Determinants of synapse diversity revealed by super-resolution quantal transmission and active zone imaging.

Neural circuit function depends on the pattern of synaptic connections between neurons and the strength of those connections. Synaptic strength is determined by both postsynaptic sensitivity to neurotransmitter and the presynaptic probability of action potential evoked transmitter release (Pr). Whereas morphology and neurotransmitter receptor number indicate postsynaptic sensitivity, presynaptic indicators and the mechanism that sets Pr remain to be defined. To address this, we developed QuaSOR, a super-resolution method for determining Pr from quantal synaptic transmission imaging at hundreds of glutamatergic synapses at a time. We mapped the Pr onto super-resolution 3D molecular reconstructions of the presynaptic active zones (AZs) of the same synapses at the Drosophila larval neuromuscular junction (NMJ). We find that Pr varies greatly between synapses made by a single axon, quantify the contribution of key AZ proteins to Pr diversity and find that one of these, Complexin, suppresses spontaneous and evoked transmission differentially, thereby generating a spatial and quantitative mismatch between release modes. Transmission is thus regulated by the balance and nanoscale distribution of release-enhancing and suppressing presynaptic proteins to generate high signal-to-noise evoked transmission.

Graphene-Enabled, Spatially Controlled Electroporation of Adherent Cells for Live-Cell Super-resolution Microscopy.

The incorporation of exogenous molecules into live cells is essential for both biological research and therapeutic applications. In particular, for the emerging field of super-resolution microscopy of live mammalian cells, it remains a challenge to deliver tailored, often cell-impermeable, fluorescent probes into live cells for target labeling. Here, utilizing the outstanding mechanical, electrical, and optical properties of graphene, we report a facile approach that enables both high-throughput delivery of fluorescent probes into adherent mammalian cells and in situ super-resolution microscopy on the same device. Approximately 90% delivery efficiencies are achieved for free dyes and dye-tagged affinity probes, short peptides, and whole antibodies, thus enabling high-quality super-resolution microscopy. Moreover, we demonstrate good spatiotemporal controls, which, in combination with the ready patternability of graphene, allow for the spatially selective delivery of two different probes for cells at different locations on the same substrate.

Publisher Correction: Oblique-plane single-molecule localization microscopy for tissues and small intact animals.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Switchable Solvatochromic Probes for Live-Cell Super-resolution Imaging of Plasma Membrane Organization.

Visualization of the nanoscale organization of cell membranes remains challenging because of the lack of appropriate fluorescent probes. Herein, we introduce a new design concept for super-resolution microscopy probes that combines specific membrane targeting, on/off switching, and environment sensing functions. A functionalization strategy for solvatochromic dye Nile Red that improves its photostability is presented. The dye is grafted to a newly developed membrane-targeting moiety composed of a sulfonate group and an alkyl chain of varied lengths. While the long-chain probe with strong membrane binding, NR12A, is suitable for conventional microscopy, the short-chain probe NR4A, owing to the reversible binding, enables first nanoscale cartography of the lipid order exclusively at the surface of live cells. The latter probe reveals the presence of nanoscopic protrusions and invaginations of lower lipid order in plasma membranes, suggesting a subtle connection between membrane morphology and lipid organization.

Oblique-plane single-molecule localization microscopy for tissues and small intact animals.

Single-molecule localization microscopy (SMLM), while well established for cultured cells, is not yet fully compatible with tissue-scale samples. We introduce single-molecule oblique-plane microscopy (obSTORM), which by directly imaging oblique sections of samples with oblique light-sheet illumination offers a deep and volumetric SMLM platform that is convenient for standard tissue samples and small intact animals. We demonstrate super-resolution imaging at depths of up to 66 µm for cells, Caenorhabditis elegans gonads, Drosophila melanogaster larval brain, mouse retina and brain sections, and whole stickleback fish.

Information-rich localization microscopy through machine learning.

Recent years have witnessed the development of single-molecule localization microscopy as a generic tool for sampling diverse biologically relevant information at the super-resolution level. While current approaches often rely on the target-specific alteration of the point spread function to encode the multidimensional contents of single fluorophores, the details of the point spread function in an unmodified microscope already contain rich information. Here we introduce a data-driven approach in which artificial neural networks are trained to make a direct link between an experimental point spread function image and its underlying, multidimensional parameters, and compare results with alternative approaches based on maximum likelihood estimation. To demonstrate this concept in real systems, we decipher in fixed cells both the colors and the axial positions of single molecules in regular localization microscopy data.

Optical characterization of surface adlayers and their compositional demixing at the nanoscale.

Under ambient conditions, the behavior of a solid surface is often dominated by a molecularly thin adsorbed layer (adlayer) of small molecules. Here we develop an optical approach to unveil the nanoscale structure and composition of small-molecule adlayers on glass surfaces through spectrally resolved super-resolution microscopy. By recording the images and emission spectra of millions of individual solvatochromic molecules that turn fluorescent in the adlayer phase, we obtain ~30 nm spatial resolution and achieve concurrent measurement of local polarity. This allows us to establish that the adlayer dimensionality gradually increases through a sequence of 0D (nanodroplets), 1D (nano-lines), and 2D (films) for liquids of increasing polarity. Moreover, we find that in adlayers, a solution of two miscible liquids spontaneously demixes into nanodroplets of different compositions that correlate strongly with droplet size and location. We thus reveal unexpectedly rich structural and compositional behaviors of surface adlayers at the nanoscale.

Spectrally Resolved and Functional Super-resolution Microscopy via Ultrahigh-Throughput Single-Molecule Spectroscopy.

