Multicolor Stochastic Optical Reconstruction Microscopy Research Articles

RegiSTORM: channel registration for multi-color stochastic optical reconstruction microscopy

BackgroundStochastic optical reconstruction microscopy (STORM), a super-resolution microscopy technique based on single-molecule localizations, has become popular to characterize sub-diffraction limit targets. However, due to lengthy image acquisition, STORM recordings are prone to sample drift. Existing cross-correlation or fiducial marker-based algorithms allow correcting the drift within each channel, but misalignment between channels remains due to interchannel drift accumulating during sequential channel acquisition. This is a major drawback in multi-color STORM, a technique of utmost importance for the characterization of various biological interactions.ResultsWe developed RegiSTORM, a software for reducing channel misalignment by accurately registering STORM channels utilizing fiducial markers in the sample. RegiSTORM identifies fiducials from the STORM localization data based on their non-blinking nature and uses them as landmarks for channel registration. We first demonstrated accurate registration on recordings of fiducials only, as evidenced by significantly reduced target registration error with all the tested channel combinations. Next, we validated the performance in a more practically relevant setup on cells multi-stained for tubulin. Finally, we showed that RegiSTORM successfully registers two-color STORM recordings of cargo-loaded lipid nanoparticles without fiducials, demonstrating the broader applicability of this software.ConclusionsThe developed RegiSTORM software was demonstrated to be able to accurately register multiple STORM channels and is freely available as open-source (MIT license) at https://github.com/oystein676/RegiSTORM.git and https://doi.org/10.5281/zenodo.5509861 (archived), and runs as a standalone executable (Windows) or via Python (Mac OS, Linux).

Targeting the Microtubule EB1-CLASP2 Complex Modulates NaV1.5 at Intercalated Discs.

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Coupling of Distinct Ion Channel Types in Neurons Mediated by AKAP79/150

M-type K+ channels, comprised of KCNQ2-5 (Kv7.2-7.5) subunits, play key roles in the regulation of neuronal excitability in the nervous system. In diverse neurons, L-type Ca2+ channels (LTCCs) drive transcriptional regulation via NFAT transcription factors, and in sensory neurons, TRPV1 cation channels excite neurons in response to heat, acidity or chemical ligands, driving nociception. The A-kinase-anchoring protein (AKAP)79/150 has been shown to orchestrate regulation of all three types of channels by PKC, PKA, calcineurin and NFATs. Using stochastic optical reconstruction microscopy (STORM) super-resolution microscopy, we have directly visualized individual signaling complexes containing AKAP79/150, these three ion channels and G protein-coupled receptors in neurons and tissue-culture cells. Using multi-color STORM, we observe AKAP150-mediated clustering of KCNQ, LTCCs and TRPV1 channels at the single-complex level. Thus, AKAP79/150 links different channel types together, raising the possibility of their functional, as well as physical, coupling. In sensory neurons, capsaicin caused PIP2 hydrolysis by TRPV1 activation. In neurons isolated from AKAP150+/+ mice, brief application of low concentrations of capsaicin (100 nM), which we believe triggers only local PIP2 depletion, induced ∼40% suppression of M-current (IM), suggesting close localization of TRPV1 and M-channels, the latter thus suppressed by TRPV1-induced local PIP2 depletion. However, in AKAP150-/- neurons, IM was not affected by this modest activation of TRPV1 channels, implying the critical role of AKAP79/150. Application of the LTCC blocker, nifedipine, but not the N-type Ca2+ channel blocker, ω-conotoxin GVIA, significantly suppressed desensitization and tachyphylaxis of TRPV1 currents, suggesting the functional coupling of LTCCs with TRPV1 channels, consistent with their physical coupling at the single-complex level seen with STORM. We thus find AKAP79/150 mediates physical and functional coupling of these three ion channels in sensory neurons, indicating physiological roles in tuning the nociceptive response to painful stimuli.

Cross-talk-free multi-color STORM imaging using a single fluorophore.

Multi-color stochastic optical reconstruction microscopy (STORM) is routinely performed; however, the various approaches for achieving multiple colors have important caveats. Color cross-talk, limited availability of spectrally distinct fluorophores with optimal brightness and duty cycle, incompatibility of imaging buffers for different fluorophores, and chromatic aberrations impact the spatial resolution and ultimately the number of colors that can be achieved. We overcome these complexities and develop a simple approach for multi-color STORM imaging using a single fluorophore and sequential labelling. In addition, we present a simple and versatile method to locate the same region of interest on different days and even on different microscopes. In combination, these approaches enable cross-talk-free multi-color imaging of sub-cellular structures.

