University of Manitoba, Cancer Care Manitoba, Winnipeg, 675 McDermot Ave, R3E 0V9, Canada.
Department of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Gronostajowa 7, 30-387, Poland.
Sci Rep. 2018 Sep 18;8(1):13971. doi: 10.1038/s41598-018-32191-x.
Structured Illumination Microscopy (SIM) is a super-resolution microscopy method that has significantly advanced studies of cellular structures. It relies on projection of illumination patterns onto a fluorescently labelled biological sample. The information derived from the sample is then shifted to a detectable band, and in the process of image calculation in Fourier space the resolution is doubled. Refractive index homogeneity along the optical path is crucial to maintain a highly modulated illumination pattern necessary for high-quality SIM. This applies in particular to thick samples consisting of large cells and tissues. Surprisingly, sample mounting media for SIM have not undergone a significant evolution for almost a decade. Through identification and systematic evaluation of a number of non-hazardous, water-soluble chemical components of mounting media, we demonstrate an unprecedented improvement in SIM-image quality. Mounting solutions presented in this research are capable of reducing abundant light scattering which constitutes the limiting factor in 3D-SIM imaging of large Hodgkin's lymphoma and embryonic stem cells as well as 10 µm tissue sections. Moreover, we demonstrate usefulness of some of the media in single molecule localisation microscopy. The results presented here are of importance for standardisation of 3D-SIM data acquisition pipelines for an expanding community of users.
结构光照明显微镜（SIM）是一种超分辨率显微镜方法，它极大地推动了细胞结构的研究。它依赖于将照明图案投影到荧光标记的生物样本上。然后，从样本中获取的信息被转移到可检测的波段，并且在傅立叶空间中的图像计算过程中，分辨率提高了一倍。沿着光路的折射率均匀性对于保持高质量 SIM 所需的高度调制照明图案至关重要。这尤其适用于由大细胞和组织组成的厚样本。令人惊讶的是，用于 SIM 的样本安装介质在近十年内没有经历重大的演变。通过鉴定和系统评估安装介质中的一些非危险的水溶性化学物质成分，我们展示了 SIM 图像质量的前所未有的改善。本研究中提出的安装解决方案能够减少大量的光散射，这是大霍奇金淋巴瘤和胚胎干细胞以及 10μm 组织切片的 3D-SIM 成像的限制因素。此外，我们还证明了一些介质在单分子定位显微镜中的有用性。这里呈现的结果对于为不断扩大的用户群体的 3D-SIM 数据采集管道的标准化具有重要意义。
