Nanoscale subcellular architecture revealed by multicolor three-dimensional salvaged fluorescence imaging

Abstract

Combining the molecular specificity of fluorescent probes with three-dimensional (3D) imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a super-resolution light microscope that enables simultaneous multicolor imaging of mammalian cells at 5-10 nm single-molecule localization precision in 3D. We show its power for cell biology research with fluorescence images that resolve the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Light microscopy of proteins in their ultrastructural context

“…While the ER did not show a characteristic NHS-ester pan-staining that would make it directly identifiable, we could visualize it by overexpressing ER-membrane localized Sec61β-GFP and immunolabeling with an anti-GFP antibody (Fig. 6b–e ), a strategy which we have previously employed successfully in optical super-resolution studies 1 , 26 . pan-ExM clearly resolves the two sides of ER tubules using a standard confocal microscope (Fig.…”

“…Fluorescence microscopy has transformed the field of cell biology through its exceptional contrast and high specificity of labeling. With the advent of super-resolution microscopy, the three-dimensional (3D) distribution of specific proteins of interest can be imaged at spatial resolutions down to ~10 nm, revealing their astounding sub-cellular organization at the nanoscale 1 . Fluorescence microscopy, unlike electron microscopy, is however fundamentally prohibited from resolving the ultrastructural context of the cell: the smallest fluorescent labels, organic dyes, are ~1 nm in diameter, a size comparable to the distance between proteins in the densely crowded cellular interior 2 .…”

It Started With a Kiss: Monitoring Organelle Interactions and Identifying Membrane Contact Site Components in Plants

“…Research in non-plant systems is demonstrating further potential for super-resolution monitoring of specific MCS proteins. For example, SIM and dSTORM (direct Stochastic Optical Reconstruction Microscopy) were used to visualize clusters of MIRO1 and MIRO2 proteins corresponding to ER-mitochondrial contact sites in mammalian cells (Modi et al, 2019), and ER-PM contact sites were recently investigated using multicolor three-dimensional salvaged fluorescence imaging (Zhang et al, 2020).…”

Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded (POSSIBLE) microscopy for ultra-superresolution imaging

“…This is largely eluded due to the diffraction of light which sets a lower bound on the resolution-limit [1,2]. However, recent microscopy techniques such as, STED [3], fPALM [4], STORM [5], PALM [6], SIM [7] GSDIM [8] SOFI [9], PAINT [10,11], SMILE [12,13], MINFLUX [14] and others techniques [15][16][17][18][19][20][21][22][23] have surpassed this limit [1,2]. The above techniques are far-from achieving truly molecular-scale resolution and thus are incapable of functional imaging with ultra-high-resolution (preferably in the range of single molecule length-scales).…”