Modern proteomic efforts aim to define protein composition and protein-protein interactions within native spatial tissue contexts to understand disease mechanisms and therapeutic responses. Two complementary strategies dominate this effort: physically resolved mapping using high-plex imaging, mass cytometry, or laser-capture microdissection, and chemically defined spatial proteomics based on enzyme or photo-proximity-mediated labeling. Photo-proximity labeling enables nanometer-scale mapping of protein neighborhoods with precise spatial control, overcoming limitations of enzyme-based approaches. This review highlights emerging light-driven spatial proteomic technologies, including targeted photocatalytic labeling and optically guided microproteomics, that combine optical precision with deep proteome coverage to generate high-resolution interactome maps. These methods enable analysis of molecular organization across in vitro and in vivo systems, including formalin-fixed, paraffin-embedded tissues, and reveal disease-relevant molecular reorganization. We discuss recent advances in photo-proteomic strategies that provide mechanistic insight into protein organization in patient tissues and support biomarker discovery in cancer, neurodegeneration, and other complex diseases.

Ongoing efforts to integrate Raman spectroscopy with machine learning for biomarker discovery is demonstrated in the current work. Biological sample preparation (fixation method, substrate & sample sandwich configuration) was developed and optimized for robust and efficient data collection with a commercial, benchtop instrument, in this case the Horiba Xplora. The effects of fixation reagents, substrate, and for 3D cultures various optical clearing methods, were investigated. It was possible to detect spectral differences among diverse cell lines in both 2D and 3D cultures as well as changes in the functional state of the cell. Also, the difference in the Raman signal corresponding to distinct cellular compartments (nucleus vs cytoplasm) is identified using clustering approach, opening the door for advanced machine learning methods. The acquired data is an initial step for model training with the ultimate goal of distinguishing cancerous and healthy cells, and future implementation of Raman spectroscopy in cancer screening.

Dimeric IgA (dIgA) can move through cells via the IgA/IgM polymeric immunoglobulin receptor (PIGR), which is expressed mainly on mucosal epithelia. Here, we studied the ability of dIgA to target commonly mutated cytoplasmic oncodrivers. Mutation-specific dIgA, but not IgG, neutralized KRASG12D within ovarian carcinoma cells and expelled this oncodriver from tumor cells. dIgA binding changed endosomal trafficking of KRASG12D from accumulation in recycling endosomes to aggregation in the early/late endosomes through which dIgA transcytoses. dIgA targeting of KRASG12D abrogated tumor cell proliferation in cell culture assays. In vivo, KRASG12D-specific dIgA1 limited the growth of KRASG12D-mutated ovarian and lung carcinomas in a manner dependent on CD8+ T cells. dIgA specific for IDH1R132H reduced colon cancer growth, demonstrating effective targeting of a cytoplasmic oncodriver not associated with surface receptors. dIgA targeting of KRASG12D restricted tumor growth more effectively than small-molecule KRASG12D inhibitors, supporting the potential of this approach for the treatment of human cancers.

Exosomes are secreted to the extracellular milieu when multivesicular endosomes (MVEs) dock and fuse with the plasma membrane. However, MVEs are also known to fuse with lysosomes for degradation. How MVEs are directed to the plasma membrane for exosome secretion rather than to lysosomes is unclear. Here we report that a conversion of phosphatidylinositol-3-phosphate (PI(3)P) to phosphatidylinositol-4-phosphate (PI(4)P) catalyzed sequentially by Myotubularin 1 (MTM1) and phosphatidylinositol 4-kinase type IIα (PI4KIIα) on the surface of MVEs mediates the recruitment of the exocyst complex. The exocyst then targets the MVEs to the plasma membrane for exosome secretion. We further demonstrate that disrupting PI(4)P generation or exocyst function blocked exosomal secretion of Programmed death-ligand 1 (PD-L1), a key immune checkpoint protein in tumor cells, and led to its accumulation in lysosomes. Together, our study suggests that the PI(3)P to PI(4)P conversion on MVEs and the recruitment of the exocyst direct the exocytic trafficking of MVEs for exosome secretion.

