Department of Nanophotonics, Ultrafast Bio- and Nanophotonics Group, INL - International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330 Braga, Portugal.
Faculty of Medicine and Health Technology, BioMediTech, Tampere University, 33014 Tampere, Finland.
Biochim Biophys Acta Mol Cell Res. 2019 Dec;1866(12):118530. doi: 10.1016/j.bbamcr.2019.118530. Epub 2019 Aug 12.
Phasor-assisted Metal Induced Energy Transfer-Fluorescence Lifetime Imaging Microscopy (MIET-FLIM) nanoscopy is introduced as a powerful tool for functional cell biology research. Thin metal substrates can be used to obtain axial super-resolution via nanoscale distance-dependent MIET from fluorescent dyes towards a nearby metal layer, thereby creating fluorescence lifetime contrast between dyes located at different nanoscale distance from the metal. Such data can be used to achieve axially super-resolved microscopy images, a process known as MIET-FLIM nanoscopy. Suitability of the phasor approach in MIET-FLIM nanoscopy is first demonstrated using nanopatterned substrates, and furthermore applied to characterize the distance distribution of the epithelial basal membrane of a biological cell from the gold substrate. The phasor plot of an entire cell can be used to characterize the full Förster resonance energy transfer (FRET) trajectory as a large distance heterogeneity within the sensing range of about 100 nm from the metal surface is present due to the extended shape of cell with curvatures. In contrast, the different proteins of nuclear lamina show strong confinement close to the nuclear envelope in nanoscale. We find the lamin B layer resides in average at shorter distances from the gold surface compared to the lamin A/C layer located in more extended ranges. This and the observed heterogeneity of the protein layer thicknesses suggests that A- and B-type lamins form distinct networks in the nuclear lamina. Our results provide detailed insights for the study of the different roles of lamin proteins in chromatin tethering and nuclear mechanics.
相幅辅助金属诱导能量转移-荧光寿命成像显微镜（MIET-FLIM）纳米显微镜被引入作为功能细胞生物学研究的强大工具。薄金属衬底可用于通过纳米级距离依赖的 MIET 从荧光染料获得轴向超分辨率，从而在位于不同纳米级距离的染料之间产生荧光寿命对比金属层。可以使用此类数据来获得轴向超分辨显微镜图像，这一过程称为 MIET-FLIM 纳米显微镜。首先使用图案化衬底证明了相幅方法在 MIET-FLIM 纳米显微镜中的适用性，并且进一步应用于从金衬底上表征生物细胞的上皮基膜的距离分布。整个细胞的相幅图可用于表征整个Förster 共振能量转移（FRET）轨迹，因为由于细胞的扩展形状和曲率，在距金属表面约 100nm 的感应范围内存在大的距离异质性。相比之下，核层的不同蛋白质在纳米级上表现出靠近核膜的强限制。我们发现与位于更扩展范围内的 lamin A/C 层相比，lamin B 层在平均情况下更靠近金表面。这种和观察到的蛋白质层厚度异质性表明 A-和 B 型 lamin 在核层中形成不同的网络。我们的结果为研究 lamin 蛋白在染色质固定和核力学中的不同作用提供了详细的见解。
