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DNA origami structures provide flexible scaffolds for the organization of single biomolecules with nanometer precision. While they find increasing use for a variety of biological applications, the functionalization with proteins at defined stoichiometry, high yield, and under preservation of protein function remains challenging. In this study, we applied single molecule fluorescence microscopy in combination with a cell biological functional assay to systematically evaluate different strategies for the site-specific decoration of DNA origami structures, focusing on efficiency, stoichiometry and protein functionality. Using an activating ligand of the T-cell receptor (TCR) as protein of interest, we found that two commonly used methodologies underperformed with regard to stoichiometry and protein functionality. While strategies employing tetravalent wildtype streptavidin for coupling of a biotinylated TCR-ligand yielded mixed populations of DNA origami structures featuring up to 3 proteins, the use of divalent (dSAv) or DNA-conjugated monovalent streptavidin (mSAv) allowed for site-specific attachment of a single biotinylated TCR-ligand. The most straightforward decoration strategy, via covalent DNA conjugation, resulted in a 3-fold decrease in ligand potency, likely due to charge-mediated impairment of protein function. Replacing DNA with charge-neutral PNA in a ligand conjugate emerged as the coupling strategy with the best overall performance in our study, as it produced the highest yield with no multivalent DNA origami structures and fully retained protein functionality. With our study we aim to provide guidelines for the stoichiometrically defined, site-specific functionalization of DNA origami structures with proteins of choice serving a wide range of biological applications.

Single-molecule Förster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. It has been used in a wide range of biophysical and molecular biological experiments, including the measurement of molecular forces, characterization of conformational dynamics of biomolecules, observation of intracellular colocalization of proteins, and determination of receptor-ligand interaction times. In a widefield microscopy configuration, experiments are typically performed using surface-immobilized probes. Here, a method combining single-molecule tracking with alternating excitation (ALEX) smFRET experiments is presented, permitting the acquisition of smFRET time traces of surface-bound, yet mobile probes in plasma membranes or glass-supported lipid bilayers. For the analysis of recorded data, an automated, open-source software collection was developed supporting (i) the localization of fluorescent signals, (ii) single-particle tracking, (iii) determination of FRET-related quantities including correction factors, (iv) stringent verification of smFRET traces, and (v) intuitive presentation of the results. The generated data can conveniently be used as input for further exploration via specialized software, e.g., for the assessment of the diffusional behavior of probes or the investigation of FRET transitions.

T-cells engage with antigen-presenting cells in search for antigenic peptides and form transient interfaces termed immunological synapses. A variety of protein-protein interactions in trans-configuration defines the topography of the synapse and orchestrates the antigen-recognition process. In turn, the synapse topography affects receptor binding rates and the mutual segregation of proteins due to size exclusion effects. For better understanding it is hence critical to map the 3D topography of the immunological synapse at high precision. Current methods, however, provide only rather coarse images of the protein distribution within the synapse, which do not reach the dimension of the protein ectodomains. Here, we applied supercritical angle fluorescence microscopy combined with defocused imaging, which allows 3-dimensional single molecule localization microscopy (3D-SMLM) at an isotropic localization precision below 15 nm. Experiments were performed on hybrid synapses between primary T-cells and functionalized glass-supported lipid bilayers. We used 3D-SMLM to quantify the cleft size within the synapse by mapping the position of the T-cell receptor (TCR) with respect to the supported lipid bilayer, yielding average distances of 18 nm up to 31 nm for activating and non-activating bilayers, respectively.

