A Direct Method for Measuring Mouse Capillary Cortical Blood Volume Using Multiphoton Laser Scanning Microscopy

Abstract

Knowledge of the blood volume per unit volume of brain tissue is important for understanding brain function in health and disease. We describe a direct method using two-photon laser scanning microscopy to obtain in vivo the local capillary blood volume in the cortex of anesthetized mouse. We infused fluorescent dyes in the circulating blood and imaged the blood vessels, including the capillaries, to a depth of 600 lm below the dura at the brain surface. Capillary cortical blood volume (CCBV) was calculated without any form recognition and segmentation, by normalizing the total fluorescence measured at each depth and integrating the collected intensities all over the stack. Theoretical justifications are presented and numerical simulations were performed to validate this method which was weakly sensitive to background noise. Then, CCBV had been estimated on seven healthy mice between 2%60.3% and 2.4%60.4%. We showed that this measure of CCBV is reproductible and that this method is highly sensitive to the explored zones in the cortex (vessel density and size). This method, which dispenses with form recognition, is rapid and would allow to study in vivo temporal and highly resolute spatial variations of CCBV under different conditions or stimulations.

How Stereological Analysis of Vascular Morphology Can Quantify the Blood Volume Fraction as a Marker for Tumor Vasculature: Comparison with Magnetic Resonance Imaging

“…The simulated 512 Â 512 Â 512 voxel binary model (Verant et al, 2007) was composed of a number of straight cylinders with varying diameters and orientations (random distribution with equal probability). The number of cylinders, as well as the minimum and maximum diameter could be chosen to yield the desired total cylinder volume.…”

“…Figure 3A). Three different models were generated from optic microscopy z-stack acquisitions of the mouse cortical vasculature (Verant et al, 2007) by binarization, closing of the profiles, and increasing the spatial resolution to achieve pixel sizes in the order of 1 mm, comparable to the spatial resolution of the histology sections of the rat cerebral and tumor vasculature analyzed in this study. Although this numeric model accurately reflects the irregular shape, length, and branching of vessels, we have no precise previous knowledge of the average vascular diameter.…”

Two-photon microscopy of cortical NADH fluorescence intensity changes: correcting contamination from the hemodynamic response

“…Although the effect of optical scattering on the TPLSM signal has been recognized and studied extensively in the case of a spatially uniform distribution of optical parameters, 23,[25][26][27] the effect of scattering from heterogeneously distributed RBCs has not been quantified. Large shadows in detected fluorescence can be seen underneath the blood vessels in virtually all in vivo TPLSM brain images, 7,18 and the highest imaging penetration depths can be obtained only by positioning the field of view away from the large pial vessels. Both hemoglobin absorption of the fluorescence emission and reduction of excitation due to RBC scattering contribute to decay of the detected signal underneath the blood vessels.…”

“…At excitation wavelengths typically used in TPLSM imaging of NADH fluorescence (700-740 nm), the optical scattering coefficient of blood (>100 mm −1 ) [10][11][12][13] is an order of magnitude larger than the scattering coefficient in the cortical tissue (∼ 10 mm −1 ), [14][15][16][17] and the significant decrease in detected fluorescence is evident underneath the blood vessels in TPLSM brain images. 18 Therefore, TPLSM imaging of NADH fluorescence changes could be affected by the blood volume and oxygenation changes starting at imaging depths of only a few tens of microns. Further progress in utilizing TPLSM imaging of NADH fluorescence for understanding brain metabolism and oxygenation is dependent on our better understanding of the confounding effect of the hemodynamic response on the measured NADH signal, especially in the case of functional brain activation with relatively small expected NADH signal changes.…”

Imaging the Structure of Macroporous Hydrogels by Two-Photon Fluorescence Microscopy

“…This limit decreases significantly in turbid biological samples. For this reason, two-photon fluorescence microscopy 26 (TPFM) that enhances the depth of penetration 27 became a leading tool for imaging cellular and subcellular events within living tissue [28][29][30][31] .…”