We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to È2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method-termed photoactivated localization microscopy-to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.

Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, too small, or occur too rapidly to see clearly with existing tools. We crafted ultra-thin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at sub-second intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and complexity of living systems.

Cell adhesions to the extracellular matrix (ECM) are necessary for morphogenesis, immunity and wound healing 1, 2. Focal adhesions are multifunctional organelles that mediate cell-ECM adhesion, force transmission, cytoskeletal regulation and signalling1 -3. Focal adhesions consist of a complex network 4 of trans-plasma-membrane integrins and cytoplasmic proteins that form a <200-nm plaque5 , 6 linking the ECM to the actin cytoskeleton. The complexity of focal adhesion composition and dynamics implicate an intricate molecular machine7 ,8 . However, focal adhesion molecular architecture remains unknown. Here we used three-dimensional super-resolution fluorescence microscopy (interferometric photoactivated localization microscopy) 9 to map nanoscale protein organization in focal adhesions. Our results reveal that integrins and actin are vertically separated by a ~40-nm focal adhesion core region consisting of multiple protein-specific strata: a membrane-apposed integrin signalling layer containing integrin cytoplasmic tails, focal adhesion kinase and paxillin; an intermediate force-transduction layer containing talin and vinculin; and an uppermost actin-regulatory layer containing zyxin, vasodilator-stimulated phosphoprotein and α-actinin. By localizing amino-and carboxy-terminally tagged talins, we reveal talin's polarized orientation, indicative of a role in organizing the focal adhesion strata. The composite multilaminar protein architecture provides a molecular blueprint for understanding focal adhesion functions.Modern understanding of cellular function is founded on the revolution in the 1950s to 1970s in visualizing cellular ultrastructure by electron microscopy 10,11 . Together with the

Despite the existence of fluorescent proteins spanning the entire visual spectrum, the bulk of modern imaging experiments continue to rely on variants of the green fluorescent protein derived from Aequorea victoria. Meanwhile, a great deal of recent effort has been devoted to engineering and improving red fluorescent proteins, and relatively little attention has been given to green and yellow variants. Here we report a novel monomeric yellow-green fluorescent protein, mNeonGreen, which is derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. This fluorescent protein is the brightest monomeric green or yellow fluorescent protein yet described, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging, and is an excellent FRET acceptor for the newest generation of cyan fluorescent proteins.

All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.

Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce “miniSOG” (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.

A key challenge when imaging living cells is how to noninvasively extract the most spatiotemporal information possible. Unlike popular wide-field and confocal methods, plane-illumination microscopy limits excitation to the information-rich vicinity of the focal plane, providing effective optical sectioning and high speed while minimizing out-of-focus background and premature photobleaching. Here we used scanned Bessel beams in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets (<0.5 μm) better suited to three-dimensional (3D) subcellular imaging. As demonstrated by imaging the dynamics of mitochondria, filopodia, membrane ruffles, intracellular vesicles and mitotic chromosomes in live cells, the microscope currently offers 3D isotropic resolution down to ~0.3 μm, speeds up to nearly 200 image planes per second and the ability to noninvasively acquire hundreds of 3D data volumes from single living cells encompassing tens of thousands of image frames.

The organization of the eukaryotic cell into discrete membrane-bound organelles allows for the separation of incompatible biochemical processes, yet the activities of these organelles must be coordinated. For example, lipid metabolism is distributed between the endoplasmic reticulum (ER) for lipid synthesis, lipid droplets (LDs) for storage and transport, mitochondria and peroxisomes for β-oxidation, and lysosomes for lipid hydrolysis and recycling1–5. Organelle contacts are increasingly understood to be vital for diverse cellular functions5–8. However, the spatial and temporal organization of organelles within the cell remains poorly characterized due to the inability of fluorescence imaging-based approaches to distinguish more than a few fluorescent labels in a single image9. Here we present a systems-level analysis of the organelle interactome using a multispectral image acquisition method that overcomes the challenge of spectral overlap in the fluorescent protein palette. We employed confocal and lattice light sheet (LLS)10 instrumentation and an imaging informatics pipeline of five steps to achieve mapping of organelle numbers/volumes/speeds/positions and dynamic inter-organelle contacts in live fibroblast cells. We describe the frequency and locality of two-, three-, four-, and five-way interactions among six different membrane-bound organelles (ER, Golgi, lysosome, peroxisome, mitochondria and LD) and show how these relationships change over time. We demonstrate that each organelle has a characteristic distribution and dispersion pattern in three-dimensional space and that there is a reproducible pattern of contacts among the six organelles, impacted by microtubule and cell nutrient status. These live-cell confocal and LLS spectral-imaging approaches are applicable to any cell system expressing multiple fluorescent probes, whether in normal conditions or when cells are exposed to disturbances such as drugs, pathogens or stress. This methodology thus offers a powerful new descriptive tool and source for hypotheses about cellular organization and dynamics.