When T-cells probe their environment for antigens, the bond between the T-cell receptor (TCR) and the peptide-loaded major histocompatibility complex (MHC) is put under tension, thereby influencing the antigen discrimination process. Yet, the quantification of such forces in the context of T-cell signaling is technically challenging. Common approaches such as traction force microscopy (TFM) employ a global readout of the force fields, e.g. by measuring the displacements of hydrogel-embedded marker beads. Recent data, however, indicated that T-cells exert tensile forces locally via TCR-enriched microvilli while scanning the surface of antigen-presenting cells. Here, we developed a traction force microscopy platform, which allows for quantifying the pulls exerted via T-cell microvilli, in both tangential and normal directions, during T-cell activation. For this, we immobilized specific T-cell activating antibodies directly on the marker beads used to read out the hydrogel deformation. Microvilli targeted the functionalized beads, as confirmed by superresolution microscopy of the local actin organization. Moreover, we found that cellular components, such as actin, TCR and CD45 reorganize upon interaction with the beads, such that actin forms a vortex-like ring structure around the beads and TCR is enriched at the bead surface, whereas, CD45 is excluded from bead-microvilli contacts.Significance statementDuring the antigen recognition process, T-cells explore and probe their environment via microvilli, which exert local pushes and pulls at the surface of the antigen presenting cell. It is currently believed that these forces influence or even enable the antigen recognition process. Here, we describe the development of a platform, which allows us to quantify the magnitude and direction of traction forces exerted locally by T cell microvilli. Simultaneous Ca2+ imaging was used to link the measured forces to the overall T cell activation status. Superresolution microscopy resolved the contact sites of bead-microvilli contact at the nanoscale: cells contacted beads via actin vortex-like structures, which excluded the phosphatase CD45 from the contacts.

T-cells detect with their T-cell antigen receptors (TCRs) the presence of rare peptide/MHC complexes (pMHCs) on the surface of antigen presenting cells (APCs). How they convert a biochemical interaction into a signaling response is poorly understood, yet indirect evidence pointed to the spatial antigen arrangement on the APC surface as a critical factor. To examine this, we engineered a biomimetic interface based on laterally mobile functionalized DNA origami platforms, which allow for nanoscale control over ligand distances without interfering with the cell-intrinsic dynamics of receptor clustering. We found that the minimum signaling unit required for efficient T-cell activation consisted of two ligated TCRs within a distance of 20 nanometers, if TCRs were stably engaged by monovalent antibody fragments. In contrast, antigenic pMHCs stimulated T-cells robustly as well-isolated entities. These results identify the minimal requirements for effective TCR-triggering and validate the exceptional stimulatory potency of transiently engaging pMHCs.

T-Lymphocytes initiate an adaptive immune response after specific binding of the T-cell receptor (TCR) to an antigenic peptide bound to the major histocompatibility complex (peptide-MHC) on an antigen presenting cell. Key events upon TCR-pMHC binding involve the phosphorylation of intracellular tyrosine residues of the TCR and recruitment of adapter molecules, which results in downstream signalling. Initial TCR phosphorylation is primarily carried out by the membrane associated protein lymphocyte specific kinase (Lck) making it a central molecule for T-cell signalling.Recent work on Lck, using superresolution microscopy techniques, has shown the organisation of Lck in nanoclusters in fixed as well as in live cells [1]. However, neither the structural mechanism nor the functional role of this non-random Lck distribution in the plasma membrane are well understood. Possible explanations for Lck clustering may be local lipid heterogeneities as well as interactions with other plasma membrane proteins or the cortical actin cytoskeleton.In order to acquire a better understanding of Lck dynamics in live cells, we carried out superresolution experiments on Lck-mEOS3.2, stably expressed in JCaM 1.6 cells. We used single molecule tracking to elucidate the diffusional behaviour of Lck. Based on the reported existence of nanoclusters, we implemented a simple and practical method to separately analyse Lck diffusion inside and outside these structures. Surprisingly, only Lck-molecules inside clusters were immobilised, whereas molecules outside of clusters exhibited pure Brownian motion. We further found that disruption of the actin cytoskeleton did not influence Lck immobilisations.[1] Rossy, J., et al., Conformational states of the kinase Lck regulate clustering in early T cell signalling. Nature immunology 14, 82-89 (2013).This work was supported by the Austrian Science Fund/FWF project P26337-B21 and P27941-B28.

The oligomeric lipid raft-associated integral protein stomatin normally localizes to the plasma membrane and the late endosomal compartment. Similar to the caveolins, it is targeted to lipid bodies (LBs) on overexpression. Endogenous stomatin also associates with LBs to a small extent. Green fluorescent protein-tagged stomatin (StomGFP) and the dominant-negative caveolin-3 mutant DGV(cav3)HA occupy distinct domains on LB surfaces but eventually intermix. Studies of StomGFP deletion mutants reveal that the region for membrane association but not oligomerization and raft association is essential for LB targeting. Blocking protein synthesis leads to the redistribution of StomGFP from LBs to LysoTracker-positive vesicles indicating a connection with the late endosomal/lysosomal pathway. Live microscopy of StomGFP reveals multiple interactions between LBs and microtubule-associated vesicles possibly representing signaling events and/or the exchange of cargo. Proteomic analysis of isolated LBs identifies adipophilin and TIP47, various lipid-specific enzymes, cytoskeletal components, chaperones, Ras-related proteins, protein kinase D2, and other regulatory proteins. The association of the Rab proteins 1, 6, 7, 10, and 18 with LBs indicates various connections to other compartments. Our data suggest that LBs are not only involved in the storage of lipids but also participate actively in the cellular signaling network and the homeostasis of lipids.

