Multi-plane, wide-field fluorescent microscopy for biodynamic imaging in vivo.

Ruheng Shi,Yuanlong Zhang,Hao Xie,Lingjie Kong,Cheng Jin,Qionghai Dai,Xinyang Li

doi:10.1364/boe.10.006625

Abstract

Wide-field fluorescent microscopy (WFFM) is widely employed in biomedical studies, due to its inherent advantages in high-speed imaging of biological dynamics noninvasively and specifically. However, WFFM suffers from the loss of axial resolution and the poor resistance to light scattering in deep tissue imaging. Here we propose a novel WFFM which has the capability in optical sectioning and volumetric imaging. We perform speckle illumination with a digital-micromirror-device for optical sectioning and employ an electrically tunable lens for defocusing modulation so as to quickly switch the image planes. We demonstrate its applications in multi-plane, wide-field imaging of biological dynamics in both zebrafish brains and mouse brains in vivo.

Highlights

Large-scale imaging of biological dynamics, at high spatio-temporal resolutions over extended three-dimensional (3D) volumes, is highly desired in biomedical studies, especially in the study of system biology
All procedures involving animals were approved by the Animal Care and Use Committees of Tsinghua University
We propose a new multi-plane wide-field fluorescent microscopy (WFFM), capable of optical sectioning and volumetric imaging

Summary

Introduction

Large-scale imaging of biological dynamics, at high spatio-temporal resolutions over extended three-dimensional (3D) volumes, is highly desired in biomedical studies, especially in the study of system biology. Neil et al firstly proposed a structured illumination based optical sectioning microscopy (SIM). In this method, three images, recorded at different grid positions with each being laterally translated by a third of the grid period, are computationally combined to calculate an optically sectioned image [7]. SIM is highly susceptible to sample motions [8] To address this problem, HiLo microscopy was proposed (“Hi” and “Lo” for the high and low spatial frequency components, respectively) [8,9,10]. In HiLo microscopy, only two images were required to achieve optical-sectioning: one for structured-illumination image; the other one for conventional uniform-illumination image Combining these two images will allow us to recover the optical sectioning image at full frequency bandwidth. DMD is a fast, bi-stable spatial light modulator consisting of an array of movable micro-mirrors, where each micro-mirror can be individually rotated ± 12 degree, corresponding to an “on” or “off” state during the illumination

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Conclusion