Photobleaching Imprinting Enhanced Background Rejection in Line-Scanning Temporal Focusing Microscopy

Abstract

Compared with two-photon point-scanning microscopy, two-photon temporal focusing microscopy (2pTFM) provides a parallel high-speed imaging strategy with optical sectioning capability. Owing to out-of-focus fluorescence induced by scattering, 2pTFM suffers deteriorated signal-to-background ratio (SBR) for deep imaging in turbid tissue, Here, we utilized the photobleaching property of fluorophore to eliminate out-of-focus fluorescence. According to different decay rates in different focal depth, we extract the in-focus signals out of backgrounds through time-lapse images. We analyzed the theoretical foundations of photobleaching imprinting of the line-scanning temporal focusing microscopy, simulated implementation for background rejection, and demonstrated the contrast enhancement in MCF-10A human mammary epithelial cells and cleared Thy1-YFP mouse brains. More than 50% of total background light rejection was achieved, providing higher SBR images of the MCF-10A samples and mouse brains. The photobleaching imprinting method can be easily adapted to other fluorescence dyes or proteins, which may have application in studies involving relatively large and nontransparent organisms.

High-Throughput, Low Background, and Wide-Field Microscopy by Flat-Field Photobleaching Imprinting Microscopy

“…Photobleaching imprinting microscopy (PIM) is a computational imaging method based on the principle that the photobleaching rate of a fluorophore is proportional to the local illumination power density. It utilizes two sets of a priori information to achieve optical sectioning and improve the SBR. − One is the photobleaching property of the fluorophore itself, and the second is the illumination pattern of the imaging system. Different from DM, where the PSF of the system needs to be calibrated in advance, PIM is based on fitting higher-order components from a series of time-lapse images.…”