Superresolution microscopy has facilitated the investigation of cellular structures at length scales far below the optical diffraction limit. When applied to the plasma membrane, the presence of a variety of protein nanoclusters was revealed, which lead to speculations whether nanoclustering was a general feature of plasma membrane proteins. Particularly in T lymphocytes, clustering of signaling proteins has been proposed to represent a fundamental mechanism for cell activation. Recently, however, doubts were raised whether imaging artifacts inherent to PALM/STORM might have influenced or even caused the observation of some of those protein clusters. To approach these concerns, we developed a method to robustly discriminate clustered from random distributions of molecules detected with single molecule localization microscopy-based techniques like PALM and STORM.1 The approach is based on deliberate variations of the labeling density of the samples and quantitative cluster analysis. Our method circumvents the problem of clustering artifacts generated by the blinking statistics of the fluorophores used. It can be readily applied to PALM and STORM experiments where either overexpressed proteins are present over a broad range of expression levels or antibody concentrations are titrated to achieve different degrees of labeling.1 Baumgart et al., Nat. Meth. 13: 661-664 (2016)

Proper supply of mammalian cells with cholesterol is crucial for membrane function and cellular survival and is regulated by several mechanisms, including cellular uptake, synthesis, storage and export. Transport of excess cholesterol from the periphery back to the liver for excretion into the bile is achieved by high-density lipoproteins (HDL). HDL particles are captured from the blood stream by the scavenger receptor class B type I (SR-BI), the so-called HDL receptor. The subsequent lipid transfer process, however, is highly speculative: it has been proposed to occur after HDL endocytosis or directly at the cell surface across an unstirred water layer or via a hydrophobic channel in the receptor. Here we show via direct imaging techniques that, upon contact with the membrane, HDL is incorporated into the hydrophobic core of the lipid bilayer and amphiphilic cargo is immediately released to the plasma membrane. Particle incorporation and cargo transfer is abolished at increased membrane cholesterol concentrations, as a consequence of the reduced membrane elasticity. Our observations reveal a mechanism for regulation of lipid uptake, which is based on sensing plasma membrane cholesterol levels: HDL-receptors tether particles close to the plasma membrane; once in proximity, elastic membrane properties regulate the particles’ fusion with the bilayer and subsequent cargo transfer. The data indicate that the plasma membrane itself signals a cell's cholesterol demand: high cholesterol levels act repulsive, low cholesterol levels fusogenic.

The plasma membrane has been hypothesized to contain nanoscopic lipid platforms, which are discussed in the context of “lipid rafts” or “membrane rafts”. Based on biochemical and cell biological studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition and heterogeneity. We present here a method which allows for the first time the direct imaging of nanoscopic stable platforms with raft-like properties diffusing in the live cell plasma membrane. Our method senses these platforms by their property to assemble a characteristic set of fluorescent marker-proteins or lipids on a time-scale of seconds. A special photobleaching protocol was used to reduce the surface density of labeled mobile platforms down to the level of well-isolated diffraction-limited spots, without altering the single spot brightness. The statistical distribution of probe molecules per platform was determined by single molecule brightness analysis. For demonstration, we used the consensus raft marker glycosylphosphatidylinositol-anchored monomeric GFP and the fluorescent lipid analogue Bodipy-GM1 which preferentially partitions into liquid ordered phases. For both markers we found cholesterol-dependent homo-association in the plasma membrane of living CHO and Jurkat T cells in the resting state, thereby demonstrating the existence of small, mobile, stable platforms containing these probes. We further applied the technology to address structural changes in the plasma membrane during fever-type heat shock: at elevated temperatures the mGFP-GPI homo-association disappeared, accompanied by an increase in the expression of the small heat shock protein Hsp27.

