Super-resolution Fluorescence Localization Microscopy Research Articles

Probing the Role of Actin in Membrane Organization

The actin cortex has long been hypothesized to organize membrane components, especially ordered membrane domains thought to play important functional roles at the plasma membrane. Recent developments in super-resolution fluorescence localization microscopy have enabled imaging of the actin cortex in chemically fixed cells through the use of fluorophores that spatially sample filaments at densities required to trace actin meshworks. This poster describes our ongoing efforts to quantify how actin impacts the domain structure of cell plasma membranes, utilizing these actin binding probes in conjunction with well characterized peptide markers of membrane domains. We aim to employ super-resolution imaging to validate past studies, which have characterized meshwork compartment sizes in several cell types based on diffusion data. The majority of our studies are conducted in B cells, where past work indicates that actin structure plays important roles in both antigen mediated B cell receptor signaling and in the tonic signaling that occurs in the absence of ligands. Through this approach, we explore the extent to which these signaling functions are impacted by coupling between actin and membrane domains.

Malic Acid Carbon Dots: From Super-resolution Live-Cell Imaging to Highly Efficient Separation.

As-synthesized malic acid carbon dots are found to possess photoblinking properties that are outstanding and superior compared to those of conventional dyes. Considering their excellent biocompatibility, malic acid carbon dots are suitable for super-resolution fluorescence localization microscopy under a variety of conditions, as we demonstrate in fixed and live trout gill epithelial cells. In addition, during imaging experiments, the so-called "excitation wavelength-dependent" emission was not observed for individual as-made malic acid carbon dots, which motivated us to develop a time-saving and high-throughput separation technique to isolate malic acid carbon dots into fractions of different particle size distributions using C18 reversed-phase silica gel column chromatography. This post-treatment allowed us to determine how particle size distribution influences the optical properties of malic acid carbon dot fractions, that is, optical band gap energies and photoluminescence behaviors.

Probing membrane‐mediated forces between proteins in cells using super‐resolution fluorescence localization imaging.

Transmembrane and peripheral membrane proteins can interact directly via mechanisms common to soluble proteins, but also can feel forces mediated through their embedding membrane through mechanisms involving membrane curvature, electrostatics, as well as their preference for phase‐like membrane domains. In this presentation, I will discuss our recent efforts to probe effective interactions between plasma membrane proteins using super‐resolution fluorescence localization microscopy paired with a quantitative cross‐correlation analysis in both live and chemically fixed cells. In particular, we find that, in a B cell line, ordered phase‐like domains can sort plasma membrane kinases and phosphatases, establishing localized hotspots for tyrosine phosphorylation. We also use peptide probes that contain membrane proximal charged residues to explore the roles of membrane electrostatics in modulating the lateral distribution of proteins on the cell surface. Overall, we argue that cells use these interaction modalities that are unique to membranes in order to influence the biochemical networks that occur on the cell surface in ways that contribute a broad array of cellular sensing and signaling cascades.Support or Funding InformationResearch was funded by the National Institutes of Health (NIH) (R01 GM110052) and the the National Science Foundation (NSF) (MCB 1552439).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phases and Fluctuations in Biological Membranes

Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma membrane lipids and proteins that resemble liquid-ordered phase separated domains in model membranes. However, this concept remains controversial due to the difficulty in directly observing these domains in intact cells. Here, we visualize ordered and disordered phase-like domains in intact B cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, establish that ordered domains are local environments that favor tyrosine phosphorylation, and present a minimal and predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling as well as a plausible mechanism of BCR activation via receptor clustering. More generally, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that may broadly impact signal transduction.