Dual Regulation of M-Type K+ Channels by AKAP79/150 Signaling Complexes

M-type K+ channels, comprised of KCNQ2-5 (Kv7.2-7.5) subunits, play key roles in the regulation of neuronal excitability. We study the regulation of M-channel activity and transcriptional expression by A-kinase-anchoring protein (AKAP)79/150. Our FRET and functional studies suggest AKAP79/150 action correlates with the PIP2-depletion mode of neuronal IM suppression, and is disrupted by functional calmodulin. The modulation of M current involves recruitment of PKC to the channels, their subsequent phosphorylation, and likely decreased affinity for PIP2. The complex orchestrated by AKAP79/150 also includes M1 muscarinic receptors, thus constituting a “signalosome” spanning the initiating receptor to channel target. We have discovered a novel mechanism regulating KCNQ2/3 transcriptional expression by neuronal activity, involving calcineurin and Nuclear Factor of Activated T-cells (NFAT) transcription factors, orchestrated by AKAP79/150. The signal requires Ca2+ influx through L-type Ca2+ channels and both local and global Ca2+ elevations. AKAP79/150 and the complex it organizes thus mediate activity-dependent M-channel transcription, which may potentially serve throughout the nervous system to limit over-excitability associated with disease states such as epilepsy. We are now utilizing stochastic optical reconstruction microscopy (STORM) offering sub-diffraction (∼20 nm) resolution, to directly visualize these AKAP79/150 signaling complexes and interactions between AKAP79/150, ion channels and receptors in neurons. STORM uses dyes that can cycle between a dark and a fluorescent state thousands of times, thus enabling detection of the precise localization of the center of these scattered spots given by cumulative integration of each cycle. Using multi-color STORM to simultaneously image AKAP150, KCNQ2-3 or KCNQ5 channels, and receptors, we observe AKAP150 to form signaling clusters with the channels and receptors at the single-complex level. We have also obtained evidence that AKAP79/150 links different channel types together, raising the possibility of their functional, as well as physical, coupling.

Stochastic Optical Reconstruction Microscopy (STORM): A Method for Superresolution Fluorescence Imaging

The relatively low spatial resolution of the optical microscope presents significant limitations for the observation of biological ultrastructure. Subcellular structures and molecular complexes essential for biological function exist on length scales from nanometers to micrometers. When observed with light, however, structural features smaller than ∼0.2 µm are blurred and are difficult or impossible to resolve. In this article, we describe stochastic optical reconstruction microscopy (STORM), a method for superresolution imaging based on the high accuracy localization of individual fluorophores. It uses optically switchable fluorophores: molecules that can be switched between a nonfluorescent and a fluorescent state by exposure to light. The article discusses photoswitchable fluorescent molecules, STORM microscope design and the imaging procedure, data analysis, imaging of cultured cells, multicolor STORM, and three-dimensional (3D) STORM. This approach is generally applicable to biological imaging and requires relatively simple experimental apparatus; its spatial resolution is theoretically unlimited, and a resolution improvement of an order of magnitude over conventional optical microscopy has been experimentally demonstrated.

Super-Resolution Imaging of AKAP79/150 Signaling Complexes using Stochastic Optical Reconstruction Microscopy (STORM)

Our previous FRET and functional studies suggest AKAP (A-kinase-anchoring-protein) 79/150 to be crucial in modulation of ion channel activity and neuronal function, by orchestrating important molecules such as protein kinases, phosphatases, G protein-coupled receptors and ion channels into signaling complexes in the plasma membrane. In this study, to directly visualize these AKAP79/150 signaling complexes and interactions between AKAP79/150, ion channels and receptors in sympathetic neurons, we utilized stochastic optical reconstruction microscopy (STORM) with sub-diffraction (∼20 nm half width) resolution. STORM uses dyes that can cycle between a dark and a fluorescent state thousands of times, thus enabling detection of the precise localization of the center of these scattered spots given by cumulative integration of each cycle. Consistent with previous immunostaining/confocal studies, STORM imaging of fixed Chinese hamster ovary (CHO) cells using fluorescently labeled antibodies against AKAP150, or KCNQ2 and KCNQ3 subunits revealed the precise plasma membrane localization for the former, and membrane and cytoplasmic distribution of the latter, at the single-molecule level. using multi-color STORM to simultaneous image AKAP150, KCNQ2-3 and Gq/11-coupled muscarinic M1 receptors, further experiments on sympathetic neurons isolated from AKAP150+/+ or AKAP150-/- mice will shed light on the spatial co-distribution of AKAP150 with its signaling partners, and whether loss of AKAP150 causes redistribution of ion channels and receptors.