Fluorescence nanoscopy has become increasingly powerful for biomedical research, but it has historically afforded a small field-of-view (FOV) around 50 µm x 50 µm at once and more recently up to ~200 µm x 200 µm. Efforts to further increase the FOV in fluorescence nanoscopy have thus far relied on the use of fabricated waveguide substrates, adding cost and sample constraints on the applications. Here we report PRism-Illumination and Microfluidics-Enhanced DNA-PAINT (PRIME-PAINT) for multiplexed fluorescence nanoscopy across millimeter-scale FOVs. Built upon the well-established prism-type total internal reflection microscopy, PRIME-PAINT achieves robust single-molecule localization with up to ~520 µm x 520 µm single FOVs and 25-40 nm lateral resolutions. Through stitching, nanoscopic imaging over mm 2 sample areas can be completed in as little as 40 minutes per target. An on-stage microfluidics chamber facilitates probe exchange for multiplexing and enhances image quality particularly for formalin-fixed paraffin-embedded (FFPE) tissue sections. We demonstrate the utility of PRIME-PAINT by analyzing ~10 6 caveolae structures in ~1,000 cells and imaging entire pancreatic cancer lesions from patient tissue biopsies. By imaging from nanometers to millimeters with multiplexity and broad sample compatibility, PRIME-PAINT will be useful for building multiscale, Google-Earth-like views of biological systems.

Metal nanoparticles can be sensitive molecular sensors due to enhanced absorption and scattering of light near a localized surface plasmon resonance (LSPR). Variations in both intrinsic properties such as the geometry and extrinsic properties such as the environment can cause heterogeneity in nanoparticle LSPR and impact the overall sensing responses. To date, however, few studies have examined LSPR and sensing heterogeneities, due to technical challenges in obtaining the full LSPR spectra of individual nanoparticles in dynamic assays. Here, we report multispectral LSPR (msLSPR), a wide-field imaging technique for real-time spectral monitoring of light scattering from individual nanoparticles across the whole field of view (FOV) at ∼0.5 nm spectral and ∼100 ms temporal resolutions. Using msLSPR, we studied the spectral and sensing properties of gold nanoparticles commonly used in LSPR assays, including spheres, rods, and bipyramids. Complemented with electron microscopy imaging, msLSPR analysis revealed that all classes of gold nanoparticles exhibited variations in LSPR peak wavelengths that largely paralleled variations in morphology. Compared with the rods and spheres, gold nanobipyramids exhibited both more uniform and stronger sensing responses as long as the bipyramids are structurally intact. Simulations incorporating the experimental LSPR properties demonstrate the negative impact of spectral heterogeneity on the overall performance of conventional, intensity-based LSPR assays and the ability of msLSPR in overcoming both particle heterogeneity and measurement noise. These results highlight the importance of spectral heterogeneity in LSPR-based sensors and the potential advantage of performing LSPR assays in the spectral domain.

Mechanisms of therapeutic resistance and vulnerability evolve in metastatic cancers as tumor cells and extrinsic microenvironmental influences change during treatment. To support the development of methods for identifying these mechanisms in individual people, here we present an omic and multidimensional spatial (OMS) atlas generated from four serial biopsies of an individual with metastatic breast cancer during 3.5 years of therapy. This resource links detailed, longitudinal clinical metadata that includes treatment times and doses, anatomic imaging, and blood-based response measurements to clinical and exploratory analyses, which includes comprehensive DNA, RNA, and protein profiles; images of multiplexed immunostaining; and 2- and 3-dimensional scanning electron micrographs. These data report aspects of heterogeneity and evolution of the cancer genome, signaling pathways, immune microenvironment, cellular composition and organization, and ultrastructure. We present illustrative examples of how integrative analyses of these data reveal potential mechanisms of response and resistance and suggest novel therapeutic vulnerabilities.