A Hands-On Freshman Seminar Course in DNA Origami

In the fall semester of 2016, the biophysics program at the University of Michigan offered a hands-on seminar course in the theory and practice of DNA origami aimed at first year undergraduate students with varying scientific backgrounds. The course began with lectures on theoretical underpinnings of self-assembly, and quickly moved into group work where students learned to design DNA origami structures using freely accessible software tools (cadnano: http://cadnano.org/ and CanDo: http://cando-dna-origami.org/). Students then assembled an instructor-designed single layer origami and characterized its assembly using electrophoresis, atomic force microscopy (AFM) and super-resolution fluorescence localization microscopy. The course ended with discussions of current research aimed at using origami and other self-organizing systems to approach fundamental problems in biology and medicine. The goal of the course is to introduce young students to several fundamental biophysical concepts and approaches. This poster will discuss the positive and negative outcomes of this class, as well as several obstacles encountered in course design and implementation.

Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes.

Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma membrane lipids and proteins that resemble liquid-ordered phase-separated domains in model membranes. This concept remains controversial and lacks direct experimental support in intact cells. Here, we visualize ordered and disordered domains in mouse B lymphoma cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, and present a minimal, predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling and a plausible mechanism of BCR activation via receptor clustering that could be generalized to other signaling pathways. Overall, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that broadly impact signal transduction.

Nanoscale Membrane Buds Induced by CTxB-GM1 in One Component Bilayer Detected by Polarized Localization Microscopy (PLM)

Membrane biomolecules binding, organization, and clustering play a vital role in cell function. For instance, Cholera toxin subunit B (CTxB) binds to the ganglioside (GM1) and preferentially partitions on highly curved membranes as an initial stage for its internalization into the cell. Further, CTxB-GM1 demonstrates confined diffusion at nanoscale sites in synthetic bilayers and cells. In our attempt to understand the mechanism of the CTxB-GM1 complex, we introduced CTxB on a quasi-one component bilayer of 99.4% POPC + 1:1 GM1/DiI while imaging membrane topology and protein dynamics by Polarized Localization Microscopy (PLM). PLM, a super-resolution technique developed in our lab, combines super-resolution fluorescence localization microscopy (FLM) with polarized total internal reflection fluorescence microscopy (pTIRFM) to reveal nanoscale membrane orientation. In particular, PLM enabled the detection of nanoscale membrane budding events that initiated upon adding CTxB. Single particle tracking (SPT) was then performed on lipid (DiI) and CTxB trajectories revealing correlated diffusion between CTxB and the membrane structure. Confined CTxB diffusion was observed at the nanoscale buds locations. In this study, we obtained super-resolution, time-lapse images of CTxB locations and the corresponding membrane topology. CTxB was recruited to the negative Gaussian curved membrane; single-event and correlation analysis demonstrated increased clustering of lipids as function of CTxB incubation time with the membrane. Finally, we explored the effect of changing CTxB concentration on the bud formation process and bud sizes. The presented study is the first known report of bud formation induced by CTxB-GM1 in one component bilayer and in the absence of lipid phases. Our work demonstrates a possible mechanism for CTxB internalization and confinement in cells. PLM and this discovery may open up new avenues in the investigation of the processes governing cell signaling triggered by bacterial toxins.

Correlative Fluorescence Super-Resolution Localization Microscopy and Platinum Replica EM on Unroofed Cells.

Platinum replicas of unroofed mammalian cells can be imaged with a transmission electron microscope (TEM) to produce high contrast, high resolution images of the structure of the cytoplasmic side of a plasma membrane. A complementary approach, super-resolution fluorescence localization microscopy, can be used to localize labeled molecules with better than 20nm precision in cells. Here, we describe a correlative method that couples these two techniques and produces images where localization microscopy data can be used to highlight specific proteins of interest within the structural context of the platinum replica TEM image. This combined method is uniquely suited to investigate the nanometer-scale structural organization of the plasma membrane and its associated organelles and proteins.