Imaging cellular structure across scales with correlated light, superresolution, and electron microscopy

Recent advances in fluorescence and electron microscopy (FM and EM) are closing the resolution gap between the two technologies, allowing cellular structures to be studied across the scales between structural and cell biology. FM and EM not only cover different scales, they provide complementary information. FM provides dynamic information, allows the imaging of large volumes, and is specific for the fluorescently labeled protein. EM, on the other hand, while imaging static and smaller volumes, offers contextual information, because the nonlabeled components of the cell are also seen. This working group presented methods and applications that bridge the gaps in resolution and integrate information from different FM and EM imaging modalities. John Briggs discussed motivations and methods for correlating FM and EM. He introduced sample preparation for transmission EM, comparing high-contrast overviews available after plastic-embedding with low-contrast, high-resolution structural views from cryo-EM. When correlative FM/EM is required, FM can be carried out before or after EM sample preparation. FM before sample preparation allows live imaging, but movements or distortions may occur between the FM and EM images. FM after sample preparation requires preservation of the FM signal during preparation. Methods and applications from different laboratories were presented (e.g., Muller-Reichert et al., 2007 ; Nixon et al., 2009 ; van Driel et al., 2009 ; Jun et al., 2011 ), as was the application of a sensitive, precise correlative FM–EM method (Kukulski et al., 2011 ) to study spatiotemporal dynamics of membrane bending during yeast endocytosis. Melike Lakadamyali presented an overview of superresolution FM techniques, such as stimulated emission depletion microscopy (STED), stochastic optical reconstruction microscopy (STORM), and photoactivation localization microscopy (PALM), and their extension to three-dimensional, multicolor, and live-cell imaging (Hell, 2009 ; Huang et al., 2010 ; Patterson et al., 2010 ). She also discussed their limitations, particularly in imaging living cells (e.g., trade-off between temporal and spatial resolution). Finally, she gave two examples of biological applications of superresolution: 1) using three-dimensional, multicolor STORM provides improved accuracy in neuronal tracing in the context of connectomics (Lakadamyali et al., 2012 ), and 2) correlating fast organelle dynamics with superresolution images of the cytoskeleton can reveal the biophysical mechanisms of organelle transport (Balint and Lakadamyali, unpublished). Stephen Smith of Stanford University's Department of Molecular and Cellular Physiology highlighted array tomography, a versatile method that can potentially reveal the synaptic connectivity of neurons at the light and electron microscopic scale (Micheva and Smith, 2007 ). Cutting ultrathin tissue sections and immunostaining them with multiple rounds of antibodies allow imaging of a large number of synaptic molecules. Using array tomography, the Smith laboratory is getting the first glimpse into the rich molecular diversity of synapses and searching for principles that govern the complex molecular architectures of synapses and synaptic circuits (Micheva et al., 2010 ). Array tomography is also compatible with EM and superresolution FM and allows correlating molecular and ultrastructural information across different scales. Finally, Erik Jorgensen of the Biology Department at the University of Utah gave an example of how molecular content of cells captured by superresolution FM can be correlated with ultrastructural information derived using scanning EM. Having previously correlated STED-based methods with EM, he described the correlation of PALM-based superresolution methods with scanning EM (Watanabe et al., 2011 ). This method allowed the imaging of specific ultrastructural features of synapses within Caenorhabditis elegans. He also introduced a three-dimensional PALM-based system capable of generating images with resolution of 20 nm in the plane of the image and 50 nm in the axial dimension.

Super-Resolution Imaging of Membrane Topology using Small Molecule Lipophilic Dye

Plasma membrane of any cell supports a vast array of biomolecules on its surface. These are crucial in response and adaptation to the external environmental cues of the cell. The process of determining these cues is done by a plethora of plasma membrane associated proteins including receptors, channels and pores. The spatial organization including compartmentalization of these proteins is critical for cellular function. In order to generate a high spatial resolution topological map of a plasma membrane we use wide-field fluorescence super-resolution imaging technique known as Stochastic optical reconstruction microscopy (STORM). STORM works on the principle of stochastic switching of single-molecule fluorophores and precise localization of the centers of these spots. Here we labeled the plasma membrane using a small molecule lipophilic carbocyanine dye, DiD, which can photoswitch under similar conditions as previously used for Cy5 photoswitching. We stained Drosophila Schneider 2 cells with DiD and imaged them under both fixed and live conditions using 3D STORM imaging and/or single-particle tracking modes. We observed plasma membrane features including clathrin coated pit-like structures. Further experiments using multicolor STORM to image DiD together with membrane proteins will shed light on how membrane protein distribution is correlated with membrane topology.

Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection.