Förster resonance energy transfer (FRET) is a powerful tool for studying molecular interactions. Its use for studying interactions involving more than two molecules, however, has been limited by spectral crosstalk among the fluorophores. Here, we report multispectral FRET (msFRET) for imaging multiple pairs of interactions in parallel by spectrally resolving single fluorescent molecules. By using a dual (positional and spectral) channel and wide-field imaging configuration, fluorophores with emission maxima as close as 6-10 nm could be reliably distinguished. We demonstrate msFRET by continuously monitoring the hybridization dynamics among 2 × 2 pairs of DNA oligos in parallel using Cy3 and Cy3.5 as donors and Cy5 and Cy5.5 as acceptors. Aside from studying molecular interactions, msFRET may also find applications in probing fluorophore photophysics during FRET and in multiplexed superresolution imaging.

Recent work suggests that Ras small GTPases interact with the anionic lipid phosphatidylserine (PS) in an isoform-specific manner, with direct implications for their biological functions. Studies on PS-Ras associations in cells, however, have relied on immuno-EM imaging of membrane sheets. To study their spatial relationships in intact cells, we have combined the use of Lact-C2-GFP, a biosensor for PS, with multicolor super resolution imaging based on DNA-PAINT. At ~20 nm spatial resolution, the resulting super resolution images clearly show the nonuniform molecular distribution of PS on the cell membrane and its co-enrichment with caveolae, as well as with unidentified membrane structures. Two-color imaging followed by spatial analysis shows that KRas-G12D and HRas-G12V both co-enrich with PS in model U2OS cells, confirming previous observations, yet exhibit clear differences in their association patterns. Whereas HRas-G12V is almost always co-enriched with PS, KRas-G12D is strongly co-enriched with PS in about half of the cells, with the other half exhibiting a more moderate association. In addition, perturbations to the actin cytoskeleton differentially impact PS association with the two Ras isoforms. These results suggest that PS-Ras association is context-dependent and demonstrate the utility of multiplexed super resolution imaging in defining the complex interplay between Ras and the membrane.

Recent advances in super resolution microscopy have enabled imaging at the 10-20 nm scale on a light microscope, providing unprecedented details of native biological structures and processes in intact and hydrated samples. Of the existing strategies, DNA points accumulation in imaging nanoscale topography (DNA-PAINT) affords convenient multiplexing, an important feature in interrogating complex biological systems. A practical limitation of DNA-PAINT, however, has been the slow imaging speed. In its original form, DNA-PAINT imaging of each target takes tens of minutes to hours to complete. To address this challenge, several improved implementations have been introduced. These include DNA-PAINT-ERS (where E = ethylene carbonate; R = repeat sequence; S = spacer), a set of strategies that leads to both accelerated DNA-PAINT imaging speed and improved image quality. With DNA-PAINT-ERS, imaging of typical cellular targets such as microtubules takes only 5-10 min. Importantly, DNA-PAINT-ERS also facilitates multiplexing and can be easily integrated into current workflows for fluorescence staining of biological samples. Here, we provide a detailed, step-by-step guide for fast and multiplexed DNA-PAINT-ERS imaging of fixed and immunostained cells grown on glass substrates as adherent monolayers. The protocol should be readily extended to biological samples of a different format (for example tissue sections) or staining mechanisms (for example using nanobodies). (c) 2022 Wiley Periodicals LLC.Basic Protocol 1: Preparation of probes for DNA-PAINT-ERSBasic Protocol 2: Sample preparation for imaging membrane targets with DNA-PAINT-ERS in fixed cellsAlternate Protocol: Immunostaining of extracted U2OS cellsBasic Protocol 3: Super resolution image acquisition and analysis

Formation of Ras multimers, including dimers and nanoclusters, has emerged as an exciting, new front of research in the 'old' field of Ras biomedicine. With significant advances made in the past few years, we are beginning to understand the structure of Ras multimers and, albeit preliminary, mechanisms that regulate their formation in vitro and in cells. Here we aim to synthesize the knowledge accrued thus far on Ras multimers, particularly the presence of multiple globular (G-) domain interfaces, and discuss how membrane nanodomain composition and structure would influence Ras multimer formation. We end with some general thoughts on the potential implications of Ras multimers in basic and translational biology.

