The emergence of the SARS-CoV-2 coronavirus has brought an unprecedented global challenge. Despite the surge in research on coronavirus biology, our understanding of various aspects of SARS-CoV-2 infection within the cellular milieu remains incomplete. The coronavirus transforms the cellular interior into an environment permissive for viral replication. This transformation is characterized by the appearance of virus-induced organelles, e.g. double-membrane vesicles (DMVs). However, obtaining detailed localization of the key SARS-CoV-2 components, specifically the viral genomic RNA (vgRNA) and viral proteins in the remodeled cellular context, has been challenging due to lack of specific contrast in electron microscopy and due to limited resolution in fluorescence microscopy.

The development of many quantum optical technologies depends on the availability of single quantum emitters with near-perfect coherence. Systematic improvement is limited by a lack of understanding of the microscopic energy flow at the single-emitter level and ultrafast timescales. Here we utilize a combination of fluorescence correlation spectroscopy and ultrafast spectroscopy to capture the sample-averaged dynamics of defects with single-particle sensitivity. We employ this approach to study heterogeneous emitters in two-dimensional hexagonal boron nitride. From milliseconds to nanoseconds, the translational, shelving, rotational and antibunching features are disentangled in time, which quantifies the normalized two-photon emission quantum yield. Leveraging the femtosecond resolution of this technique, we visualize electron-phonon coupling and discover the acceleration of polaronic formation on multi-electron excitation. Corroborated with theory, this translates to the photon fidelity characterization of cascaded emission efficiency and decoherence time. Our work provides a framework for ultrafast spectroscopy in heterogeneous emitters, opening new avenues of extreme-scale characterization for quantum applications. Combining fluorescence correlation spectroscopy and ultrafast spectroscopy, the sample-averaged dynamics of defects are studied with single-particle sensitivity in two-dimensional hexagonal boron nitride heterogeneous emitters.

Single-molecule localization microscopy combined with optimized point spread function (PSF) engineering is a powerful tool for understanding the 3D spatial organization of cellular entities with a resolution of a few tens of nanometers. Usually, phase masks are designed assuming a freely rotating molecule to encode the axial position in the PSF shape with the best precision. However, new labeling strategies can reduce the mobility of the fluorophore for which the resulting PSF shape is highly dependent on the orientation of the molecule, leading to biases in 3D position measurement when this is not considered.

Stimulated Emission (StE) remains relatively unused as an image-forming signal despite having potential advantages over fluorescence in speed, coherence, and ultimately resolution. Several ideas for the radiation pattern and directionality of StE remain prevalent, namely whether a single molecule would radiate StE itself in a pure dipole pattern, or whether its emission direction depends on the driving field. Previous StE imaging has been carried out in transmission, which would collect signal either way. Here, we introduce the StE driving field (the probe) at an angle, using total internal reflection to avoid incident probe light and its specular reflections in our detection path. In this non-collinear detection configuration which also collects some fluorescence from the sample, we observe fluorescence depletion even in the spectral window where an increase in detected signal from StE would be expected if StE radiated like a simple classical dipole. Because simultaneous direct measurement of the fluorescence represents a calibration of the potential size of StE were it spatially patterned like a classical dipole emitter, our study clarifies a critical characteristic of StE for optimal microscope design, optical cooling, and applications using small arrays of emitters.

Approximately 1 billion people globally are infected by the parasite Toxoplasma gondii. While most infections are asymptomatic, serious disease can occur in the immunocompromised and in fetuses. Despite the organism’s significance to human health, there are many gaps in our understanding of its basic biology. The parasite uses an apical complex (AC) comprised of secretory organelles and proteinaceous structures to drive invasion of virtually any animal cell. However, fundamental details of AC organization and function are unknown, with direct observation hindered by the fact that the AC is approximately a single diffraction-limited volume in size (in the visible). The motor MyosinH is indispensable for parasite invasion and shown to associate with the conoid, a barrel of uniquely-arranged tubulin filaments within the AC that protrudes during host cell invasion. However, characterizing the distribution of MyosinH and its localization with nanoscale precision is difficult given the small spatial extent of the AC. To circumvent this, we fuse the ALFA tag to MyosinH and utilize 3D single-molecule localization microscopy with the double-helix point spread function to determine the position of the protein with 12 nm lateral and 22 nm axial localization precision, revealing the nanoscale details of the shape and size of the MyosinH superstructure in the AC. We similarly image tubulin in the parasites to provide context at the spatial scale necessary to determine how the MyosinH distribution organizes with respect to the AC as a whole. The resulting two-color 3D super-resolved images of conoid-protruded and conoid-retracted parasites determine with high resolution how this vital motor protein changes spatial organization in preparation for host cell invasion, bringing nanoscale detail to a biological process with vast public health implications.