Chemomechanical Interactions Enhance IgE-FcεRI Signaling in Mast Cells

Cells respond to their physical environment and to chemical stimuli in terms of collective molecular interactions that are regulated in time and space. Small molecules may engage specific receptors to initiate a transmembrane signal, and the system amplifies this nanoscale interaction to microscale assemblies within the cell and often to longer length scales involving surrounding tissue and ultimately the whole organism. A striking example of signal integration over multiple length scales is the allergic immune response. IgE receptors (FceRI) on mast cells are the gatekeepers of this response, and this system has proven to be a valuable model for investigating receptor-mediated cellular activation. Spanning the range of cellular responses we use super resolution fluorescence localization microscopy to investigate the earliest signaling events and ligands patterned in micron size features together with confocal microscopy to investigate early and later events. We are characterizing distinctive regulatory roles played by the actin cytoskeleton at different stages. We are further investigating spatial redistributions of the cellular integrins that relate to the enhancement of signaling observed for cells spreading on surfaces.

Super-Resolution Microscopy Reveals Presynaptic Localization of the ALS/FTD Related Protein FUS in Hippocampal Neurons.

Fused in Sarcoma (FUS) is a multifunctional RNA-/DNA-binding protein, which is involved in the pathogenesis of the neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A common hallmark of these disorders is the abnormal accumulation of mutated FUS protein in the cytoplasm. Under normal conditions FUS is confined to the nuclear compartment, in neurons, however, additional somatodendritic localization can be observed. In this study, we carefully analyzed the subcellular localization of endogenous FUS at synaptic sites of hippocampal neurons which are among the most affected cell types in FTD with FUS pathology. We could confirm a strong nuclear localization of FUS as well as its prominent and widespread neuronal expression throughout the adult and developing rat brain, particularly in the hippocampus, the cerebellum and the outer layers of the cortex. Intriguingly, FUS was also consistently observed at synaptic sites as detected by neuronal subcellular fractionation as well as by immunolabeling. To define a pre- and/or postsynaptic localization of FUS, we employed super-resolution fluorescence localization microscopy. FUS was found to be localized within the axon terminal in close proximity to the presynaptic vesicle protein Synaptophysin1 and adjacent to the active zone protein Bassoon, but well separated from the postsynaptic protein PSD-95. Having shown the presynaptic localization of FUS in the nervous system, a novel extranuclear role of FUS at neuronal contact sites has to be considered. Since there is growing evidence that local presynaptic translation might also be an important mechanism for plasticity, FUS – like the fragile X mental retardation protein FMRP – might act as one of the presynaptic RNA-binding proteins regulating this machinery. Our observation of presynaptic FUS should foster further investigations to determine its role in neurodegenerative diseases such as ALS and FTD.

Optimized two-color super resolution imaging of Drp1 during mitochondrial fission with a slow-switching Dronpa variant.

We studied the single-molecule photo-switching properties of Dronpa, a green photo-switchable fluorescent protein and a popular marker for photoactivated localization microscopy. We found the excitation light photoactivates as well as deactivates Dronpa single molecules, hindering temporal separation and limiting super resolution. To resolve this limitation, we have developed a slow-switching Dronpa variant, rsKame, featuring a V157L amino acid substitution proximal to the chromophore. The increased steric hindrance generated by the substitution reduced the excitation light-induced photoactivation from the dark to fluorescent state. To demonstrate applicability, we paired rsKame with PAmCherry1 in a two-color photoactivated localization microscopy imaging method to observe the inner and outer mitochondrial membrane structures and selectively labeled dynamin related protein 1 (Drp1), responsible for membrane scission during mitochondrial fission. We determined the diameter and length of Drp1 helical rings encircling mitochondria during fission and showed that, whereas their lengths along mitochondria were not significantly changed, their diameters decreased significantly. These results suggest support for the twistase model of Drp1 constriction, with potential loss of subunits at the helical ends.