Highly branched gold (Au) nanostructures with sharp tips are considered excellent substrates for surface-enhanced Raman scattering (SERS)-based sensing technologies. Here, a simple synthetic route for producing Au or Au-Ag bimetallic mesostructures with multiple sharpened tips in the presence of carbon quantum dots (CQDs) is presented. The morphologies of these mesostructured plasmonic nanoparticles (MSPNs) can be controlled by adjusting the concentration of CQDs, reaction temperatures, and seed particles. The optimal molar ratio for [HAuCl4 ]/[CQDs] is found to be ≈25. At this molar ratio, the diameters of MSPNs can be tuned from 80 to 200 nm by changing the reaction temperature from 25 to 80 °C. In addition, it is found that hierarchical MSPNs consisting of multiple Au nanocrystals can be formed over the entire seed particle surface. Finally, the SERS activity of these MSPNs is examined through the detection of rhodamine 6G and methylene blue. Of the different mesostructures, the bimetallic MSPNs have the highest sensitivity with the ability to detect 10-7  m of rhodamine 6G and 10-6  m of methylene blue. The properties of these MSPN particles, made using a novel synthetic process, make them excellent candidates for SERS-based chemical sensing applications.

Fluorophores are powerful tools for interrogating biological systems. Carbon nanotubes (CNTs) have long been attractive materials for biological imaging due to their near-infrared excitation and bright, tunable optical properties. The difficulty in synthesizing and functionalizing these materials with precision, however, has hampered progress in this area. Carbon nanohoops, which are macrocyclic CNT substructures, are carbon nanostructures that possess ideal photophysical characteristics of nanomaterials, while maintaining the precise synthesis of small molecules. However, much work remains to advance the nanohoop class of fluorophores as biological imaging agents. Herein, we report an intracellular targeted nanohoop. This fluorescent nanostructure is noncytotoxic at concentrations up to 50 μM, and cellular uptake investigations indicate internalization through endocytic pathways. Additionally, we employ this nanohoop for two-photon fluorescence imaging, demonstrating a high two-photon absorption cross-section (65 GM) and photostability comparable to a commercial probe. This work further motivates continued investigations into carbon nanohoop photophysics and their biological imaging applications.

A major goal of cancer research is to understand how mutations distributed across diverse genes affect common cellular systems, including multiprotein complexes and assemblies. Two challenges-how to comprehensively map such systems and how to identify which are under mutational selection-have hindered this understanding. Accordingly, we created a comprehensive map of cancer protein systems integrating both new and published multi-omic interaction data at multiple scales of analysis. We then developed a unified statistical model that pinpoints 395 specific systems under mutational selection across 13 cancer types. This map, called NeST (Nested Systems in Tumors), incorporates canonical processes and notable discoveries, including a PIK3CA-actomyosin complex that inhibits phosphatidylinositol 3-kinase signaling and recurrent mutations in collagen complexes that promote tumor proliferation. These systems can be used as clinical biomarkers and implicate a total of 548 genes in cancer evolution and progression. This work shows how disparate tumor mutations converge on protein assemblies at different scales.

