Owing to its tissue-penetration ability, multi-photon fluorescence microscopy allows for the high-resolution, non-invasive imaging of deep tissue ; the recently developed three-photon microscopy (3PM) has extended the depth of high-resolution, non-invasive functional imaging of mouse brains to beyond 1.0 mm. However, the low repetition rate of femtosecond lasers that are normally used in 3PM limits the temporal resolution of point-scanning three-photon microscopy. To increase the volumetric imaging speed of 3PM, we propose a combination of an axially elongated needle-like Bessel-beam with three-photon excitation (3PE) to image biological samples with an extended depth of focus. We demonstrate the higher signal-to-background ratio (SBR) of the Bessel-beam 3PM compared to the two-photon version both theoretically and experimentally. Finally, we perform simultaneous calcium imaging of brain regions at different axial locations in live fruit flies and rapid volumetric imaging of neuronal structures in live mouse brains. These results highlight the unique advantage of conducting rapid volumetric imaging with a high SBR in the deep brain using scanning Bessel-3PM.

How pancreatic β-cells acquire function in vivo is a long-standing mystery due to the lack of technology to visualize β-cell function in living animals. Here, we applied a high-resolution two-photon light-sheet microscope for the first in vivo imaging of Ca2+activity of every β-cell in Tg (ins:Rcamp1.07) zebrafish. We reveal that the heterogeneity of β-cell functional development in vivo occurred as two waves propagating from the islet mantle to the core, coordinated by islet vascularization. Increasing amounts of glucose induced functional acquisition and enhancement of β-cells via activating calcineurin/nuclear factor of activated T-cells (NFAT) signaling. Conserved in mammalians, calcineurin/NFAT prompted high-glucose-stimulated insulin secretion of neonatal mouse islets cultured in vitro. However, the reduction in low-glucose-stimulated insulin secretion was dependent on optimal glucose but independent of calcineurin/NFAT. Thus, combination of optimal glucose and calcineurin activation represents a previously unexplored strategy for promoting functional maturation of stem cell-derived β-like cells in vitro.

Due to its ability of optical sectioning and low phototoxicity, z -stacking light-sheet microscopy has been the tool of choice for in vivo imaging of the zebrafish brain. To image the zebrafish brain with a large field of view, the thickness of the Gaussian beam inevitably becomes several times greater than the system depth of field (DOF), where the fluorescence distributions outside the DOF will also be collected, blurring the image. In this paper, we propose a 3D deblurring method, aiming to redistribute the measured intensity of each pixel in a light-sheet image to in situ voxels by 3D deconvolution. By introducing a Hessian regularization term to maintain the continuity of the neuron distribution and using a modified stripe-removal algorithm, the reconstructed z -stack images exhibit high contrast and a high signal-to-noise ratio. These performance characteristics can facilitate subsequent processing, such as 3D neuron registration, segmentation, and recognition.