Department of Immunology, Medical University of Warsaw, 02-097 Warsaw, Poland.
The Doctoral School of the Medical University of Warsaw, Medical University of Warsaw, 02-097 Warsaw, Poland.
Int J Mol Sci. 2021 Jun 23;22(13):6730. doi: 10.3390/ijms22136730.
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.
超分辨率显微镜 (SRM) 技术的快速发展为在纳米尺度上检查细胞和组织细节开辟了新途径。由于与特定标记方法的兼容性、体内成像以及相对容易的样品制备，SRM 似乎是一种替代繁琐的电子显微镜技术的有价值的方法。然而，SRM 并非没有缺点，最明显的缺点是荧光信号的快速猝灭、对球差和光散射的敏感性，这些通常限制了成像深度至几微米。最近提出的和经过稳健优化的一系列组织光学透明化 (TOC) 技术使生物样本变得透明，这大大增加了可用于成像而不会损失分辨率的组织厚度。因此，SRM 和 TOC 是自然协同的技术，这些技术的适当结合可能会立即以纳米分辨率揭示整个器官的三维结构。因此，已经开始努力引入大规模体积 SRM；在这篇综述中，我们讨论了在 SRM 样品制备过程中可能有利的 TOC 方法。因此，特别强调了那些增强荧光强度保持、提供分子探针均匀分布以及大大降低球差的 TOC 方法。最后，我们回顾了成功应用于研究生物系统的 SRM 和 TOC 的例子。
