Single-scan HiLo with line-illumination strategy for optical section imaging of thick tissues.

Abstract

Optical sectioning has been widely employed for inhibiting out-of-focus backgrounds in three-dimensional (3D) imaging of biological samples. However, point scanning imaging or multiple acquisitions for wide-field optical sectioning in epi-illumination microscopy remains time-consuming for large-scale imaging. In this paper, we propose a single-scan optical sectioning method based on the hybrid illumination (HiLo) algorithm with a line-illumination strategy. Our method combines HiLo background inhibition with confocal slit detection. It thereby offers a higher optical sectioning capability than wide-field HiLo and line-confocal imaging without extra modulation and multiple data acquisition. To demonstrate the optical-sectioning capability of our system, we imaged a thin fluorescent plane and different fluorescence-labeled mouse tissue. Our method shows an excellent background inhibition in thick tissue and thus potentially provides an alternative tool for 3D imaging of large-scale biological tissue.