Sontag Emily M
Department of Biological Sciences, Marquette University, Milwaukee, WI, USA.
Methods Mol Biol. 2025;2958:83-98. doi: 10.1007/978-1-0716-4714-1_6.
The ability to image biological samples beyond the diffraction limit (super-resolution) has opened a world of new discoveries in molecular biology. Super-resolution fluorescence microscopy requires specific fluorophores that often require immunostaining with labeled antibodies to achieve the necessary parameters for imaging. Budding yeast Saccharomyces cerevisiae is a common model organism used in cell biology, genetics, and molecular biology. However, yeast are difficult to immunostain due to the cell wall that hinders antibody penetration. In this chapter, I describe the methods we use for immunostaining yeast to visualize the nuclear membrane with the necessary efficiency for imaging by super-resolution fluorescence microscopy. I discuss approaches and optimizations to prepare samples for different imaging techniques including Structured Illumination Microscopy (SIM) and Stochastic Optical Reconstruction Microscopy (STORM). Super-resolution imaging can then be used to determine the precise location of proteins within the nuclear membrane, or if perinuclear proteins reside inside or outside the nucleus. The methods can be extended to other techniques requiring antibody penetration into yeast cells.
超越衍射极限对生物样本进行成像（超分辨率成像）的能力开启了分子生物学领域新发现的大门。超分辨率荧光显微镜需要特定的荧光团，通常需要用标记抗体进行免疫染色以达到成像所需的参数。出芽酵母酿酒酵母是细胞生物学、遗传学和分子生物学中常用的模式生物。然而，由于细胞壁阻碍抗体渗透，酵母很难进行免疫染色。在本章中，我将描述我们用于对酵母进行免疫染色的方法，以便通过超分辨率荧光显微镜以必要的效率观察核膜。我将讨论为不同成像技术（包括结构光照显微镜（SIM）和随机光学重建显微镜（STORM））制备样本的方法和优化措施。然后，超分辨率成像可用于确定蛋白质在核膜内的精确位置，或者核周蛋白是位于细胞核内部还是外部。这些方法可以扩展到其他需要抗体渗透到酵母细胞中的技术。
