Multicolor Three-dimensional Whole-cell Imaging 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. In this work, we have developed multicolor three-dimensional (3D) stochastic optical reconstruction microscopy (STORM) as a tool to probe molecular structures and their interactions on sub-diffraction length scales. STORM achieves sub-diffraction limit image resolution by using photoswitchable fluorescent probes to separate the spatially overlapping images of individual probes in time. Only a small subset of probes was activated at any given time, allowing us to resolve individual activated probes and determine their positions with high precision. A super-resolution image was then constructed by plotting the measured probe positions accumulated over time. With this we have generated 3D whole cell images, several micrometers thick, 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 mammalian cells and studied the spatial relationship between mitochondria and the microtubule cytoskeleton. The 3D STORM images clearly resolved the hollow mitochondria outer membrane structures obscured in conventional fluorescence images. Distinct mitochondrial morphologies were observed, ranging from thin elongated tubes to globular compartments. Interestingly, while globular mitochondria are relatively dispersed in size, from 200 nm to 1500 nm, the tubular structures are more uniform in diameter, taking a narrow distribution around 200 nm. The images also displayed several distinct interaction modes between mitochondria and microtubules. Notably, elongated mitochondria were observed to “inchworm” along microtubules with discrete attachment sites, while such an interaction mode was completely unresolvable with conventional fluorescence. Super-resolution optical microscopy techniques such as STORM promise to significantly expand the understanding of biological structures and their interactions on a molecular level.