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

Chaowei Zhuang,Yuanlong Zhang,Hao Xie,Qionghai Dai,Xinyang Li,Lingjie Kong

doi:10.3389/fchem.2020.618131

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.

Highlights

Two-photon fluorescence microscopy has become a powerful tool in biomedical deep-tissue imaging for its advantages in high spatial resolution, deep penetration, and optical sectioning capability (Denk et al, 1990; Zipfel et al, 2003; Helmchen and Denk, 2005)
We integrate the intensity in each fluorescent slice and the results indicate that the signal-to-background ratio (SBR) of the linescanning temporal focusing microscopy (LTFM) and LTFM-photobleaching imprinting microscopy (PIM) are obtained 0.4 for LTFM and 2.3 for LTFMPIM, demonstrates that the LTFM-PIM (l = 1) could suppress nearly five times the out-of-focus fluorescence (Figure 3)
The results demonstrate that the out-of-focus fluorescence is almost eliminated by the PIM technology, improving the SBR effectively

Summary

Introduction

Two-photon fluorescence microscopy has become a powerful tool in biomedical deep-tissue imaging for its advantages in high spatial resolution, deep penetration, and optical sectioning capability (Denk et al, 1990; Zipfel et al, 2003; Helmchen and Denk, 2005). Due to the point scanning strategy, the imaging speed of the two-photon microscopy is limited by the inertia of mechanical scanners and the laser repetition rate (Kong et al, 2015) To solve this problem, temporal focusing microscopy (TFM) has reportedly achieved parallel excitation in samples. WTFM has a larger illuminated area, the two-photon excitation efficiency is decreased severely, according to power-law dependence on light intensity in multiphoton processes (Dana et al, 2013) Benefiting from both spatial focusing and pulse width modulation, LTFM has superior performance in imaging speed, field-of-view, depth, and axial confinement, so it has been shown to have wide applications in large-scale imaging of biological dynamics (Li et al, 2017; Park et al, 2017).

Methods

Results

Discussion

Conclusion