We have developed an assay for quantitative analysis of the interaction between a fluorescently marked protein (prey) and a membrane protein (bait) using microstructured surfaces covered with biotinylated ligands (antibodies) targeted against the bait. The proof-of-concept was demonstrated for the interaction between CD4, a major co-receptor in T-cell signalling, and Lck, a protein tyrosine kinase essential for early T cell signalling. Here we present improvements and a more precise characterization of the method as well as the applicability of the assay for the analysis of protein interactions within lipid rafts in the inner and outer leaflet of the plasma membrane. We stably expressed fluorescently labelled raft and non-raft proteins in the human T24 cell line as prey proteins and determined the degree of interaction with the antibody-targeted bait proteins CD59 (GPI-anchored protein, raft marker) and CD71 (Transferrin-receptor, non-raft marker), respectively. We found strong interaction of CD59 with putative raft markers including various GPI-GFP constructs, the inner-leaflet associated proteins Lck and Flottilin1 and a Pleckstrin-Homology domain fused to GFP. Importantly, we did not find interaction of CD59 with CD71-GFP and other potential non-raft proteins. When CD71 was used as the bait protein we did not find interaction with the putative raft markers. While the detected absence of CD71 from and the presence of CD59 in lipid rafts confirm current knowledge, it is still very unclear if a lipid-raft dependent coupling of proteins and certain especially negatively charged lipids across the plasma-membrane bilayer exists. Thus, our micropatterning assay will be of great interest to address this question.

The organization of the cellular plasma membrane at a nanoscopic length scale is believed to affect the association of distinct sets of membrane proteins for the regulation of multiple signaling pathways. Based on in vitro results, conflicting models have been proposed which postulate the existence of stable or highly dynamic platforms of membrane lipids and proteins; commonly, these structures are termed membrane rafts. The lack of experimental evidence confirming the existence of putative rafts in living cells has yielded increasing skepticism, casting doubt on a major portion of the recent literature. Here we directly imaged and further characterized lipid rafts in the plasma membrane of living CHO cells by single molecule TIRF microscopy. Using a novel recording scheme for “Thinning Out Clusters while Conserving Stoichiometry of Labeling” (1), molecular homo-association of GPI-anchored mGFP was detected at 37°C and ascribed to specific enrichment in lipid platforms. The mobile mGFP-GPI homo-associates were found to be stable on a seconds timescale and dissolved after cholesterol depletion using methyl-beta-cyclodextrin or cholesterol-oxidase.Having confirmed the association of mGFP-GPI to stable membrane rafts, we attempted to use an externally applied marker to test this hypothesis. We used Bodipy-GM1, a probe that was recently reported to be enriched in the liquid-ordered phase of plasma membrane vesicles. When applied to CHO cells at different surface staining, we found that also Bodipy-GM1 homo-associated in a cholesterol-dependent manner, thus providing further evidence for the existence of membrane rafts.(1). Moertelmaier, M., Brameshuber, M., Linimeier, M., Schutz, G.J. & Stockinger, H. Thinning out clusters while conserving stoichiometry of labeling. Appl Phys Lett 87, 263903 (2005).

Genomic research is nowadays based on high throughput analytical techniques. Microarray assays are commonly used to determine DNA content of heterogeneous mixtures up to full genome scale. For low amounts of sample material this method, however, requires time consuming and error prone PCR based amplification steps. Here, we present an assay with the ability to characterize the cDNA content of a low number of cells using ultra-sensitive fluorescence microscopy. For detection, a newly developed chip reader was used. The instrument is based on a modified fluorescence microscope with single dye sensitivity. The highly sensitive CCD detector is operated in TDI mode, which allows avoiding overhead times for sample positioning and signal integration. This enabled the scanning of areas of 1x0.2cm 2 within 50 seconds at a pixel size of 200nm. At this resolution, single dye molecules can be reliably detected with an average signal to background noise ratio of ~42. For DNA hybridization experiments, oligonucleotides were covalently linked to a newly developed aldehyde surface. Subsequently, fluorescence labeled complementary oligonucleotides were hybridized at various concentrations. Down to femto-molar oligonucleotide concentrations, specific signals were detected. At 10fM concentration signals of individual specifically hybridized oligonucleotide molecules were resolvable. This assay provides the conceptual basis for expression profiling of low amounts of sample material without signal amplification.