Early stages of deadly respiratory diseases including COVID-19 are challenging to elucidate in humans. Here, we define cellular tropism and transcriptomic effects of SARS-CoV-2 virus by productively infecting healthy human lung tissue and using scRNA-seq to reconstruct the transcriptional program in "infection pseudotime" for individual lung cell types. SARS-CoV-2 predominantly infected activated interstitial macrophages (IMs), which can accumulate thousands of viral RNA molecules, taking over 60% of the cell transcriptome and forming dense viral RNA bodies while inducing host profibrotic (TGFB1, SPP1) and inflammatory (early interferon response, CCL2/7/8/13, CXCL10, and IL6/10) programs and destroying host cell architecture. Infected alveolar macrophages (AMs) showed none of these extreme responses. Spike-dependent viral entry into AMs used ACE2 and Sialoadhesin/CD169, whereas IM entry used DC-SIGN/CD209. These results identify activated IMs as a prominent site of viral takeover, the focus of inflammation and fibrosis, and suggest targeting CD209 to prevent early pathology in COVID-19 pneumonia. This approach can be generalized to any human lung infection and to evaluate therapeutics.

Today, biomolecular nanoparticles are prevalent as diagnostic tools and molecular delivery carriers, and it is particularly useful to examine individuals within a sample population to quantify the variations between objects and directly observe the molecular dynamics involving these objects. Using interferometric scattering as a highly sensitive label-free detection scheme, we recently developed the interferometric scattering anti-Brownian electrokinetic (ISABEL) trap to hold a single nanoparticle in solution for extended optical observation. In this perspective, we describe how we implemented this trap, how it extends the capabilities of previous ABEL traps, and how we have begun to study individual carboxysomes, a fascinating biological carbon fixation nanocompartment. By monitoring single nanocompartments for seconds to minutes in the ISABEL trap using simultaneous interferometric scattering and fluorescence spectroscopy, we have demonstrated single-compartment mass measurements, cargo-loading trends, and redox sensing inside individual particles. These experiments benefit from rich multiplexed correlative measurements utilizing both scattering and fluorescence with many exciting future capabilities within reach.

Stimulated emission (StE) remains relatively unused as an image-forming signal despite having potential advantages over fluorescence in speed, coherence, and ultimately resolution. Several ideas for the radiation pattern and directionality of StE remain prevalent, namely, whether a single molecule would radiate StE itself in a pure dipole pattern, or whether its emission direction depends on the driving field. Previous StE imaging has been carried out in transmission, which would collect signal either way. Here, we introduce the StE driving field (the probe) at an angle, using total internal reflection to avoid incident probe light and its specular reflections in our detection path. In this non-collinear detection configuration that also collects some fluorescence from the sample, we observe fluorescence depletion even in the spectral window where an increase in detected signal from StE would be expected if StE radiated like a simple classical dipole. Because simultaneous direct measurement of the fluorescence represents a calibration of the potential size of StE were it spatially patterned like a classical dipole emitter, our study clarifies a critical characteristic of StE for optimal microscope design, optical cooling, and applications using small arrays of emitters.

Super-resolved cryogenic correlative light and electron microscopy is a powerful approach which combines the single-molecule specificity and sensitivity of fluorescence imaging with the nanoscale resolution of cryogenic electron tomography. Key to this method is active control over the emissive state of fluorescent labels to ensure sufficient sparsity to localize individual emitters. Recent work has identified fluorescent proteins (FPs) that photoactivate or photoswitch efficiently at cryogenic temperatures, but long on-times due to reduced quantum yield of photobleaching remain a challenge for imaging structures with a high density of localizations. In this work, we explore the photophysical properties of the red photoactivatable FP PAmKate and identify a 2-color process leading to enhanced turn-off of active emitters, improving localization rate. Specifically, after excitation of ground state molecules, we find that a transient state forms with a lifetime of ∼2 ms under cryogenic conditions, which can be bleached by exposure to a second wavelength. We measure the response of the transient state to different wavelengths, demonstrate how this mechanism can be used to improve imaging, and provide a blueprint for the study of other FPs at cryogenic temperatures.