Localization microscopy: mapping cellular dynamics with single molecules

Resolution describes the smallest details within a sample that can be recovered by a microscope lens system. For optical microscopes detecting visible light, diffraction limits the resolution to ∼200-250 nm. In contrast, localization measures the position of an isolated object using its image. Single fluorescent molecules can be localized with an uncertainty of a few tens of nanometres, and in some cases less than one nanometre. Superresolution fluorescence localization microscopy (SRFLM) images and localizes fluorescent molecules in a sample. By controlling the visibility of the fluorescent molecules with light, it is possible to cause a sparse subset of the tags to fluoresce and be spatially separated from each other. A movie is acquired with a camera, capturing images of many sets of visible fluorescent tags over a period of time. The movie is then analysed by a computer whereby all of the single molecules are independently measured, and their positions are recorded. When the coordinates of a sufficient number of molecules are collected, an image can be rendered by plotting the coordinates of the localized molecules. The spatial resolution of these rendered images can be better than 20 nm, roughly an order of magnitude better than the diffraction limited resolution. The invention of SRFLM has led to an explosion of related techniques. Through the use of specialized optics, the fluorescent signal can be split into multiple detection channels. These channels can capture additional information such as colour (emission wavelength), orientation and three-dimensional position of the detected molecules. Measurement of the colour of the detected fluorescence can allow researchers to distinguish multiple types of fluorescent tags and to study the interaction between multiple molecules of interest. Three-dimensional imaging and determination of molecular orientations offer insight into structural organization of the sample. SRFLM is compatible with living samples and has helped to illuminate many dynamic biological processes, such as the trajectories of molecules within living cells. This review discusses the concept and process of SRFLM imaging and investigates recent advances in SRFLM functionality. Since its announcement in 2006, SRFLM has been quickly adopted and modified by many researchers to help investigate questions whose answers lie below the diffraction limit. The versatility of the SRFLM technique has great promise for improving our understanding of cell biology at the molecular level.

Phases and Fluctuations in Biological Membranes

Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma membrane lipids and proteins that resemble liquid-ordered phase separated domains in model membranes. However, this concept remains controversial due to the difficulty in directly observing these domains in intact cells. Here, we visualize ordered and disordered phase-like domains in intact B cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, establish that ordered domains are local environments that favor tyrosine phosphorylation, and present a minimal and predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling as well as a plausible mechanism of BCR activation via receptor clustering. More generally, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that may broadly impact signal transduction.

Super-Resolution Localization Microscopy Identifies Distinct Stages of Antigen-Induced IgE Receptor Cross-Linking and Immobilization in Rbl-2H3 Mast Cells

Cross-linking of immunoglobulin E (IgE) bound to its receptor, FceRI, by multivalent antigen initiates a transmembrane signaling cascade essential for mast cell activation and important for inflammatory immune responses and allergic disease. In this study, we apply super-resolution fluorescence localization microscopy to record receptor organization and dynamics on live RBL-2H3 mast cells undergoing antigen-mediated signaling, allowing us to measure nanoscale clustering and diffusion of FceRI simultaneously. Through comparison of cross-linking-induced changes in these properties as a function of time, we are able to resolve two distinct temporal phases of receptor clustering and immobilization. Additionally, we correlate the time-dependence of the distinct phases with a functional signaling response, Ca2+ mobilization. In the first phase of receptor clustering and immobilization, receptors slow dramatically with a relatively small average increase in clustering, and individual receptors appear to transiently associate with small clusters. This first phase occurs before Ca2+ mobilization and concurrently with initial signaling steps. At later times, receptor-rich clusters become increasingly dense while receptors remain predominately immobilized. These latter behaviors are observed at times following the initiation of the Ca2+ response, and we conclude that although cross-linking is necessary for commencement of downstream signaling, receptor assembly into large, densely packed clusters at later times is likely associated with termination of the stimulated response. These findings motivate future study of the physical interactions that give rise to the observed changes in receptor organization and mobility, and how these translate into cellular functions. In ongoing experiments, we are exploring the requirements of signaling for receptor cross-linking through the use of antigens with controlled structure and valency, and we will correlate our observations with functional responses such as Ca2+ mobilization and receptor association with downstream signaling partners.

Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution.

Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of <20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures.