As an elegant integration of the spatial and temporal dimensions of single-molecule fluorescence, single-molecule localization microscopy (SMLM) overcomes the diffraction-limited resolution barrier of optical microscopy by localizing single molecules that stochastically switch between fluorescent and dark states over time. While this type of super-resolution microscopy (SRM) technique readily achieves remarkable spatial resolutions of ∼10 nm, it typically provides no spectral information. Meanwhile, current scanning-based single-location approaches for mapping the positions and spectra of single molecules are limited by low throughput and are difficult to apply to densely labeled (bio)samples. In this Account, we summarize the rationale, design, and results of our recent efforts toward the integration of the spectral dimension of single-molecule fluorescence with SMLM to achieve spectrally resolved SMLM (SR-SMLM) and functional SRM ( f-SRM). By developing a wide-field scheme for spectral measurement and implementing single-molecule fluorescence on-off switching typical of SMLM, we first showed that in densely labeled (bio)samples it is possible to record the fluorescence spectra and positions of millions of single molecules synchronously within minutes, giving rise to ultrahigh-throughput single-molecule spectroscopy and SR-SMLM. This allowed us to first show statistically that for many dyes, single molecules of the same species exhibit near identical emission in fixed cells. This narrow distribution of emission wavelengths, which contrasts markedly with previous results at solid surfaces, allowed us to unambiguously identify single molecules of spectrally similar dyes. Crosstalk-free, multiplexed SRM was thus achieved for four dyes that were merely 10 nm apart in emission spectrum, with the three-dimensional SRM images of all four dyes being automatically aligned within one image channel. The ability to incorporate single-molecule fluorescence measurement with SMLM was next utilized to achieve f-SRM. By encoding functional information into the spectral responses of environment-sensing fluorescent probes, f-SRM transcends the structural information provided by typical SRM techniques and reveals the spatiotemporal distribution of physicochemical parameters with single-molecule sensitivity and nanoscale spatial resolution. As one example, by employing the solvatochromic dye Nile Red to sense local chemical polarity, we revealed nanoscale heterogeneity in the membranes of live mammalian cells. This enabled us to unveil substantial polarity differences between the plasma membrane and the membranes of nanoscale intracellular organelles, a result we determined to be due to differences in local cholesterol levels. With the addition of cholesterol or cholera toxin, we further observed the formation of low-polarity, raftlike nanodomains in the plasma membrane. In another study, we generalized SR-SMLM to fluorogenic single-molecule reactions. As a wide-field technique, SR-SMLM readily captures the emission spectra of individual product fluorescent molecules that are stochastically produced from nonfluorescent reactants at random locations over large sample areas, and therefore, it provides the unique possibility to spectrally identify and characterize single product molecules in a high-throughput fashion. Using the ring-opening reaction of a photochromic spiropyran as an example, we demonstrated that the capability to resolve the emission spectra of single product molecules could unveil rich, multipath reaction pathways. In summary, by integrating the spatial, temporal, and spectral dimensions of single-molecule fluorescence, SR-SMLM and f-SRM add rich spectral and functional dimensions to SRM and thus open up new ways of probing biological and chemical systems at the single-molecule and nanoscale levels.

Spectrally Resolved, Functional Super-Resolution Microscopy Reveals Nanoscale Compositional Heterogeneity in Live-Cell Membranes.

By recording the full fluorescence spectra and super-resolved positions of ∼106 individual polarity-sensing solvatochromic molecules, we reveal compositional heterogeneity in the membranes of live mammalian cells with single-molecule sensitivity and ∼30 nm spatial resolution. This allowed us to unveil distinct polarity characteristics of the plasma membrane and the membranes of nanoscale intracellular organelles, a result we found to be due to differences in cholesterol levels. Within the plasma membrane, we observed the formation of low-polarity, raft-like nanodomains upon cholesterol addition or cholera-toxin treatment, but found this nanoscale phase separation absent in native cells. The ultimate sensitivity achieved through examining the spectra of individual molecules thus opens the door to functional interrogations of intracellular physicochemical parameters at the nanoscale.

Direct Optical Visualization of Graphene and Its Nanoscale Defects on Transparent Substrates.

The discovery and rise of graphene were historically enabled by its ∼10% optical contrast on specialized substrates like oxide-capped silicon. However, substantially lower contrast is obtained on transparent substrates. Moreover, it remains difficult to visualize nanoscale defects in graphene, including voids, cracks, wrinkles, and multilayers, on most device substrates. We report the use of interference reflection microscopy (IRM), a facile, label-free optical microscopy method originated in cell biology, to directly visualize graphene on transparent inorganic and polymer substrates at 30-40% image contrast per graphene layer. Our noninvasive approach overcomes typical challenges associated with transparent substrates, including insulating and rough surfaces, enables unambiguous identification of local graphene layer numbers and reveals nanoscale structures and defects with outstanding contrast and throughput. We thus demonstrate in situ monitoring of nanoscale defects in graphene, including the generation of nanocracks under uniaxial strain, at up to 4× video rate.

Graphene-enabled electron microscopy and correlated super-resolution microscopy of wet cells.

The application of electron microscopy to hydrated biological samples has been limited by high-vacuum operating conditions. Traditional methods utilize harsh and laborious sample dehydration procedures, often leading to structural artefacts and creating difficulties for correlating results with high-resolution fluorescence microscopy. Here, we utilize graphene, a single-atom-thick carbon meshwork, as the thinnest possible impermeable and conductive membrane to protect animal cells from vacuum, thus enabling high-resolution electron microscopy of wet and untreated whole cells with exceptional ease. Our approach further allows for facile correlative super-resolution and electron microscopy of wet cells directly on the culturing substrate. In particular, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and well correlated with super-resolution results.