information in the time domain. ,− It relies on the fluorescence lifetime of fluorescent probes, which represents the average duration during which excited-state photons remain in the excited state before returning to the ground state. − By employing time-gating technology as a temporal filter, the long-lifetime emission (ranging from microseconds to milliseconds) from the probes is captured following the reduction of autofluorescence or scattered light (0.1–5 ns). − Time-resolved imaging greatly enhances imaging sensitivity and SNR by effectively mitigating the impact of absorption, scattering, and background autofluorescence induced by excitation light. − This technique has the potential to decrease false-positive and false-negative signals in biological detection. The SNR increases with extended acquisition time, reaching its peak after the complete decay of autofluorescence.…”

Chirality-Regulated Clusteroluminescence in Polypeptides

“…Organic luminescent materials have been extensively employed in the fields of sensors and biological imaging. − However, the practical application of traditional organic luminescent materials in biomedicine-related fields is limited due to their poor biocompatibility and biodegradability. In recent years, several nonconjugated and nonaromatic small molecules and polymers have been discovered with visible emission in the clustering state, namely clusteroluminescence (CL). − Compared to traditional organic luminescent materials, the emission of these clusteroluminogens (CLgens) is generated by the through-space interaction between small molecules and polymers, and the CLgens always exhibit low toxicity and good biocompatibility. − Up to present, there remain challenges in CL investigations on the mechanism and practical applications. − First, the structure–property relationship for CLgens at the primary and secondary structure levels is highly required for the regulation of CL wavelength and intensity.…”