Inhibitors of SCF-Skp2/Cks1 E3 Ligase Block Estrogen-Induced Growth Stimulation and Degradation of Nuclear p27kip1: Therapeutic Potential for Endometrial Cancer

In many human cancers, the tumor suppressor, p27(kip1) (p27), a cyclin-dependent kinase inhibitor critical to cell cycle arrest, undergoes perpetual ubiquitin-mediated proteasomal degradation by the E3 ligase complex SCF-Skp2/Cks1 and/or cytoplasmic mislocalization. Lack of nuclear p27 causes aberrant cell cycle progression, and cytoplasmic p27 mediates cell migration/metastasis. We previously showed that mitogenic 17-β-estradiol (E2) induces degradation of p27 by the E3 ligase Skp1-Cullin1-F-Box- S phase kinase-associated protein2/cyclin dependent kinase regulatory subunit 1 in primary endometrial epithelial cells and endometrial carcinoma (ECA) cell lines, suggesting a pathogenic mechanism for type I ECA, an E2-induced cancer. The current studies show that treatment of endometrial carcinoma cells-1 (ECC-1) with small molecule inhibitors of Skp2/Cks1 E3 ligase activity (Skp2E3LIs) stabilizes p27 in the nucleus, decreases p27 in the cytoplasm, and prevents E2-induced proliferation and degradation of p27 in endometrial carcinoma cells-1 and primary ECA cells. Furthermore, Skp2E3LIs increase p27 half-life by 6 hours, inhibit cell proliferation (IC50, 14.3μM), block retinoblastoma protein (pRB) phosphorylation, induce G1 phase block, and are not cytotoxic. Similarly, using super resolution fluorescence localization microscopy and quantification, Skp2E3LIs increase p27 protein in the nucleus by 1.8-fold. In vivo, injection of Skp2E3LIs significantly increases nuclear p27 and reduces proliferation of endometrial epithelial cells by 42%-62% in ovariectomized E2-primed mice. Skp2E3LIs are specific inhibitors of proteolytic degradation that pharmacologically target the binding interaction between the E3 ligase, SCF-Skp2/Cks1, and p27 to stabilize nuclear p27 and prevent cell cycle progression. These targeted inhibitors have the potential to be an important therapeutic advance over general proteasome inhibitors for cancers characterized by SCF-Skp2/Cks1-mediated destruction of nuclear p27.

Super-Resolution Imaging of IgE Receptor Clustering Initiated by Structurally-Defined Ligands in Rbl Mast Cells

Cross-linking of IgE bound to its receptor, FceRI, by multivalent ligands initiates a transmembrane signaling cascade that activates allergic and inflammatory responses in mast cells. Super-resolution fluorescence localization microscopy is an effective tool for quantitative nanoscale imaging of the distribution and ligand-induced redistribution of FceRI. We use structurally-defined trivalent ligands specific for anti-dinitrophenyl (DNP) IgE, for which the ligand size and valency is precisely controlled, to cross-link IgE-FceRI. These ligands are based on a Y-shaped double-stranded DNA scaffold with DNP groups conjugated to each of the three 5’ ends. The distance between DNP groups is fixed by the length of single-stranded oligonucleotides annealed to form the Y-shaped structure. As described previously for these Yn-DNP3 ligands, (Sil et al., 2007, ACS Chem Biol) some (but not other) cell signaling pathways depend on inter-DNP distances, ranging from 5 – 15 nm, which constrain the proximity of ligand-bound IgE-receptors. We are investigating the organization and dynamics of ligand-induced IgE-receptor clustering to gain new information about interactions of cross-linked IgE-receptors within the membrane that accompany or limit transmembrane signaling. We image fixed and live cells and compare Yn-DNP3 ligands with the heterogeneous ligand, DNP20-BSA. IgE is labeled with a photo-switchable probe and IgE-receptor clustering is quantified in super-resolution images with spatial correlation functions. In living cells, we record single-receptor trajectories, and receptor mobility is monitored in real time through analysis of populations of tracks. Both DNP20-BSA and Y16-DNP3 cause a sharp decrease in mobility upon ligand addition, but produce IgE-receptor clusters that appear distinctive in size and density. Y16-DNP3–stimulated changes occur on a shorter time scale when matched for DNP concentration with DNP20-BSA. Continuing work with Yn-DNP3 ligands will probe clustering-dependent IgE-receptor association with membrane structures and downstream signaling partners.

