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

Background

Cerebral blood vessels, such as veins, arteries, and capillaries, form the cerebrovascular network and are responsible for a variety of functions, including ion exchange regulation, molecular and oxygen transport, and waste removal [1]. Cerebral blood flow (CBF), particularly in capillaries, provides vital information about the central nervous system’s functionality. For instance, abnormalities in CBF patterns can indicate conditions such as ischemic stroke when blood flow slows down, brain tumors when flow patterns alter, or neurodegenerative diseases like Alzheimer's when CBF gradually declines [2,3]. Thus, CBF assessment can reveal how the brain’s autoregulation system maintains stable blood flow despite systemic changes in blood pressure. Various techniques, such as computed tomography (CT) [4], magnetic resonance imaging (MRI) [5], and fluorescence imaging [6,7] have been developed for measuring CBF. However, real-time measurement of blood flow rates in individual vessels using these existing technologies is challenging, especially in capillaries (<10 μm), due to their limited spatial resolution [8]. While CT and MRI provide whole-brain perfusion measurements, they lack the spatial resolution required to resolve capillary-scale flow dynamics [9,10]. Optical techniques such as laser speckle imaging and Doppler optical coherence tomography improve temporal resolution but do not achieve single-capillary sensitivity or single-particle detection [8,11]. Multiphoton laser-scanning microscopy (MPLSM) techniques can measure flow rates when a two-photon fluorescence dye provides contrast against red blood cells (RBCs). However, these approaches require line-scanning over vessel segments and are limited by scan speed, which prevents pixel-level flow readout and reduces temporal precision [12,13]. Furthermore, intensity-based imaging methods cannot directly quantify nanocarrier concentration or number in real time.

Fluorescence correlation spectroscopy (FCS) is a single-molecule-sensitive technique that measures and correlates the fluctuations of fluorescent molecules diffusing through a defined detection volume, often called the confocal volume. The correlation from fluctuations in fluorescent molecules provides information such as the molecules' diffusion time, surface binding, and the number of molecules within the confocal volume. The strength of FCS lies in its ability to distinguish the uncorrelated background from the correlated signal [14,15]. FCS has previously been widely applied for in vitro applications, including in microfluidic devices, but has now been expanded to study flow and diffusion dynamics in thick biological tissues using multiphoton excitation. By leveraging deeper penetration and reducing out-of-focus excitation artifacts of two-photon (2P) microscopy, in vivo FCS was used to measure pixel-by-pixel blood flow velocity in live mice. For example, Xu et al. employed FCS to measure CBF in live mice using optimized two-photon fluorophores with high spatial and temporal resolution at a depth of 300 μm within brain tissue [16]. Further developments in cerebral blood flow measurements were reported by Xiaojin et al., who used near-infrared-emitting DNA-stabilized silver nanoclusters (DNA-AgNCs) [17]. The liposomes-encapsulated near-NIR DNA-AgNCs showed a better spectral crosstalk with the blood vessel-lighting dye FITC. A recently published article by the same author shows that flow rate measurements were further improved by chronically labeling the cerebrovasculature with adeno-associated viral vectors (AAV) encoding albumin-mNeonGreen (Alb-mNG) expression [18]. A nanocarrier composed of loaded DNA-AgNCs, encapsulated in liposomes and loaded into cationic mesoporous silica nanoparticles, was used as an FCS probe for measuring cerebral blood flow. These studies established the feasibility of quantitative, real-time measurements of nanocarrier dynamics in the cerebrovasculature. Despite these published articles, guidance on the reproducibility of in vivo two-photon FCS remains challenging due to the complexity of the experimental procedures. Many experimental parameters, such as excitation and emission spectra for 2P dyes, confocal volume calibration, cranial window preparation, and correlation-based velocity extraction, among others, need to be optimized and are briefly described in the published articles. However, troubleshooting techniques to minimize motion artifacts are not discussed in detail in such articles. Hence, a step-by-step experimental procedure is needed to ensure the reproducibility of in vivo FCS measurements for the quantification of nanocarrier number in the cerebrovasculature.

The protocol described here provides detailed, reproducible experimental procedures for performing in vivo two-photon fluorescence correlation spectroscopy (2P-FCS) to measure the number of nanocarriers and their flow velocity in the mouse cerebral microvasculature in real time. This includes optimization of two-photon excitation, microfluidic calibration, cranial window preparation, and flow-diffusion autocorrelation modeling to determine absolute particle concentration and transport dynamics in real time. The benefit of this protocol is that it works well for a wide range of fluorescent nanocarriers, including dextran conjugates, liposomes, and polymeric nanoparticles. This technique can be used with various two-photon imaging systems that have photon-counting detectors. It is especially beneficial for researchers studying nanomedicine pharmacokinetics, cerebral blood flow, vascular transport dynamics, and in vivo nanoparticle clearance. Compared to earlier published in vivo FCS protocols, this one offers (i) a microfluidic calibration process to convert correlation decay times to absolute flow velocities, (ii) practical strategies to reduce motion artifacts in live animals, and (iii) a detailed data analysis framework for extracting both particle number and velocity from the same autocorrelation curve. Beyond cerebral blood flow measurement, this protocol can be adapted to investigate nanocarrier pharmacokinetics, blood–brain barrier transport, vascular permeability, tumor microvascular dynamics, and nanoparticle clearance in real time. By providing a standardized methodological framework, this protocol facilitates broader adoption of in vivo two-photon FCS for quantitative vascular and nanomedicine research.