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

Abstract

Inverted selective plane illumination microscopy (iSPIM) enables fast, large field-of-view, long term imaging with compatibility with conventional sample mounting. However, the imaging quality can be deteriorated in thick tissues due to sample scattering. Three strategies have been adopted in this paper to optimize the imaging performance of iSPIM on thick tissue imaging: electronic confocal slit detection (eCSD), structured illumination (SI) and the two combined. We compared the image contrast when using SPIM, confocal SPIM (using eCSD alone), SI SPIM (using SI alone) or confocal-SI SPIM (combining both methods) on images of gelatin phantom and highly-scattering fluorescently-stained human tissue. We demonstrate that all the three methods showed remarkable contrast enhancement on both samples compared to iSPIM alone, and SI SPIM and the combined confocal-SI mode outperformed confocal SPIM in contrast enhancement. Moreover, the use of SI at high pattern frequencies outperformed confocal SPIM in terms of optical sectioning capability. However, image signal-to-noise ratio (SNR) was decreased at high pattern frequencies when imaging scattering samples with SI SPIM. By combining eCSD with SI to reduce background signal and noise, the superior optical sectioning performance of SI could be achieved while also maintaining high image SNR.

Tissue Dynamics Spectroscopic Imaging: Functional Imaging of Heterogeneous Cancer Tissue

“…The iSPIM system is constructed around a commercially available diSPIM platform (ASI) and has been described in previous publications (45,46). In brief, two immersion objectives (CTO, ASI/Special Optics, 15.3X -17.9X) are orthogonally mounted above the sample, with each at a 45° angle from the norm, enabling traditional sample preparation.…”

Spatially modulated illumination allows for light sheet fluorescence microscopy with an incoherent source and compressive sensing

“…Structuring the light in the detection plane is a powerful method to improve the axial resolution of a microscope [18] as well as its lateral resolution [19]. This concept has been adopted to increase resolution and contrast in SPIM [20][21][22][23] even beyond the diffraction limit [24].…”

Enhanced resolution 3D digital cytology and pathology with dual-view inverted selective plane illumination microscopy

“…Currently, both OTLS and LSTM are designed for singleview imaging. In contrast, a specific feature of the diSPIM design [24,25] is that the illumination and detection paths can switch roles and capture orthogonal images of the sample by turn, contributing to more isotropic resolution in 3D by fusing and deconvolving the two views. Though multi-view deconvolution has been used before to obtain isotropic resolution, it was either applied to small and transparent samples (micrometer size) [15,23,[26][27][28]; large, but sparsely labeled structures [29]; required beads embedded as registration fiduciaries [20]; or required complicated imaging configuration and sample preparation for sample rotation and multi-view acquisition [30].…”