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

Bihe Hu,Daniel Bolus,J Quincy Brown

doi:10.1364/boe.8.005546

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.

Highlights

Fluorescence microscopy with optical sectioning ability provides unique benefits of high contrast, high molecular sensitivity, and depth information, which has proved useful in cell study [1], developmental biology [2], neuroscience [3], as well as clinical applications [4,5,6]
structured illumination (SI), and the combined mode all exhibited superior contrast enhancement over SPIM mode alone in gelatin phantom (Fig. 4), especially SI SPIM and confocal-SI SPIM, which were about 2.8 times better compared to SPIM alone
We found that the combination of electronic confocal slit detection (eCSD) and SI dramatically improved illumination pattern modulation depth at high pattern frequencies compared to SI SPIM alone, helping to recover more signal at higher frequencies

Summary

Introduction

Fluorescence microscopy with optical sectioning ability provides unique benefits of high contrast, high molecular sensitivity, and depth information, which has proved useful in cell study [1], developmental biology [2], neuroscience [3], as well as clinical applications [4,5,6]. Compared to some traditional configurations of point detection approaches such as CLSM or 2P microscopy where a PMT is usually used, better signal to noise ratio (SNR) can be achieved at similar frame rates due to optically efficient widefield detection and the use of high QE, low-noise wide-field cameras [1]. All these features strongly benefit imaging of thick samples, such as embryos, brain tissue, and diagnostic cancer biopsies

Results

Discussion

Conclusion