In Vivo Neurodynamics Mapping via High‐Speed Two‐Photon Fluorescence Lifetime Volumetric Projection Microscopy

Abstract Monitoring the morphological and biochemical information of neurons and glial cells at high temporal resolution in three‐dimensional (3D) volumes of in vivo is pivotal for understanding their structure and function, and quantifying the brain microenvironment. Conventional two‐photon fluorescence lifetime volumetric imaging speed faces the acquisition speed challenges of slow serial focal tomographic scanning, complex post‐processing procedures for lifetime images, and inherent trade‐offs among contrast, signal‐to‐noise ratio, and speed. This study presents a two‐photon fluorescence lifetime volumetric projection microscopy using an axially elongated Bessel focus and instant frequency‐domain fluorescence lifetime technique, and integrating with a convolutional network to enhance the imaging speed for in vivo neurodynamics mapping. The proposed method is validated by monitoring intracellular Ca²⁺ concentration throughout whole volume, tracking microglia movement and microenvironmental changes following thermal injury in the zebrafish brain, analyzing structural and functional variations of gap junctions in astrocyte networks, and measuring the Ca²⁺ concentration in neurons in mouse brains. This innovative methodology enables quantitative in vivo visualization of neurodynamics and the cellular processes and interactions in the brain.