High-speed volumetric two-photon fluorescence imaging of neurovascular dynamics

Abstract

Understanding the structure and function of vasculature in the brain requires us to monitor distributed hemodynamics at high spatial and temporal resolution in three-dimensional (3D) volumes in vivo. Currently, a volumetric vasculature imaging method with sub-capillary spatial resolution and blood flow-resolving speed is lacking. Here, using two-photon laser scanning microscopy (TPLSM) with an axially extended Bessel focus, we capture volumetric hemodynamics in the awake mouse brain at a spatiotemporal resolution sufficient for measuring capillary size and blood flow. With Bessel TPLSM, the fluorescence signal of a vessel becomes proportional to its size, which enables convenient intensity-based analysis of vessel dilation and constriction dynamics in large volumes. We observe entrainment of vasodilation and vasoconstriction with pupil diameter and measure 3D blood flow at 99 volumes/second. Demonstrating high-throughput monitoring of hemodynamics in the awake brain, we expect Bessel TPLSM to make broad impacts on neurovasculature research.

Non-Diffracting Light Wave: Fundamentals and Biomedical Applications

“…Moreover, the non-diffracting beam features shape-preserving for a distance much longer than the Rayleigh distance, offering the ability to extend the illumination FOV in the LSFM application [11,136]. The Bessel focus TPLSM allows volumetric imaging of neurovascular dynamics while preserving the high lateral resolution and multi-color capability [25]. With the advancement of bright and efficient two-photon fluorescent sensors, Bessel TPLSM simultaneously images neuro-vasculature with neurons and glia to produce a high-throughput database for unveiling the dynamics of neurovascular in normal and diseased brains.…”

“…One possible means to overcome scattering is using the nondiffracting beam, which preserves the shape over a distance longer than the Gaussian Rayleigh range. Here, the theory, generation, and significant features of many "non-diffracting" beams are reviewed, with an emphasis on the biomedical applications of those fascinating beams, including the multimodal biomedical imaging [11,[23][24][25][26], optical micromanipulation [10,27,28], and optical transfection [29][30][31]. Specifically, we introduce the use of non-diffracting beams to overcome the challenges arising from various applications, including two-photon microscopy, Raman spectroscopy, and optical manipulation.…”

Long-term Microglial Phase-specific Dynamics During Single Vessel Occlusion and Recanalization

“…Besides, consistent with the previous observation, the cluster resolved to redistribute single microglia territory to regulate brain parenchyma microenvironment. Furthermore, combining pharmacologically manipulating these redistributed proliferation cells, such as cell depletion, with more advanced optical methods (Fan et al, 2020), it is expected to illuminate their role in the subsequent long-term pathological development.…”

Location Matters: Navigating Regional Heterogeneity of the Neurovascular Unit

“…The capabilities of MPM (Urban et al, 2017 ; Li B. et al, 2020 ) combined with cell-specific genetically encoded activity reporters suited for probing either calcium or voltage changes (GECIs and GEVIs) (Lin and Schnitzer, 2016 ) has allowed the simultaneous recording of many cell populations involved in NVC [neurons (Urban et al, 2017 ), roles of astrocytes in the control of arteriole diameter, increase in local blood flow (Takano et al, 2006 ; Tran and Gordon, 2015 ), microglia as important regulators of blood flow during NVC (Hierro-Bujalance et al, 2018 ; Császár et al, 2021 ), pericytes control blood flow direction at capillary junctions, maintenance of capillary flow resistance and metabolic exchanges (Berthiaume et al, 2018 ; Gonzales et al, 2020 )] and/or BBB permeability (Knowland et al, 2014 ). Along with cell-specific imaging, MPM can record local volumetric hemodynamic changes [blood flow and red blood cell velocity (Urban et al, 2017 )] from surface pial vessels down to deep capillaries (Fan et al, 2020 ) with dedicated circulating fluorescent contrast agents (Miller et al, 2017 ). The unique combination of approaches offered by MPM makes it a tool of choice for investigating the complex interplay between cellular functions and vascular dynamics under awake conditions.…”