Correlative light and electron microscopy (CLEM) is a powerful tool for defining the ultrastructural context of molecularly-labeled biological specimens, particularly when superresolution fluorescence microscopy (SRM) is used for CLEM. Current CLEM, however, is limited by the stark differences in sample preparation requirements between the two modalities. For CLEM using SRM, the small region of interest (ROI) of either or both modalities also leads to low success rate and imaging throughput. To overcome these limitations, here we present a CLEM workflow based on a novel focused ion beam/scanning electron microscope (FIB/SEM) compatible with common SRM for imaging biological specimen with ultrahigh 3D resolution and improved imaging throughput. By using a reactive oxygen source in a plasma FIB (PFIB) and a rotating sample stage, the novel FIB/SEM was able to achieve several hundreds of micrometer large area 3D analysis of resin embedded cells through a process named oxygen serial spin mill (OSSM). Compared with current FIB mechanisms, OSSM offers gentle erosion, highly consistent slice thickness, reduced charging during SEM imaging, and improved SEM contrast without increasing the dose of post-staining and fixation. These characteristics of OSSM-SEM allowed us to pair it with interferometric photoactivated localization microscopy (iPALM), a recent SRM technique that affords 10–20 nm isotropic spatial resolution on hydrated samples, for 3D CLEM imaging. We demonstrate a CLEM workflow generalizable to using other SRM strategies using mitochondria in human osteosarcoma (U2OS) cells as a model system, where immunostained TOM20, a marker for the mitochondrial outer membrane, was used for iPALM. Owing to the large scan area of OSSM-SEM, it is now possible to select as many FOVs as needed for iPALM and conveniently re-locate them in EM, this improving the imaging throughput. The significantly reduced dose of post-fixation also helped to better preserve the sample ultrastructures as evidenced by the excellent 3D registration between OSSM-SEM and iPALM images and by the accurate localization of TOM20 (by iPALM) to the peripheries of mitochondria (by OSSM-SEM). These advantages make OSSM-SEM an ideal modality for CLEM applications. As OSSM-SEM is still in development, we also discuss some of the remaining issues and the implications to biological imaging with SEM alone or with CLEM.

Mechanisms of therapeutic resistance manifest in metastatic cancers as tumor cell intrinsic alterations and extrinsic microenvironmental influences that can change during treatment. To support the development of methods for the identification of these mechanisms in individual patients, we present here an Omic and Multidimensional Spatial (OMS) Atlas generated from four serial biopsies of a metastatic breast cancer patient during 3.5 years of therapy. This resource links detailed, longitudinal clinical metadata including treatment times and doses, anatomic imaging, and blood-based response measurements to exploratory analytics including comprehensive DNA, RNA, and protein profiles, images of multiplexed immunostaining, and 2- and 3-dimensional scanning electron micrographs. These data reveal aspects of therapy-associated heterogeneity and evolution of the cancer’s genome, signaling pathways, immune microenvironment, cellular composition and organization, and ultrastructure. We present illustrative examples showing how integrative analyses of these data provide insights into potential mechanisms of response and resistance, and suggest novel therapeutic vulnerabilities.

DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) facilitates multiplexing in superresolution microscopy but is practically limited by slow imaging speed. We have developed DNA-PAINT-ERS, where E=ethylene carbonate, R=repeating sequence, and S=spacer, for fast and multiplexed superresolution imaging with DNA-PAINT. Here we describe detailed procedures for DNA-PAINT-ERS including reagent preparation, sample labeling, as well as image acqusition and analysis.

Summary Metastatic cancers often respond to treatment initially but almost universally escape therapeutic control through molecular mechanisms intrinsic to tumor cells, as well as extrinsic influences from immune cells, stroma, and structural microenvironments. We explore the extent to which we can learn these mechanisms and associated therapeutic vulnerabilities by linking comprehensive molecular and multiscale imaging analyses of tumor biopsies to detailed clinical information for a patient with metastatic breast cancer. Our analyses of three serial biopsies include DNA, RNA, and protein profiling, plus quantification of tumor microenvironment composition using multiplex immunostaining and electron microscopy. Features from these analyses are linked to treatments and treatment-response measurements, including CT and PET images, circulating tumor DNA and tumor protein levels in blood, and treatment-limiting readouts of blood counts and liver function. Importantly, the measurement modalities unique to this study complemented routine diagnostics to suggest actionable mechanisms of response and resistance for each treatment phase.

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