The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelle where the replication of viral genomic RNA (vgRNA) occurs. To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain vgRNA clusters along with viral double-stranded RNA (dsRNA) clusters and the replication enzyme, encapsulated by membranes derived from the host endoplasmic reticulum (ER). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of ER labels and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are enclosed by DMVs at early infection stages which then merge into vesicle packets as infection progresses. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.

Single-molecule superresolution microscopy is a powerful tool for the study of biological structures on size scales smaller than the optical diffraction limit. Imaging samples at cryogenic temperatures (77 K) reduces the quantum yield of photobleaching for many fluorescent labels, yielding localization precisions below 10 nm. Cryogenic imaging further enables correlation with cryogenic electron tomography. A key limitation in applying methods such as PALM and STORM to samples maintained at 77 K is the limited number of fluorophores known to undergo efficient turn-on and turn-off mechanisms necessary to control the sparsity of active emitters. We find that mApple, a red-emitting fluorescent protein, undergoes a novel turn-off mechanism in response to simultaneous illumination with two colors of light. This turn-off mechanism enables localization of many individual molecules in initially bright samples, but the final density of localizable emitters is limited by relatively inefficient turn-on (photoactivation). Bulk excitation and emission spectroscopy shows that mApple has access to two distinct emissive states as well as dark states accessible optically or through changes in pH. The bright and stable emission of mApple enables widefield collection of single-molecule emission spectra, which highlight the complex nature and environmental sensitivity of states observed in red fluorescent proteins.

Anti-Brownian traps confine single molecules or particles in free solution by closed-loop feedback forces, allowing detailed characterization of photophysical and transport properties. We have recently extended this approach to tracking the particles with interferometric scattering at near infrared wavelengths. This extension allows trapping non-fluorescent particles and performing simultaneous modulated fluorescence measurements in the Interferometric Scattering Anti-Brownian ELectrokinetic (ISABEL) trap. Here we use the interferometric scattering signal in the ISABEL trap to measure the scattering cross-sections of single carboxysomes, bacterial nanocompartments involved in carbon fixation. With a core-shell model, we can calculate the total mass and internal loading of single nano-objects.

The development of many optical quantum technologies depends on the availability of solid-state single quantum emitters with near-perfect optical coherence. However, a standing issue that limits systematic improvement is the significant sample heterogeneity and lack of mechanistic understanding of microscopic energy flow at the single emitter level and ultrafast timescales. Here we develop solution-phase single-particle pump-probe spectroscopy with photon correlation detection that captures sample-averaged dynamics in single molecules and/or defect states with unprecedented clarity at femtosecond resolution. We apply this technique to single quantum emitters in two-dimensional hexagonal boron nitride, which suffers from significant heterogeneity and low quantum efficiency. From millisecond to nanosecond timescales, the translation diffusion, metastable-state-related bunching shoulders, rotational dynamics, and antibunching features are disentangled by their distinct photon-correlation timescales, which collectively quantify the normalized two-photon emission quantum yield. Leveraging its femtosecond resolution, spectral selectivity and ultralow noise (two orders of magnitude improvement over solid-state methods), we visualize electron-phonon coupling in the time domain at the single defect level, and discover the acceleration of polaronic formation driven by multi-electron excitation. Corroborated with results from a theoretical polaron model, we show how this translates to sample-averaged photon fidelity characterization of cascaded emission efficiency and optical decoherence time. Our work provides a framework for ultrafast spectroscopy in single emitters, molecules, or defects prone to photoluminescence intermittency and heterogeneity, opening new avenues of extreme-scale characterization and synthetic improvements for quantum information applications.

