Recent advances in far-field fluorescence microscopy have led to substantial improvements in image resolution, achieving a near-molecular resolution of 20 -30 nm in the two lateral dimensions. Three-dimensional (3D) nanoscale-resolution imaging, however, remains a challenge. Here, we demonstrate 3D stochastic optical reconstruction microscopy (STORM) by determining both axial and lateral positions of individual fluorophores with nanometer accuracy using optical astigmatism. Iterative, stochastic activation of photo-switchable probes enables high-precision 3D localization of each probe and thus the construction of a 3D image without scanning the sample. Using this approach, we achieved an image resolution of 20 -30 nm in the lateral dimensions and 50 -60 nm in the axial dimension. This development allowed us to resolve the 3D morphology of nanoscopic cellular structures.Due to its non-invasive nature and multi-color capability, far-field optical microscopy offers three-dimensional (3D) imaging of biological specimens with minimal perturbation and biomolecular specificity when combined with fluorescent labeling. These advantages make fluorescence microscopy one of the most widely used imaging methods in biology. The diffraction barrier, however, limits the imaging resolution of conventional light microscopy to 200 -300 nm in the lateral dimensions, leaving many intracellular organelles and molecular structures unresolvable. Recently, the diffraction limit has been surpassed and lateral imaging resolutions of 20 -50 nm have been achieved by several "super-resolution" far-field microscopy techniques, including stimulated emission depletion (STED) and its related RESOLFT microscopy (1,2), saturated structured illumination microscopy (SSIM) (3), STORM (4,5), photoactivated localization microscopy (PALM) (6,7) and other methods using similar principles (8-10).While these techniques have improved 2D image resolution, resolving most organelles and cellular structures requires high-resolution imaging in all three dimensions. Threedimensional fluorescence imaging is most commonly performed using confocal or multiphoton microscopy, the axial resolution of which is typically in the range of 500 -800 nm, two to three times worse than the lateral resolution (11,12). The axial imaging resolution can be improved to roughly 100 nm by 4Pi and I 5 M microscopy (13)(14)(15) resolution as high as 30 -50 nm has been obtained by employing stimulated emission depletion along the axial direction using the 4Pi illumination geometry, but the same scheme does not provide super resolution in the lateral dimensions (1).Here we demonstrate 3D STORM imaging with a spatial resolution that is 10 times better than the diffraction limit in all three dimensions without invoking sample or optical beam scanning. STORM and PALM rely on single-molecule detection (16) and exploit the photoswitchable nature of certain fluorophores to temporally separate the otherwise spatially overlapping images of numerous molecules, thereby allowing the high-pre...

SUMMARY The spatiotemporal organization and dynamics of chromatin play critical roles in regulating genome function. However, visualizing specific, endogenous genomic loci remains challenging in living cells. Here, we demonstrate such an imaging technique by repurposing the bacterial CRISPR/Cas system. Using an EGFP-tagged endonuclease-deficient Cas9 protein and a structurally optimized small guide (sg) RNA, we show robust imaging of repetitive elements in telomeres and coding genes in living cells. Furthermore, an array of sgRNAs tiling along the target locus enables the visualization of non-repetitive genomic sequences. Using this method, we have studied telomere dynamics during elongation or disruption, the subnuclear localization of the MUC4 loci, the cohesion of replicated MUC4 loci on sister chromatids, and their dynamic behaviors during mitosis. This CRISPR imaging tool has potential to significantly improve the capacity to study the conformation and dynamics of native chromosomes in living human cells.

Recent advances in far-field optical nanoscopy have enabled fluorescence imaging with spatial resolution of 20 -50 nm. Multicolor super-resolution imaging, however, remains challenging. In this report, we introduce a family of photo-switchable fluorescent probes and demonstrate multicolor stochastic optical reconstruction microscopy (STORM). Each probe consists of a photo-switchable "reporter" fluorophore that can be cycled between fluorescent and dark states, and an "activator" that facilitates photo-activation of the reporter. Combinatorial pairing of reporters and activators allows the creation of probes with many distinct colors. Iterative, color-specific activation of sparse subsets of these probes allows their localization with nanometer accuracy, enabling the construction of a super-resolution STORM image. Using this approach, we demonstrate multi-color imaging of DNA model samples and mammalian cells with 20 -30 nm resolution. This technique will facilitate direct visualization of molecular interactions at the nanometer scale.As one of the most versatile imaging modalities in biology, fluorescence microscopy allows noninvasive imaging of cells and tissues with molecular specificity. The availability of fluorescent probes in many colors and the ability to label specific gene products enable visualization of molecular interactions in biological samples. However, the spatial resolution of optical microscopy, classically limited by the diffraction of light to ∼ 300 nm, is inconveniently situated 1 -2 orders of magnitude above the typical molecular length scales in cells. Various "super-resolution" optical imaging techniques have been developed to overcome this limit (1,2). Among these methods, stimulated emission depletion microscopy (STED) and reversible saturable optically linear fluorescent transition (RESOLFT) techniques (2,3), saturated structured illumination microscopy (SSIM) (4), stochastic optical reconstruction microscopy (STORM) (5) and photoactivated localization microscopy (PALM) (6,7) have achieved 20 -50 nm resolution in the far field and promise to preserve the inherent noninvasive imaging capability of optical microscopy. In certain cases, binding kinetics or translational motions of individual molecules have also been used to paint high-resolution structures in cells (8)(9)(10) (17), localization accuracies as high as 0.1 -1 nm can be achieved for bright fluorescent or scattering objects (22)(23)(24)(25). By employing photo-switchable probes, the fluorescence emission profile of individual fluorophores can be modulated in time such that only an optically resolvable subset of fluorophores are activated at any moment, allowing their localization with high accuracy. Over the course of multiple activation cycles, the positions of numerous fluorophores are determined and used to construct a high-resolution STORM image (5-7). The development of multicolor STORM thus depends on the construction of bright, photoswitchable probes with distinct colors.To search for chromatically distinguishab...