HiLo Based Line Scanning Temporal Focusing Microscopy for High-Speed, Deep Tissue Imaging.

Ruheng Shi,Yuanlong Zhang,Tiankuang Zhou,Lingjie Kong

doi:10.3390/membranes11080634

Ruheng Shi, Yuanlong Zhang + Show 2 more

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https://doi.org/10.3390/membranes11080634

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Abstract

High-speed, optical-sectioning imaging is highly desired in biomedical studies, as most bio-structures and bio-dynamics are in three-dimensions. Compared to point-scanning techniques, line scanning temporal focusing microscopy (LSTFM) is a promising method that can achieve high temporal resolution while maintaining a deep penetration depth. However, the contrast and axial confinement would still be deteriorated in scattering tissue imaging. Here, we propose a HiLo-based LSTFM, utilizing structured illumination to inhibit the fluorescence background and, thus, enhance the image contrast and axial confinement in deep imaging. We demonstrate the superiority of our method by performing volumetric imaging of neurons and dynamical imaging of microglia in mouse brains in vivo.

Highlights

Optical-sectioning capability is highly desired in biomedical imaging, as biological cells and tissues are generally in three-dimensions
We demonstrate the superiority of our method by volumetric imaging of neurons and dynamical imaging of microglia in mouse brains in vivo
To depress tissue scattering and ensure high temporal resolution in wide-field, opticalsectioning imaging, here we propose a HiLo-based line scanning temporal focusing microscopy (LSTFM), which can enhance the image contrast and axial confinement

Summary

Introduction

Optical-sectioning capability is highly desired in biomedical imaging, as biological cells and tissues are generally in three-dimensions. Multiphoton microscopy was developed for deep tissue imaging, which achieves optical-sectioning through nonlinear localized excitation and deeper penetration, benefiting from the lesser scattering of longer excitation wavelengths [4,5,6,7]. As point-scanning methods, these techniques usually have limitations in temporal resolutions To solve this problem, spinning-disk confocal microscopy and multifocal two photon microscopy were proposed, which improve the temporal resolution by generating multifocal points through lenslet arrays or spatial light modulators (SLM), which, increase the complexity of the microscope setups [8,9,10,11]. Scattering of emitted fluorescence would introduce cross-talk, and sacrifice the image contrast

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