Quantifying the Effect of BCR Clustering on Plasma Membrane Organization

The B cell antigen receptor (BCR) is an integral part of the adaptive immune system that communicates binding of antigen in the extracellular environment through the plasma membrane. Antigen binding to the BCR results in phosphorylation of intracellular tyrosine activating motifs (ITAMs), which subsequently bind to and activate numerous proteins involved in BCR regulation. Interestingly, many of the proteins regulating the early stages of the BCR signaling pathway are linked to the inner leaflet by saturated lipid anchors which tend to be associated with liquid-ordered membrane phase in model membranes. Also, the BCR becomes transiently detergent resistant following antigen-induced BCR clustering, suggesting that BCR clusters become coupled to membrane order following stimulation. In this work, we aim to characterize how BCR clustering could reorganize plasma membrane lipids by quantifying co-localization of BCR with fluorescent markers of liquid-ordered and liquid-disordered phases. We utilize two-color super-resolution fluorescence localization microscopy (STORM and PALM) in live and chemically fixed CH27 B cells to simultaneously image BCR and membrane anchored proteins, and we quantify their co-clustering using correlation functions. Our results from chemically fixed cells show that proteins anchored to the plasma membrane inner leaflet through saturated acyl-chain lipid modifications exhibit increased co-localization with BCR upon antigen stimulation, whereas those without lipid modifications or those anchored through branched acyl-chain modifications are not significantly co-localized with BCR before or after stimulation. These results are contributing to our long term goal of elucidating the role of lipid mediated interactions in the regulation of BCR signaling.

Imaging Nanometer-Sized α-Synuclein Aggregates by Superresolution Fluorescence Localization Microscopy

The morphological features of α-synuclein (AS) amyloid aggregation in vitro and in cells were elucidated at the nanoscale by far-field subdiffraction fluorescence localization microscopy. Labeling AS with rhodamine spiroamide probes allowed us to image AS fibrillar structures by fluorescence stochastic nanoscopy with an enhanced resolution at least 10-fold higher than that achieved with conventional, diffraction-limited techniques. The implementation of dual-color detection, combined with atomic force microscopy, revealed the propagation of individual fibrils in vitro. In cells, labeled protein appeared as amyloid aggregates of spheroidal morphology and subdiffraction sizes compatible with in vitro supramolecular intermediates perceived independently by atomic force microscopy and cryo-electron tomography. We estimated the number of monomeric protein units present in these minute structures. This approach is ideally suited for the investigation of the molecular mechanisms of amyloid formation both in vitro and in the cellular milieu.

Probing the Organization and Dynamics of Lipid Probes in Phase Separated Supported Bilayers using Super-Resolution Fluorescence Localization Microscopy

Vesicles containing two coexisting liquid phases are widely used to study lateral heterogeneity of membrane components. We recently developed a method to deposit unilamellar supported bilayers on an agarose cushion that conserves the macroscopic phase separation and circular domain morphology usually found in free-floating giant vesicles. We are using these supported bilayers in conjunction with single particle tracking and super-resolution fluorescence localization imaging to probe domain structure, probe mobility, and probe confinement in well defined model membranes. We use the fluorescent lipid analogs DiD and DiIC12 to probe the liquid-disordered phase, while we use Alexa-647 labeled Cholera Toxin B subunit bound to the ganglioside GM1 to label the liquid-ordered phase. By reconstructing images of accumulated localized single probe centers, we observe domain morphology at ∼20nm lateral resolution. By calculating mean squared displacements (MSD) from an ensemble of single molecule trajectories, we measure diffusion coefficients for the different probes as a function of membrane composition and temperature. As expected, we observe confinement of probes when they partition strongly into the discontinuous phase, and measured MSD vs. time curves are consistent with confinement within circular domains of the size given by our reconstructed images. We are currently investigating the organization and dynamics of probes in membranes undergoing critical fluctuations, and are applying these same imaging and analysis techniques to quantify the diffusion of these same probes in the plasma membranes of living cells.