Multicolor Super-resolution Microscopy Research Articles

MultiMatch: geometry-informed colocalization in multi-color super-resolution microscopy

With recent advances in multi-color super-resolution light microscopy, it is possible to simultaneously visualize multiple subunits within biological structures at nanometer resolution. To optimally evaluate and interpret spatial proximity of stainings on such an image, colocalization analysis tools have to be able to integrate prior knowledge on the local geometry of the recorded biological complex. We present MultiMatch to analyze the abundance and location of chain-like particle arrangements in multi-color microscopy based on multi-marginal optimal unbalanced transport methodology. Our object-based colocalization model statistically addresses the effect of incomplete labeling efficiencies enabling inference on existent, but not fully observable particle chains. We showcase that MultiMatch is able to consistently recover existing chain structures in three-color STED images of DNA origami nanorulers and outperforms geometry-uninformed triplet colocalization methods in this task. MultiMatch generalizes to an arbitrary number of color channels and is provided as a user-friendly Python package comprising colocalization visualizations.

Mechanical-scan-free multicolor super-resolution imaging with diffractive spot array illumination.

Point-scanning microscopy approaches are transforming super-resolution imaging. Despite achieving parallel high-speed imaging using multifocal techniques, efficient multicolor imaging methods with high-quality illumination are currently lacking. In this paper, we present for the first time Mechanical-scan-free multiColor Super-resolution Microscopy (MCoSM) with spot array illumination, which enables mechanical-scan-free super-resolution imaging with adjustable resolution and a good effective field-of-view based on spatial light modulators. Through 100-2,500 s super-resolution spot illumination with different effective fields of view for imaging, we demonstrate the adjustable capacity of MCoSM. MCoSM extends existing spectral imaging capabilities through a time-sharing process involving different color illumination with phase-shift scanning while retaining the spatial flexibility of super-resolution imaging with diffractive spot array illumination. To demonstrate the prospects of MCoSM, we perform four-color imaging of fluorescent beads at high resolution. MCoSM provides a versatile platform for studying molecular interactions in complex samples at the nanoscale level.

Nanoscopic Spatial Association between Ras and Phosphatidylserine on the Cell Membrane Studied with Multicolor Super Resolution Microscopy.

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.

Fluctuation‐based Fluorescence Labeling and Biosensing for Multi‐color Super‐resolution Imaging

Multi-color super-resolution microscopy has become a desirable method for exploring the dynamic interaction and compartmentalization of biomolecules in living cells. Among numerous super-resolution microscopy concepts, photochromic stochastic optical fluctuation imaging (pcSOFI) has been widely recognized and applied in the life sciences due to advantages such as use of off-the-shelf equipment and high temporal resolution. Furthermore, with reversibly switchable fluorescent proteins (RSFP), pcSOFI could be used to localize biomolecules in high resolution in living systems. Previously, we discovered a phenomenon called fluorescence fluctuation increase by contact (FLINC), in which the RSFP Dronpa can specifically enhance the ability of fluorescence fluctuations of non-RSFP TagRFP-T when they are spatially close to each other. We developed FLINC-based A kinase-activity reporter (AKAR) and achieved super-resolution PKA activity imaging by pcSOFI in living cells. However, FLINC-based sensors contain two FPs, presenting challenges in multi-color super-resolution imaging. To overcome this issue, we identified mutations that disrupt fluorescence of Dronpa but maintain FLINC. We then introduced a Dronpa-removed FLINC (DrFLINC) as an enhanced red label, in combination with pcSOFI, to localize biomolecules in super-resolution in different compartments of living cells. Applying this new red fluorescent label in dual-color pcSOFI, we revealed that type II PKA regulatory subunit-α (RIIα) mainly located on microtubule in MIN6 beta cells. Furthermore, we developed DrFLINC-AKAR and demonstrated the multiplexed super-resolution imaging to reveal the regulatory context of PKA activity microdomains in living cells.

Instant multicolor super-resolution microscopy with deep convolutional neural network.

