Ultrafast two-photon fluorescence imaging of cerebral blood circulation in the mouse brain in vivo

Abstract

Significance Characterizing blood flow by tracking individual red blood cells as they move through vessels is essential for understanding vascular function. With high spatial resolution, two-photon fluorescence microscopy is the method of choice for imaging blood flow at the cellular level. However, its application is limited to a low flow speed regimen in anesthetized animals by its slow focus scanning mechanism. Using an ultrafast scanning module, we demonstrated two-photon fluorescence imaging of blood flow at 1,000 two-dimensional frames and 1,000,000 one-dimensional line scans per second in the brains of awake mice. These ultrafast measurements enabled us to study hemodynamic and fluid mechanical regimens beyond the reach of conventional methods.

High-throughput deep tissue two-photon microscopy at kilohertz frame rates

“…Blood flow rates are determined from the dominant orientation of RBC kymographs, which, traditionally, can be measured on single blood vessels by rapid 1D line scanning 2 . More recently, FACED-based 2PM 17 was shown to enable kilohertz imaging of a few vessel segments at a time. Here, by virtue of the high throughput of SMU scanning, we routinely imaged more than 10 vessel segments at a time, allowing connections between multiple capillaries, venules, and arteries to be monitored simultaneously, and a wide diversity of RBC flow speeds to be quantified [Fig.…”

A deep learning approach for improving two-photon vascular imaging speeds

“…Neurovascular function is disrupted by disorders such as stroke, Alzheimer's and other neurodegenerative diseases, and diabetes, with lasting effects that are not fully understood 1,2 . Advances in multiphoton fluorescence microscopy (MPM) have enabled imaging with capillary-level resolution in vivo, and this noninvasive tool could be used to monitor capillary-level changes over time in cerebral vasculature as a potential predictor of disease progression/prognosis [3][4][5][6] . A constraint with MPM, however, is the slow acquisition process that is necessary for producing high quality, three-dimensional images with a traditional point scanning multiphoton imaging setup.…”

A FACED lift for cerebral blood flow imaging

“…In PNAS, Meng et al. ( 1 ) introduce a powerful ultrafast method to improve in vivo measurement of cerebral blood flow in microvascular networks, which will significantly advance the applicability of two-photon microscopy for quantifying microvascular perfusion.…”

“…Meng et al. ( 1 ) have overcome the first two of these three limitations. The authors apply their recently introduced technology, named FACED (free-space angular chirp enhanced delay), in two-photon microscopy for cerebral blood flow measurements.…”

“…In PNAS, Meng et al. ( 1 ) now use this approach to acquire ultrafast line scans tracking the movement of RBCs in the cerebral cortex of mice ( Fig. 1 B ).…”