Three dimensional HiLo-based structured illumination for a digital scanned laser sheet microscopy (DSLM) in thick tissue imaging

Abstract

Abstract:Laser sheet based microscopy has become widely accepted as an effective active illumination method for real time three-dimensional (3D) imaging of biological tissue samples. The light sheet geometry, where the camera is oriented perpendicular to the sheet itself, provides an effective method of eliminating some of the scattered light and minimizing the sample exposure to radiation. However, residual background noise still remains, limiting the contrast and visibility of potentially interesting features in the samples. In this article, we investigate additional structuring of the illumination for improved background rejection, and propose a new technique, "3D HiLo" where we combine two HiLo images processed from orthogonal directions to improve the condition of the 3D reconstruction. We present a comparative study of conventional structured illumination based demodulation methods, namely 3Phase and HiLo with a newly implemented 3D HiLo approach and demonstrate that the latter yields superior signal-tobackground ratio in both lateral and axial dimensions, while simultaneously suppressing image processing artifacts.

Improvement of axial resolution and contrast in temporally focused widefield two-photon microscopy with structured light illumination

“…Subsequently, the optically sectioned image is obtained by combining the two with an adjusting factor so that the transition from low to high frequencies occur smoothly [19]. HiLo microscopy has been widely used in the context of background rejection for light-sheet microscopy [19,20], TFM [21,22] and depth-resolved microrheology [23]. …”

Three‐dimensional image cytometer based on widefield structured light microscopy and high‐speed remote depth scanning

“…Subsequently, the optically sectioned image is obtained by combining these two filtered images with an adjustable scalar factor so that the transition from low to high frequencies occur smoothly . HiLo microscopy has been widely used in the context of the background rejection for light‐sheet microscopy , temporally focused WF two photon microscopy , and depth‐resolved microrheology . Comparing HiLo approach with other structured light methods, most other structured light methods require taking more images.…”

Improved contrast in inverted selective plane illumination microscopy of thick tissues using confocal detection and structured illumination

“…Structured illumination (SI) has long been used to obtain optical sectioning in wide-field microscopy by projecting a single-spatial-frequency grid pattern onto the focus plane at multiple phase shifts [21], with higher frequencies providing better optical sectioning. Using SI in addition to LSFM can further lower the contribution of scattered photons to the image background [22][23][24][25]. However, there are two limitations when using SIM.…”