Multicolor super-resolution (SR) microscopy plays a critical role in cell biology research and can visualize the interactions between different organelles and the cytoskeleton within a single cell. However, more color channels bring about a heavier budget for imaging and sample preparation, and the use of fluorescent dyes of higher emission wavelengths leads to a worse spatial resolution. Recently, deep convolutional neural networks (CNNs) have shown a compelling capability in cell segmentation, super-resolution reconstruction, image restoration, and many other aspects. Taking advantage of CNN's strong representational ability, we devised a deep CNN-based instant multicolor super-resolution imaging method termed IMC-SR and demonstrated that it could be used to separate different biological components labeled with the same fluorophore, and generate multicolor images from a single super-resolution image in silico. By IMC-SR, we achieved fast three-color live-cell super-resolution imaging with ~100 nm resolution over a long temporal duration, revealing the complicated interactions between multiple organelles and the cytoskeleton in a single COS-7 cell.

Influenza A viruses use multivalent sialic acid clusters for cell binding and receptor activation

Influenza A virus (IAV) binds its host cell using the major viral surface protein hemagglutinin (HA). HA recognizes sialic acid, a plasma membrane glycan that functions as the specific primary attachment factor (AF). Since sialic acid alone cannot fulfill a signaling function, the virus needs to activate downstream factors to trigger endocytic uptake. Recently, the epidermal growth factor receptor (EGFR), a member of the receptor-tyrosine kinase family, was shown to be activated by IAV and transmit cell entry signals. However, how IAV’s binding to sialic acid leads to engagement and activation of EGFR remains largely unclear. We used multicolor super-resolution microscopy to study the lateral organization of both IAV’s AFs and its functional receptor EGFR at the scale of the IAV particle. Intriguingly, quantitative cluster analysis revealed that AFs and EGFR are organized in partially overlapping submicrometer clusters in the plasma membrane of A549 cells. Within AF domains, the local AF concentration reaches on average 10-fold the background concentration and tends to increase towards the cluster center, thereby representing a multivalent virus-binding platform. Using our experimentally measured cluster characteristics, we simulated virus diffusion on a flat membrane. The results predict that the local AF concentration strongly influences the distinct mobility pattern of IAVs, in a manner consistent with live-cell single-virus tracking data. In contrast to AFs, EGFR resides in smaller clusters. Virus binding activates EGFR, but interestingly, this process occurs without a major lateral EGFR redistribution, indicating the activation of pre-formed clusters, which we show are long-lived. Taken together, our results provide a quantitative understanding of the initial steps of influenza virus infection. Co-clustering of AF and EGFR permit a cooperative effect of binding and signaling at specific platforms, thus linking their spatial organization to their functional role during virus-cell binding and receptor activation.

Photoactivatable Rhodamine Spiroamides and Diazoketones Decorated with "Universal Hydrophilizer" or Hydroxyl Groups.

Photoactivatable rhodamine spiroamides and spirocyclic diazoketones emerged recently as synthetic markers applicable in multicolor super-resolution microscopy. However, their applicability in single molecule localization microscopy (SMLM) is often limited by aggregation, unspecific adhesion, and low reactivity caused by insufficient solubility and precipitation from aqueous solutions. We report here two synthetic modifications increasing the polarity of compact polycyclic and hydrophobic labels decorated with a reactive group: attachment of 3-sulfo-l-alanyl-beta-alanine dipeptide (a "universal hydrophilizer") or allylic hydroxylation in photosensitive rhodamine diazoketones (and spiroamides). The super-resolution images of tubulin and keratin filaments in fixed and living cells exemplify the performance of "blinking" spiroamides derived from N, N, N', N'-tetramethyl rhodamine.

Probing nano-organization of astroglia with multi-color super-resolution microscopy.