We report on the application of a novel fluorescence-microscope based scanning device with single molecule sensitivity to microarray readout. The device is based on a CCD camera operated in time delay and integration (TDI) mode synchronized with the movement of a sample scanning stage, enabling continuous data acquisition. The implementation of a focus hold system keeps the sample in focus during scanning. Results from ultra-sensitive high-resolution microarray readout provide clear evidence for the superiority of the novel detection method over conventional microarray readout systems in particular regarding to sensitivity and dynamic range. Minute sample amount and lacking amplification methods will demand for this ultra-sensitive readout technology in future genomic and proteomic research.

Selective uptake of high density lipoprotein (HDL)-associated cholesteryl-esters (CE) by hepatocytes is a crucial process for the removal of cholesterol from the circulation. Together with triglycerides, CE is transported in the hydrophobic core of HDL particles; free cholesterol, phospholipids, and apolipoproteins build up the particles' amphiphilic surface monolayer. The lipid transfer process itself is highly speculative: it is still unclear how lipids are removed from the circulation and transferred from HDL - a main carrier of cholesterol in the blood stream - to cells.In this study we provide a mechanistic understanding of the cargo exchange process between HDL and biomembranes. The interaction between HDL and synthetic lipid membranes was investigated with force spectroscopy and high speed atomic force microscopy; the transfer of single cargo molecules was directly visualized using a combined and simultaneously operating fluorescence and force microscope. Experimental evidence points to the fact that i) cargo transfer requires contact; ii) only amphiphilic cargo is transferred; iii) upon contact the particle incorporates into the hydrophobic core of the bilayer where it can diffuse. In particular, we compared the transfer of the fluorescently labelled lipids DiI, and Bodipy-labelled cholesterol and cholesteryl-ester. Live cell experiments confirmed the data obtained on the synthetic systems and on GPMV's (giant plasma membrane vesicles). Particle incorporation and cargo transfer was abolished at increased membrane cholesterol levels, as a consequence of the reduced membrane elasticity. These observations reveal a mechanism for regulation of lipid uptake based on sensing plasma membrane cholesterol levels. The function of the corresponding receptor SR-B1 is primarily an anchor to hold the particle close to the plasma membrane; once in proximity, elastic properties of the membrane regulate the fusion of the particle.

Store Operated Calcium Entry (SOCE) is a crucial mechanism for many cellular signalling processes. The two major proteins involved in SOCE are Orai1 (located in the plasma membrane) and STIM1 (located in the membrane of the endoplasmatic reticulum (ER)). Upon depletion of the calcium stores in the ER, the STIM1 co-clusters with the Orai1 in the plasma membrane which results in a calcium influx into the cell.In order to investigate the distribution of the Orai1 in the plasma membrane we used Photoactivation Localization Microscopy (PALM). PALM is a technique that allows overcoming the diffraction limit by photo-activating only a small subset of fluorophores with a laser pulse. At shallow illumination conditions, the active fluorophores are spatially well separated and can be localized with a precision of a few ten nm. Sequential activation, readout and photobleaching allows for recording a complete image of the sample. The Orai1 subunits were fused to photoactivatable GFP (paGFP) and expressed in Chinese hamster ovary cells. PALM revealed submicrometer clusters of Orai1, which show a high degree of colocalization with STIM1-mCherry, as confirmed by two-color microscopy.

We recently developed a method termed TOCCSL (Moertelmaier,APL-2005) (‘Thinning out Clusters while Conserving Stoichiometry of Labeling’) which allowed for the first time the direct imaging of nanoscopic stable platforms with raft-like properties diffusing in the live cell plasma membrane (Brameshuber,JBC-2010). Our method sensed these platforms by their property to assemble the putative raft markers glycosylphosphatidylinositol-anchored monomeric GFP (mGFP-GPI) and GM1-Bodipy on a time-scale of seconds on the cell membrane of living CHO and Jurkat T-cells. In order to resolve platforms we used a special photobleaching protocol to reduce the surface density of labeled mobile platforms down to the level of well-isolated diffraction-limited spots, without altering the single spot brightness. The statistical distribution of probe molecules per platform was determined by single-molecule brightness analysis. To further validate our method we extended TOCCSL by utilizing two-color co-localization in combination with stoichiometric photobleaching (Ruprecht,SoftMatter-2010). Glycosylphosphatidylinositol-anchored SNAP-tag (GPI-SNAP) was stably expressed by CHO cells and labeled with SNAP-AF488 and SNAP-AF647 at an equimolar ratio. For both colors we were able to observe a homogeneous surface staining with a density of 300-1000 molecules/μm². To reduce the surface density we applied TOCCSL parallel to both color channels. An appropriate recovery time yielded a stoichiometrically reduced surface density enabling the localization of single fluorescent GPI-SNAPs in both color channels with a resolution down to a few ten nanometers. After correction of chromatic aberration we used an automatic algorithm to detect pairs of co-localized GPI-SNAPs within a diffraction limited distance. The number of co-localized GPI-SNAPs and their stability was found to be comparable to our previous measured mGFP-GPI data: around 30% of GPI-SNAPs homo-associated with a lifetime of seconds and disappeared after cholesterol-oxidase treatment - thus supporting the lipid raft concept.

