Ultrafast two-photon fluorescence imaging of cerebral blood circulation in the mouse brain in vivo
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 one thousand two-dimensional frames and one million one-dimensional line scans per second in the primary visual cortex of awake mice, using FACED (free-space angular chirp enhanced delay). These ultrafast measurements enabled us to study hemodynamic and fluid mechanical regimens beyond the reach of conventional methods. This work was published on PNAS, check it out here!
Mesoscale calcium data analysis with constrained non-negative matrix factorization (CNMF)
With the extra-large FOV, a mesoscope equipped with a Bessel focus generation module enabled activity measurements of large neural ensembles over multiple cortical areas (spanning primary visual cortex and somatosensory cortex) simultaneously in vivo. We modified constrained nonnegative matrix factorization for microendoscopic data (CNMF-E) to first segment ROIs based on local contrast of fluorescence intensity rather than local correlation and then model the neuropil background with a spatially smooth low-rank matrix. The optimized CNMF-E method detected 9,247 active neurons within this volume and extracted their somatic calcium transients without background neuropil activity.
Read more about this work on Nature Methods!
High-throughput two-photon fluorescence microendoscopy for deep-brain volumetric imaging at synaptic resolution
The highly scattering mammalian brain makes microendoscopy, with an optical probe such as a gradient index (GRIN) lens embedded into brain tissue to provide optical relay, the method of choice for imaging neurons and neural activity in deeply buried brain structures. Incorporating a Bessel focus scanning module into two-photon fluorescence microendoscopy, we extended the excitation focus axially and improved its lateral resolution. Scanning the Bessel focus in 2D, we imaged volumes of neurons at high-throughput while resolving fine structures such as synaptic terminals. We applied this approach to the volumetric anatomical imaging of dendritic spines and axonal boutons in the mouse hippocampus, and functional imaging of GABAergic neurons in the mouse lateral hypothalamus in vivo.
To learn more, check out our eLife paper!