Astroglia are essential for brain development, homeostasis, and metabolic support. They also contribute actively to the formation and regulation of synaptic circuits, by successfully handling, integrating, and propagating physiological signals of neural networks. The latter occurs mainly by engaging a versatile mechanism of internal Ca2+ fluctuations and regenerative waves prompting targeted release of signaling molecules into the extracellular space. Astroglia also show substantial structural plasticity associated with age- and use-dependent changes in neural circuitry. However, the underlying cellular mechanisms are poorly understood, mainly because of the extraordinary complex morphology of astroglial compartments on the nanoscopic scale. This complexity largely prevents direct experimental access to astroglial processes, most of which are beyond the diffraction limit of optical microscopy. Here we employed super-resolution microscopy (direct stochastic optical reconstruction microscopy; dSTORM), to visualize astroglial organization on the nanoscale, in culture and in thin brain slices, as an initial step to understand the structural basis of astrocytic nano-physiology. We were able to follow nanoscopic morphology of GFAP-enriched astrocytes, which adapt a flattened shape in culture and a sponge-like structure in situ, with GFAP fibers of varied diameters. We also visualized nanoscopic astrocytic processes using the ubiquitous cytosolic astrocyte marker proteins S100β and glutamine synthetase. Finally, we overexpressed and imaged membrane-targeted pHluorin and lymphocyte-specific protein tyrosine kinase (N-terminal domain) -green fluorescent protein (lck-GFP), to better understand the molecular cascades underlying some common astroglia-targeted fluorescence imaging techniques. The results provide novel, albeit initial, insights into the cellular organization of astroglia on the nanoscale, paving the way for function-specific studies. © 2017 Wiley Periodicals, Inc.

Direct Observation of Ordered and Disordered Membrane Domains in B Cell Plasma Membranes using Multi-Color Super-Resolution Fluorescence Microscopy and Application to B Cell Receptor Signaling

Many immune receptors are hypothesized to be correlated with domains of unique membrane composition, sometimes termed “lipid rafts,” which modulate activity of the receptor during the immune response. This compositional heterogeneity is hypothesized to be analogous to liquid ordered/ liquid disordered (lo/ld) phase separation observed in giant unilamellar vesicles and in vesicles harvested from the plasma membrane. However, their existence and behavior has been difficult to measure directly due to the small size of these domains and the small difference between the composition of the domain and the rest of the plasma membrane. Here, we utilize multi-color super-resolution microscopy (STORM and PALM) to quantitate the local density of various ld or lo preferring membrane probes around B cell receptor (BCR) clusters. We show in control measurements that ld or lo preferring membrane probes have differential partitioning around clusters of proteins having strong phase preference. Clusters of lo preferring cholera toxin subunit B are enriched in lo probe and depleted of ld probe. Inversely, clusters of an ld preferring transmembrane peptide are depleted of lo probe and enriched in ld probe. We apply this technique to BCR, which is thought to anchor a raft domain during antigen binding. We find that BCR clusters in chemically fixed and live cells are enriched in lo probe and depleted of ld probe, indicating a lo-like composition is anchored around BCR clusters. We find that this anchored domain is sensitive to the ambient temperature and receptor phosphorylation. These experiments also quantitate the contribution of membrane composition on the interaction between Lyn and the BCR. These results show that lipid-mediated forces can play important roles in organizing proteins during signaling processes.

Translation Microscopy (TRAM) for super-resolution imaging.

Super-resolution microscopy is transforming our understanding of biology but accessibility is limited by its technical complexity, high costs and the requirement for bespoke sample preparation. We present a novel, simple and multi-color super-resolution microscopy technique, called translation microscopy (TRAM), in which a super-resolution image is restored from multiple diffraction-limited resolution observations using a conventional microscope whilst translating the sample in the image plane. TRAM can be implemented using any microscope, delivering up to 7-fold resolution improvement. We compare TRAM with other super-resolution imaging modalities, including gated stimulated emission deletion (gSTED) microscopy and atomic force microscopy (AFM). We further developed novel ‘ground-truth’ DNA origami nano-structures to characterize TRAM, as well as applying it to a multi-color dye-stained cellular sample to demonstrate its fidelity, ease of use and utility for cell biology.

A Hypsochromic Shift Observed in Far-Red Cyanine Dyes Leads to Artifacts in Quantitative Super-Resolution Imaging