Understanding the complexity of the cellular environment will benefit from the ability to unambiguously resolve multiple cellular components, simultaneously and with nanometer-scale spatial resolution. Multicolor super-resolution fluorescence microscopy techniques have been developed to achieve this goal, yet challenges remain in terms of the number of targets that can be simultaneously imaged and the crosstalk between color channels. Herein, we demonstrate multicolor stochastic optical reconstruction microscopy (STORM) based on a multi-parameter detection strategy, which uses both the fluorescence activation wavelength and the emission color to discriminate between photo-activatable fluorescent probes. First, we obtained two-color super-resolution images using the near-infrared cyanine dye Alexa 750 in conjunction with a red cyanine dye Alexa 647, and quantified color crosstalk levels and image registration accuracy. Combinatorial pairing of these two switchable dyes with fluorophores which enhance photo-activation enabled multi-parameter detection of six different probes. Using this approach, we obtained six-color super-resolution fluorescence images of a model sample. The combination of multiple fluorescence detection parameters for improved fluorophore discrimination promises to substantially enhance our ability to visualize multiple cellular targets with sub-diffraction-limit resolution.

Imaging Redistribution Dynamics of IgE Receptors and Signaling Partners at the Nanoscale with Storm

In mast cells, crosslinking of IgE bound to the FcεR1 receptor with multivalent antigen initiates cell activation and consequent inflammatory responses. Clustered IgE-FcεR1 interacts with early signaling partners such as Lyn, Syk, and LAT, initiating a signaling cascade. Our previous scanning electron microscopy (SEM) studies quantified the nanoscale co-redistribution of FcRI with Lyn, Syk, and LAT within the plasma membrane upon antigen stimulation. In this study, we use stochastic optical reconstruction microscopy (STORM) to localize proteins on the cell surface with sub-diffraction (∼20nm half width) resolution using a simple fluorescence microscope set up with TIRF illumination. We present, in snapshots, the time-dependence of FcRI reorganization after antigen addition in chemically fixed cells and quantify the extent of clustering using the pair autocorrelation function. The results of STORM measurements agree well with conclusions from SEM experiments. With multi-color STORM, we image the distributions of multiple protein species on the same cell and calculate the degree of protein co-clustering, before and after antigen stimulation, using the pair cross-correlation function. Ongoing experiments are aimed at quantifying the co-redistribution of FcRI with early signaling partners. We have also extended this technique to imaging living cells at room temperature to measure FcεRI redistribution in real time and study the organization and dynamics of proteins undergoing stimulated responses. Our results confirm that STORM is an effective tool for quantitative imaging of cellular components at the nanoscale, and our ongoing studies are providing new information to clarify the physical basis for spatial assembly of specific proteins in the plasma membrane during early signaling events.

Mapping Neuronal Connectivity Using Stochastic Optical Reconstruction Microscopy (Storm): The Brainstorm Project

The human brain is a highly sophisticated circuit consisting of hundreds of billions of neurons that are interconnected by an even larger number of synapses. This dense network of neurons and their connections holds key information to understanding normal brain function and perhaps what underlies its disorders. Obtaining a physical map of the brain's connectivity, however, is highly challenging due to the small size and high density of neuronal processes within a given volume. Therefore, in order to generate a map of neuronal connectivity a technique that can provide high spatial resolution and molecular specificity is needed.We are using 3D multi-color stochastic optical reconstruction microscopy (STORM) in order to trace neuronal networks in culture at high spatial resolution. In order to capture an entire network of connections, we are using an automated, motorized piezo stage to image large areas in x-y (≥ 120x120 μm) as well as in z (≥ 2 μm). With this technique we can not only outline neuronal morphology at 30 μm lateral and 50 μm axial resolution, but we can also image synaptic content with high molecular specificity and identify synaptic connections. These techniques will be greatly useful for generating connectional maps of neurons in the mammalian brain and help obtain a physical understanding behind brain function.

Multicolor super-resolution imaging with photo-switchable fluorescent probes.

Recent advances in far-field optical nanoscopy have enabled fluorescence imaging with a spatial resolution of 20 to 50 nanometers. Multicolor super-resolution imaging, however, remains a challenging task. Here, we introduce a family of photo-switchable fluorescent probes and demonstrate multicolor stochastic optical reconstruction microscopy (STORM). Each probe consists of a photo-switchable "reporter" fluorophore that can be cycled between fluorescent and dark states, and an "activator" that facilitates photo-activation of the reporter. Combinatorial pairing of reporters and activators allows the creation of probes with many distinct colors. Iterative, color-specific activation of sparse subsets of these probes allows their localization with nanometer accuracy, enabling the construction of a super-resolution STORM image. Using this approach, we demonstrate multicolor imaging of DNA model samples and mammalian cells with 20- to 30-nanometer resolution. This technique will facilitate direct visualization of molecular interactions at the nanometer scale.