We developed a method termed TOCCSL (‘Thinning out Clusters while Conserving Stoichiometry of Labeling’) which allows direct imaging of nanoscopic stable platforms with raft-like properties diffusing in the live cell plasma membrane. Our method senses these platforms by their property to assemble a characteristic set of fluorescent marker-proteins/lipids on a time-scale of seconds. A special photobleaching protocol is used to reduce the surface density of labeled mobile platforms down to the level of well-isolated diffraction-limited spots, without altering the single spot brightness. The statistical distribution of probe molecules per platform is determined by single molecule brightness analysis. As consensus raft marker we chose monomeric GFP linked via a GPI (glycosyl-phosphatidylinositol) anchor to the cell membrane. We found cholesterol-dependent homo-association of mGFP-GPI in the plasma membrane of living CHO cells, thereby demonstrating the existence of small, mobile, stable platforms hosting these probes. This strong homo-association could be released by addition of 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) or 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), two oxidized lipids typically present in oxidatively modified low density lipoprotein. We found a clear dose-response relationship of mGPF-GPI nanodomain disintegration upon addition of POVP, correlating with the signal of an apoptosis marker. Applying similar concentrations of lyso-lipid did not dissolve nanodomains. To understand the mechanism behind nanodomain disintegration we inhibited the activation of acid sphingomyelinase (aSMase), which activates several apoptotic signaling pathways via ceramide generation. Inhibition of aSMase by NB-19 before addition of POVPC completely abolished the nanodomain-disintegration effect of oxidized phospholipids, thereby proofing a rather indirect effect of oxidized phospholipids on lipid nanodomains. Additionally we addressed the role of aSMase activity in the release of mGPF-GPI homo-association upon cholesterol extraction - no correlation was found.In conclusion we could determine the mechanism how oxidized lipid species disrupt mGFP-GPI nanodomains in the live plasma membrane.

The plasma membrane has been hypothesized to contain nanoscopic lipid platforms, also termed lipid or membrane rafts. Based on biochemical and cell biological studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition and heterogeneity. We present here a method which allows for the first time the demonstration that single rafts diffuse as stable platforms in the live cell plasma membrane. Our method senses rafts by their property to assemble a characteristic set of fluorescent marker-proteins or lipids on a time-scale of seconds. The special photobleaching protocol TOCCSL (Thinning Out Clusters while Conserving Stoichiometry of Labeling1) was used to reduce the surface density of labeled mobile rafts down to the level of well-isolated diffraction-limited spots, without altering the single spot brightness. The statistical distribution of probe molecules per raft was determined by single molecule brightness analysis. For demonstration, we used the consensus markers Bodipy-GM1, a fluorescent lipid analogue, and glycosylphosphatidylinositol-anchored monomeric GFP. For both markers we found cholesterol-dependent association in the plasma membrane of living CHO and Jurkat T cells in the resting state, indicating the presence of small, mobile, stable rafts hosting these probes. We further applied the technology to address structural changes in the plasma membrane during fever-type heat shock: at elevated temperatures mGFP-GPI homo-association disappeared, accompanied by an increase in the expression of the small heat shock protein Hsp27.1. Moertelmaier, M., Brameshuber, M., Linimeier, M., Schutz, G. J. & Stockinger, H. Thinning out clusters while conserving stoichiometry of labeling. Appl Phys Lett 87, 263903 (2005).