An accurate and universal approach for short-exposure-time microscopy image enhancement.

Fluorescence microscopy imaging has become an essential technique in the biology and biomedical science which can provide comprehensive visualization of many biological processes, and the exposure time is one of the most critical parameters for fluorescence microscopy imaging. Short-exposure-time (SET) imaging overcomes the limitations of photo-bleaching and photo-toxicity, allowing comprehensive visualization of the biological processes. Unfortunately, SET images deteriorate the signal to noise ratio and the image quality when compared with the long-exposure-time (LET) images. Therefore, we introduce a data-driven microscopy image enhancement network (MIEN) to improve the quality of SET microscopy images without requiring any manual intervention, facilitating the production of high-resolution and high contrast images. The universal property and accuracy of the proposed network are validated on 38,500 real fluorescence microscopy images, which contain different object contents and are collected from various exposure time ratios and fluorescence microscopes platforms. Experimental results demonstrate that the proposed MIEN model is effective to enhance the quality of SET fluorescence microscopy images, and can be used to observe delicate changes in cells, tissues and organs with low photo-bleaching and photo-toxicity.

In vivo imaging of ß-cell function reveals glucose-mediated heterogeneity of ß-cell functional development.

How pancreatic ß-cells acquire function in vivo is a long-standing mystery due to the lack of technology to visualize ß-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 ß-cell in Tg (ins:Rcamp1.07) zebrafish. We reveal that the heterogeneity of ß-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 ß-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 ß-like cells in vitro.

Rapid volumetric imaging with Bessel-Beam three-photon microscopy.

Owing to its tissue-penetration ability, multi-photon fluorescence microscopy allows for the high-resolution, non-invasive imaging of deep tissue in vivo; 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 in vivo using scanning Bessel-3PM.