The nanometer-scale spatial organization of immune receptors plays a role in cell activation and suppression. While the connection between this spatial organization and cell signaling events is emerging from cell culture experiments, how these results translate to more physiologically relevant settings like the tumor microenvironment remains poorly understood due to the challenges of high-resolution imaging in vivo. Here we perform super-resolution immunofluorescence microscopy of human melanoma tissue sections to examine the spatial organization of the immune checkpoint inhibitor programmed cell death 1 (PD-1). We show that PD-1 exhibits a variety of organizations ranging from nanometer-scale clusters to more uniform membrane labeling. Our results demonstrate the capability of super-resolution imaging to examine the spatial organization of immune checkpoint markers in the tumor microenvironment, suggesting a future direction for both clinical and immunology research.

The small Ultra-Red Fluorescent Protein (smURFP) represents a new class of fluorescent protein with exceptional photostability and brightness derived from allophycocyanin in a previous directed evolution. Here, we report the smURFP crystal structure to better understand properties and enable further engineering of improved variants. We compare this structure to the structures of allophycocyanin and smURFP mutants to identify the structural origins of the molecular brightness. We then use a structure-guided approach to develop monomeric smURFP variants that fluoresce with phycocyanobilin but not biliverdin. Furthermore, we measure smURFP photophysical properties necessary for advanced imaging modalities, such as those relevant for two-photon, fluorescence lifetime, and single-molecule imaging. We observe that smURFP has the largest two-photon cross-section measured for a fluorescent protein, and that it produces more photons than organic dyes. Altogether, this study expands our understanding of the smURFP, which will inform future engineering toward optimal FPs compatible with whole organism studies.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human coronavirus within 20 years that gave rise to a life-threatening disease and the first to reach pandemic spread. To make therapeutic headway against current and future coronaviruses, the biology of coronavirus RNA during infection must be precisely understood. Here, we present a robust and generalizable framework combining high-throughput confocal and super-resolution microscopy imaging to study coronavirus infection at the nanoscale. Using the model human coronavirus HCoV-229E, we specifically labeled coronavirus genomic RNA (gRNA) and double-stranded RNA (dsRNA) via multi-color RNA immunoFISH and visualized their localization patterns within the cell. The 10-nm resolution achieved by our approach uncovers a striking spatial organization of gRNA and dsRNA into three distinct structures and enables quantitative characterization of the status of the infection after antiviral drug treatment. Our approach provides a comprehensive imaging framework that will enable future investigations of coronavirus fundamental biology and therapeutic effects.

Point Spread Function (PSF) engineering is an effective method to increase the sensitivity of single-molecule fluorescence images to specific parameters. Classical phase mask optimization approaches have enabled the creation of new PSFs that can achieve, for example, localization precision of a few nanometers axially over a capture range of several microns with bright emitters. However, for complex high-dimensional optimization problems, classical approaches are difficult to implement and can be very time-consuming for computation. The advent of deep learning methods and their application to single-molecule imaging has provided a way to solve these problems. Here, we propose to combine PSF engineering and deep learning approaches to obtain both an optimized phase mask and a neural network structure to obtain the 3D position and 3D orientation of fixed fluorescent molecules. Our approach allows us to obtain an axial localization precision around 30 nanometers, as well as an orientation precision around 5 degrees for orientations and positions over a one micron depth range for a signal-to-noise ratio consistent with what is typical in single-molecule cellular imaging experiments.

Biological nano-objects rapidly diffuse in the solution phase, impeding our efforts to monitor their physical and chemical properties for extended periods of time. To overcome this, we developed the Interferometric Scattering Anti-Brownian ELectrokinetic (ISABEL) Trap, which counteracts Brownian motion for an extended time by tracking a particle’s location via its scattering and rapidly applying positional feedback with electrokinetic forces. Recently, we improved the flexibility of these experiments by shifting the scattering detection beam to the near-infrared and opening the visible region for flexible and specific fluorescence measurements. These capabilities allow us to monitor the physical and chemical properties of the carboxysome, a ~100nm bacterial microcompartment responsible for CO2 fixation by the enzyme Rubisco. With the ISABEL trap, we can rapidly interleave 405 and 488 nm fluorescence excitation beams to measure the redox properties inside individual carboxysomes using the redox reporting GFP mutant, roGFP2. The capabilities provided by the ISABEL trap allow us to design solution-phase single-particle experiments for a variety of biological nanoscale objects.