Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution

Abstract

The ability to directly visualize nanoscopic cellular structures and their spatial relationship in all three dimensions will greatly enhance our understanding of molecular processes in cells. Here, we demonstrated multicolor three-dimensional (3D) stochastic optical reconstruction microscopy (STORM) as a tool to quantitatively probe cellular structures and their interactions. To facilitate STORM imaging, we generated photoswitchable probes in several distinct colors by covalently linking a photoswitchable cyanine reporter and an activator molecule to assist bioconjugation. 3D localization was performed in conjunction with focal plane scanning and correction for refractive index mismatch to obtain whole-cell images with a spatial resolution of 20–30 nm and 60–70 nm in the lateral and axial dimensions, respectively. Using this approach, we imaged the entire mitochondrial network in fixed monkey kidney BS-C-1 cells, and studied the spatial relationship between mitochondria and microtubules. The 3D STORM images revealed mitochondrial morphologies as well as mitochondria-microtubule contacts that were obscured in conventional fluorescence images.

Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus

“…The DH-PSF has an operational depth of field of 2 μm, limited by the PSF design itself. If the target structure spans an axial range greater than 2 μm, the sample would need to be scanned axially (52) to use the DH-PSF for 3D SPRAIPAINT. Aberrations associated with imaging deeply into mismatched media would also need to be considered.…”

“…Because we place the focal plane of the microscope approximately 300 nm from the glass-water interface and image single molecules within 300 nm of this focal plane, the focal shift is mostly linear (52,57). We therefore use a z correction factor of n water ∕n oil ¼ 0.88 to convert measured z positions to true z positions, and this correction factor is accurate enough to produce circular membrane cross sections in our 3D data ( Fig.…”

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

“…These probes can be activated in sparse numbers over time such that their images are optically resolvable and can be precisely localized 4 . These techniques have been exploited to image sub-cellular structures [5][6][7] , visualize protein (co)-organization [8][9][10] and quantify protein stoichiometry [11][12][13][14][15] .…”

Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI)

“…Recently, superresolution microscopy at 11 Hz has been demonstrated by using SIM, achieving a 2-fold increased lateral resolution (14). All super-resolution methods are capable of enhancing resolution in 3D, but often at the expense of major technical demands or modifications to the microscope (15)(16)(17).…”