Université de Bordeaux, Laboratoire Photonique Numérique et Nanosciences, UMR 5298, F-33400, Talence, France.
CNRS and Institut d'Optique, LP2N UMR 5298, F-33400, Talence, France.
Nat Methods. 2018 Jun;15(6):449-454. doi: 10.1038/s41592-018-0005-3. Epub 2018 Apr 30.
Fluorescence localization microscopy has achieved near-molecular resolution capable of revealing ultra-structures, with a broad range of applications, especially in cellular biology. However, it remains challenging to attain such resolution in three dimensions and inside biological tissues beyond the first cell layer. Here we introduce SELFI, a framework for 3D single-molecule localization within multicellular specimens and tissues. The approach relies on self-interference generated within the microscope's point spread function (PSF) to simultaneously encode equiphase and intensity fluorescence signals, which together provide the 3D position of an emitter. We combined SELFI with conventional localization microscopy to visualize F-actin 3D filament networks and reveal the spatial distribution of the transcription factor OCT4 in human induced pluripotent stem cells at depths up to 50 µm inside uncleared tissue spheroids. SELFI paves the way to nanoscale investigations of native cellular processes in intact tissues.
荧光定位显微镜已经实现了接近分子水平的分辨率，能够揭示超微结构，具有广泛的应用，特别是在细胞生物学领域。然而，在三维空间和第一层细胞以外的生物组织中实现如此高的分辨率仍然具有挑战性。在这里，我们介绍了 SELFI，这是一种在多细胞样本和组织中进行三维单分子定位的框架。该方法依赖于显微镜的点扩散函数（PSF）内产生的自干涉，同时对同相和强度荧光信号进行编码，这两者共同提供了发射器的三维位置。我们将 SELFI 与传统的定位显微镜相结合，用于可视化 F-肌动蛋白的三维细丝网络，并揭示转录因子 OCT4 在人诱导多能干细胞中的空间分布，这些细胞位于未被清除的组织球体内部深度达 50μm。SELFI 为在完整组织中对天然细胞过程进行纳米级研究铺平了道路。