Far-red cyanine dyes such as Alexa 647, Cy5, and Atto 647 are favored for multi-color super-resolution microscopy due to their long wavelength fluorescence and their favorable reversible photo-switching characteristics. Conjugation of these dyes to proteins can result in unwanted fluorescence effects such as shifts in absorption bands and fluorescence quenching (1). We additionally observe a ∼200 nm hypsochromic shift in the emission from a small fraction of antibody fragments conjugated to far-red cyanine dyes when probes are excited at 532 nm or 560 nm. This state is nearly undetectable in bulk fluorescent measurements, but is easily detected in live cell super-resolution experiments where we observe a long lived fluorescent state that is resistant to bleaching. This population is present in pre-conjugated secondary antibodies purchased from commercial sources, as well as in antibody fragments conjugated under dilute conditions. Unfortunately, this blue-shifted emission closely resembles that of the red-shifted state of the popular mEOS2/mEOS3.2 photo-switchable probes, making it difficult to distinguish these labels in an imaging experiment. We will describe our efforts to devise practical methods to reduce possible artifacts associated with this state in multi-color super-resolution fluorescence localization measurements.1. Gruber, H.J. et al. Anomalous Fluorescence Enhancement of Cy3 and Cy3.5 versus Anomalous Fluorescence Loss of Cy5 and Cy7 upon Covalent Linking to IgG and Noncovalent Binding to Avidin. Bioconjugate Chem. 11, 696-704 (2000).

Using Multi-Color Super-Resolution Microscopy to Probe the Organization of Dyadic Proteins within Rat Cardiac Myocytes

Cardiac excitation-contraction coupling relies on calcium release from internal stores (the sarcoplasmic reticulum -SR) via ryanodine receptors (RyRs), which are intracellular calcium channels. Most RyRs are organized into clusters within narrow dyadic junctions between sarcolemmal and sarcoplasmic reticulum (SR) membranes. It is still unclear how junctions are formed and maintained. One protein that has been implicated in dyadic junction formation is the cardiac isoform of the junctophilin family, junctophilin-2 (JPH2). With the increased resolution available in multi-color super-resolution microscopy it is now possible to investigate the nanoscale relationship between proteins such as RyRs and JPH2 which are found within the junction. Using single fluorophore localization based super-resolution microscopy we have studied the distribution of a number of such proteins in rat ventricular myocytes.Analysing the peripheral distributions of the cardiac ryanodine receptor (RyR) and a junctional protein, junctophilin-2 (JPH2), we have found that JPH2 was almost completely associated with RyR clusters. Estimates of co-localization of ∼89% between JPH2 and RyRs confirmed near complete colocalization within peripheral couplons. The shape of associated RyR clusters and JPH2 clusters were very similar, suggesting that JPH2 is dispersed throughout RyR clusters and that the packing of JPH2 into junctions and the assembly of RyR clusters are tightly linked. Our data shows, with nanoscale resolution, that junctophilin is not constrained to specific anchoring domains within the junction, but is interspersed throughout the entire area occupied by RyRs.These results demonstrate the utility of fluorescent multi-colour super-resolution immuno-labeling to investigate protein proximity at the nanometer scale. The ability to resolve the association of different molecules into the same biological structure at the nanoscale (here the dyad junction) should give new insight into the assembly and function of macromolecular signaling complexes.

Quantitative Multicolor Super-Resolution Microscopy Reveals Tetherin HIV-1 Interaction

Virus assembly and interaction with host-cell proteins occur at length scales below the diffraction limit of visible light. Novel super-resolution microscopy techniques achieve nanometer resolution of fluorescently labeled molecules. The cellular restriction factor tetherin (also known as CD317, BST-2 or HM1.24) inhibits the release of human immunodeficiency virus 1 (HIV-1) through direct incorporation into viral membranes and is counteracted by the HIV-1 protein Vpu. For super-resolution analysis of HIV-1 and tetherin interactions, we established fluorescence labeling of HIV-1 proteins and tetherin that preserved HIV-1 particle formation and Vpu-dependent restriction, respectively. Multicolor super-resolution microscopy revealed important structural features of individual HIV-1 virions, virus assembly sites and their interaction with tetherin at the plasma membrane. Tetherin localization to micro-domains was dependent on both tetherin membrane anchors. Tetherin clusters containing on average 4 to 7 tetherin dimers were visualized at HIV-1 assembly sites. Combined biochemical and super-resolution analysis revealed that extended tetherin dimers incorporate both N-termini into assembling virus particles and restrict HIV-1 release. Neither tetherin domains nor HIV-1 assembly sites showed enrichment of the raft marker GM1. Together, our super-resolution microscopy analysis of HIV-1 interactions with tetherin provides new insights into the mechanism of tetherin-mediated HIV-1 restriction and paves the way for future studies of virus-host interactions.

10.2172/1673450

10.2172/1673450