Can Developments in Tissue Optical Clearing Aid Super-Resolution Microscopy Imaging?

Paweł Matryba,Jakub Gołąb,Monika Pawłowska,Jacek Tomczuk,Kacper Łukasiewicz

doi:10.3390/ijms22136730

Abstract

The rapid development of super-resolution microscopy (SRM) techniques opens new avenues to examine cell and tissue details at a nanometer scale. Due to compatibility with specific labelling approaches, in vivo imaging and the relative ease of sample preparation, SRM appears to be a valuable alternative to laborious electron microscopy techniques. SRM, however, is not free from drawbacks, with the rapid quenching of the fluorescence signal, sensitivity to spherical aberrations and light scattering that typically limits imaging depth up to few micrometers being the most pronounced ones. Recently presented and robustly optimized sets of tissue optical clearing (TOC) techniques turn biological specimens transparent, which greatly increases the tissue thickness that is available for imaging without loss of resolution. Hence, SRM and TOC are naturally synergistic techniques, and a proper combination of these might promptly reveal the three-dimensional structure of entire organs with nanometer resolution. As such, an effort to introduce large-scale volumetric SRM has already started; in this review, we discuss TOC approaches that might be favorable during the preparation of SRM samples. Thus, special emphasis is put on TOC methods that enhance the preservation of fluorescence intensity, offer the homogenous distribution of molecular probes, and vastly decrease spherical aberrations. Finally, we review examples of studies in which both SRM and TOC were successfully applied to study biological systems.

Highlights

Significant developments in microscopy instrumentation have recently pushed the field of biomedical imaging beyond numerous limits
The application of tissue optical clearing (TOC) for super-resolution microscopy (SRM) studies opens new research opportunities that already allow for the SRM imaging of entire cells and even fly brains, which might be extended to entire rodent or even primate [110] organs in the future
A successful combination of these techniques, requires in-depth knowledge regarding the limitations of particular SRM techniques and characteristic features of the TOC to be exploited

Summary

Introduction

Significant developments in microscopy instrumentation have recently pushed the field of biomedical imaging beyond numerous limits. As “nature abhors a vacuum”, this obvious limitation of the electron microscopy stimulated developments of light microscopy in a direction that enables successful imaging of nanometer-sized structures, while retaining the vast majority of its natural advantages, e.g., cell-specific staining approaches, ease of sample preparation and the possibility to perform live imaging, just to name a few. There are multiple computational techniques which increase the resolution during post-processing, such as: deconvolution, pixel reassignment in image scanning microscopy or more sophisticated image reconstruction algorithms, including those employing machine learning. Another approach to resolve microscopical structures in the specimen is by bypassing the diffraction limit with expansion microscopy (ExM), which might be listed as either a TOC (due to resultant transparency) or SRM technique

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