Super-resolution microscopy allows biological systems to be studied at the nanoscale, but has been restricted to providing only positional information. Here, we show that it is possible to perform multi-dimensional super-resolution imaging to determine both the position and the environmental properties of single-molecule fluorescent emitters. The method presented here exploits the solvatochromic and fluorogenic properties of nile red to extract both the emission spectrum and the position of each dye molecule simultaneously enabling mapping of the hydrophobicity of biological structures. We validated this by studying synthetic lipid vesicles of known composition. We then applied both to super-resolve the hydrophobicity of amyloid aggregates implicated in neurodegenerative diseases, and the hydrophobic changes in mammalian cell membranes. Our technique is easily implemented by inserting a transmission diffraction grating into the optical path of a localization-based super-resolution microscope, enabling all the information to be extracted simultaneously from a single image plane.

Proteins fold into a single structural ensemble but can also misfold into many diverse structures including small aggregates and fibrils, which differ in their toxicity. The aggregate surface properties play an important role in how they interact with the plasma membrane and cellular organelles, potentially inducing cellular toxicity, however, these properties have not been measured to date due to the lack of suitable methods. Here, we used a spectrally resolved, super-resolution imaging method combined with an environmentally sensitive fluorescent dye to measure the surface hydrophobicity of individual aggregates formed by the protein α-synuclein (αS), whose aggregation is associated with Parkinson’s disease. We show that the surface of soluble oligomers is more hydrophobic than fibrils and populates a diverse range of coexisting states. Overall, our data show that the conversion of oligomers to fibril-like aggregates and ultimately to fibrils results in a reduction in both hydrophobicity and the variation in hydrophobicity. This funneling characteristic of the energy landscape explains many of the observed properties of αS aggregates and may be a common feature of aggregating proteins.

The T cell receptor (TCR) initiates the elimination of pathogens and tumors by T cells. To avoid damage to the host, the receptor must be capable of discriminating between wild-type and mutated self and nonself peptide ligands presented by host cells. Exactly how the TCR does this is unknown. In resting T cells, the TCR is largely unphosphorylated due to the dominance of phosphatases over the kinases expressed at the cell surface. However, when agonist peptides are presented to the TCR by major histocompatibility complex proteins expressed by antigen-presenting cells (APCs), very fast receptor triggering, i.e., TCR phosphorylation, occurs. Recent work suggests that this depends on the local exclusion of the phosphatases from regions of contact of the T cells with the APCs. Here, we developed and tested a quantitative treatment of receptor triggering reliant only on TCR dwell time in phosphatase-depleted cell contacts constrained in area by cell topography. Using the model and experimentally derived parameters, we found that ligand discrimination likely depends crucially on individual contacts being ∼200 nm in radius, matching the dimensions of the surface protrusions used by T cells to interrogate their targets. The model not only correctly predicted the relative signaling potencies of known agonists and nonagonists but also achieved this in the absence of kinetic proofreading. Our work provides a simple, quantitative, and predictive molecular framework for understanding why TCR triggering is so selective and fast and reveals that, for some receptors, cell topography likely influences signaling outcomes.

Single-molecule localization microscopy, typically based on total internal reflection illumination, has taken our understanding of protein organization and dynamics in cells beyond the diffraction limit. However, biological systems exist in a complicated three-dimensional environment, which has required the development of new techniques, including the double-helix point spread function (DHPSF), to accurately visualize biological processes. The application of the DHPSF approach has so far been limited to the study of relatively small prokaryotic cells. By matching the refractive index of the objective lens immersion liquid to that of the sample media, we demonstrate DHPSF imaging of up to 15-μm-thick whole eukaryotic cell volumes in three to five imaging planes. We illustrate the capabilities of the DHPSF by exploring large-scale membrane reorganization in human T cells after receptor triggering, and by using single-particle tracking to image several mammalian proteins, including membrane, cytoplasmic, and nuclear proteins in T cells and embryonic stem cells.

Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i.e., by the presence of a supporting surface. In this study, we implemented single-molecule light-sheet microscopy, which enables single receptors to be directly visualized at any plane in a cell to study protein dynamics and organization in live, resting T cells. The light sheet enabled the acquisition of high-quality single-molecule fluorescence images that were comparable to those of total internal reflection fluorescence microscopy. By comparing the apical and basal surfaces of surface-contacting T cells using single-molecule light-sheet microscopy, we found that most coated-glass surfaces and supported lipid bilayers profoundly affected the diffusion of membrane proteins (T cell receptor and CD45) and that all the surfaces induced calcium influx to various degrees. Our results suggest that, when studying resting T cells, surfaces are best avoided, which we achieve here by suspending cells in agarose.

vLUME is a virtual reality software package designed to render large three-dimensional single-molecule localization microscopy datasets. vLUME features include visualization, segmentation, bespoke analysis of complex local geometries and exporting features. vLUME can perform complex analysis on real three-dimensional biological samples that would otherwise be impossible by using regular flat-screen visualization programs.Super-resolution microscopy based on three-dimensional single-molecule localization microscopy (3D-SMLM) is now well established 1,2 , and its widespread adoption has led to the development of more than 36 software packages dedicated to quantitative evaluation of the spatial and temporal detection of fluorophore photoswitching 3 . While the initial emphasis in the 3D-SMLM field has clearly been on improving resolution and data quality, there is now a marked absence of 3D visualization approaches that enable the straightforward, high-fidelity exploration of this type of data. Inspired by the horological phosphorescence points that illuminate watch-faces in the dark, we present vLUME (visualization of the local universe in a micro environment, pronounced 'volume'), an immersive virtual reality (VR)-based visualization software package purposefully designed to render large 3D-SMLM datasets. It is free for academic use. vLUME enables robust visualization, segmentation, annotation and quantification of millions of fluorescence puncta from any 3D-SMLM technique. vLUME has an intuitive user interface and is compatible with all commercial gaming VR hardware (Oculus Rift/Rift S and HTC Vive/Vive Pro; Supplementary Video 1). Although other microscopy data (that is, confocal) visualization tools have previously explored VR technology using volumetric representations 4,5 , vLUME has been specifically and purposefully created for SMLM. It accelerates the analysis of highly complex 3D point-cloud data and the rapid identification of defects that are otherwise neglected in global quality metrics. (A comparison with other VR and non-VR tools can be found in Supplementary Table 1.)vLUME is a point-cloud based 3D-SMLM data visualization tool able to render all pointillism-based multidimensional datasets. It differs from other 3D tools for 3D-SMLM visualization such as ViSP 6 by providing a complete VR interactive environment and intuitive interface for life scientists, dedicated to data visualization, segmentation and analysis. Users load multidimensional particle-list datasets into vLUME (.csv files; Fig. 1a), such as those generated by commonly used 3D-SMLM software 7,8 . This allows users to comprehend the spatial and temporal relation between

The composition of soluble toxic protein aggregates formed in vivo is currently unknown in neurodegenerative diseases, due to their ultra-low concentration in human biofluids and their high degree of heterogeneity. We introduce the structure-specific chemical antibody; a Y shaped, bioinspired small molecule with a dimeric region to mimic avidity, and an attachment region to mimic the Fc region. Our probe, capture molecule for amyloid precipitation (CAP-1), consists of a derivative of Pittsburgh compound B (dimer) to target the cross -sheets of amyloids and a biotin moiety for surface immobilization. By coupling CAP-1 to magnetic beads, we targeted the amyloid structure of protein aggregates in human cerebrospinal fluid, isolated them for analysis and then characterised them using single-molecule fluorescence imaging and mass spectrometry. AP allows unbiased determination of the molecular composition and structural features of the in vivo aggregates, formed in neurodegenerative diseases, that are present in biofluids.

DECT of the abdomen, chest wall, and spine revealed extensive MSU deposits in costal cartilages and, to a lesser extent, intervertebral disks in the male patients with gout in our study. These were quantified volumetrically. However, age-matched control subjects showed similar deposits, indicating this was not a disease-specific finding. Thus, MSU deposition in the axial skeleton may be physiologic in middle-aged men.