Protocol for In Vivo Two-Photon FCS to Measure Nanocarrier Number and Flow Velocity in Mouse Cerebral...

Real-time measurement of blood flow and nanocarrier transport in the cerebral microvasculature is crucial for understanding neurovascular physiology and nanocarrier-based drug delivery. Existing techniques lack the ability to measure blood flow rates in individual vessels with high spatial and temporal resolution in real time. Two-photon fluorescence correlation spectroscopy (2P-FCS) provides a powerful approach for monitoring tracer molecules within a small confocal observation volume. This enables the simultaneous determination of particle number and flow dynamics in vivo. Here, we present a detailed protocol for in vivo 2P-FCS measurements in the mouse cerebral microvasculature. The protocol includes preparation of the cranial window, delivery of fluorescent dextran tracers for vascular visualization, and FCS measurements. It also includes two-photon imaging of the cerebrovascular network and acquisition and analysis of fluorescence correlation data. The protocol describes calibration of the confocal volume diameter and optimization of two-photon excitation parameters. This workflow enables real-time measurement of tracer concentration and flow velocity in individual cerebral microvessels with high spatial and temporal resolution. The method can be adapted to study blood flow dynamics, nanoparticle transport, and microvascular physiology in a variety of in vivo imaging systems equipped with multiphoton microscopy and FCS capabilities. Key features • Protocol for performing in vivo 2P-FCS for real-time measurements of nanocarrier flow and concentration in the mouse cerebrovasculature through an acute cranial window. • Provides guidance on calibrating the confocal volume diameter and optimizing near-infrared (NIR) laser power and excitation wavelengths of fluorophores. • Includes procedures for two-photon imaging of cerebral blood vessels. • Applicable to studies of cerebral blood flow, nanoparticle transport, and microvascular dynamics using multiphoton microscopy systems equipped with FCS modules.