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


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. We performed 3D localization 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 resolved mitochondrial morphologies as well as mitochondria-microtubule contacts that were obscured in conventional fluorescence images.

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Figure 1: Synthesis of covalently linked photoswitchable activator-reporter pairs.
Figure 2: 3D STORM images of mitochondria in a whole mammalian cell.
Figure 3: Two-color 3D STORM images of mitochondria and microtubules.
Figure 4: Two-color STORM images showing interactions between tubular mitochondria (magenta) and microtubules (green) in three BS-C-1 cells.


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This work is supported by in part by the US National Institutes of Health (to X.Z.). X.Z. is a Howard Hughes Medical Institute Investigator.

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Correspondence to Xiaowei Zhuang.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1-7, Supplementary Data and Supplementary Methods (PDF 2596 kb)

Supplementary Video 1

A 3D presentation of the mitochondria shown in Figure 2a. Different viewing perspectives of the 3D image are presented by rotating the image. In both x-y and x-z viewing perspectives, consecutive 100 nm thick sections are also shown. The x-y sections start from the bottom of the cells toward top and the x-z sections start from the upper part of the image. Scale bar: 3 μm. (MOV 2526 kb)

Supplementary Video 2

A 3D presentation of the mitochondrion and microtubules shown in Figure 3e. Different viewing perspectives of the 3D STORM image (left) are presented by rotating the image. A 3D surface rendered model (right) is also shown to facilitate understanding the spatial relationship between the mitochondrion and microtubules. The model is created by manually tracing the six microtubules and the surface of the mitochondrion in each of the 20 nm thick z sections (30 sections in total), a method that is commonly used in modeling of electron microscopy tomography data. The individual surface segments were then 3D rendered by using the ImageSurfer software ( (MOV 2007 kb)

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Huang, B., Jones, S., Brandenburg, B. et al. Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat Methods 5, 1047–1052 (2008).

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