Biology subjects

Wohland, T.

Publications and source records attributed to Wohland, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Vision Transformers as a Framework for Imaging Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is widely used for quantifying molecular dynamics in live cells. However, conventional FCS relies on the autocorrelation function (ACF), a lossy transformation that discards higher-order information, mathematically couples distinct biophysical parameters, and requires long measurements (~50,000 frames, or ~1 minute) that obscure short-lived transient events. To overcome this information bottleneck, we introduce ImFCSViT, a spatiotemporal Vision Transformer-based framework that bypasses the ACF representation entirely, operating directly on raw fluorescence image stacks. By introducing a dedicated normalization token to preserve global intensity statistics, our approach simultaneously extracts four biophysical parameters (diffusion coefficient D, particle density N, particle brightness B, and PSF structure factor {sigma}0) in a single forward pass. Requiring only 2.5 s of data, and potentially down to 1 s (a 1-2 order reduction in data requirement), our approach accurately recovers diffusion coefficients while utilizing its spatial field-of-view to reveal spatially heterogeneous, slow-diffusing sub-populations in lipid bilayers and live-cell plasma membranes that are invisible to classical FCS. By leveraging the data-driven representational learning capabilities of deep learning, we present a flexible, generalizable, and powerful tool for mapping complex, multi-parameter spatiotemporal dynamics in biological systems.

biophysics↗

bioRxiv2025

Two-colour Imaging Fluorescence Correlation Spectroscopy (ImFCS) probes the influence of the juxta-membrane actin-cortex

Here we extend Imaging Fluorescence Correlation Spectroscopy (ImFCS), together with bright, photostable fluorophores, to quantify how the juxta-membranous actin cortex influences plasma membrane dynamics. This problem is technically challenging because the membrane is only a few nanometres thick, whereas the axial resolution of live-cell fluorescence imaging is on the order of 100 nm. We address this by engineering a membrane-anchored actin reporter that combines a weakly interacting actin-binding domain with bright, photostable fluorophores optimized for two-colour ImFCS. This design enables the generation of simultaneous, co-registered diffusion maps where the mobility of the reporter serves as a dynamic sensor for the local density of the cortical actin network. The actin-binding reporter displays diffusion coefficients spanning more than two orders of magnitude, in stark contrast to an otherwise similar reporter that cannot bind actin, which diffuses much faster and within a much narrower range. Thus, the mobility of the reporter becomes a sensitive spatial readout of actin-mediated constraints. By comparing, on a pixel-by-pixel basis, the diffusion of the actin-binding reporter with that of the non-binding mutant, we construct an actin-occupancy map that quantifies the fraction of the membrane functionally influenced by cortical actin. Using this approach, we find that the juxta-membrane actin cortex modulates transport over more than 80% of the cell surface. Our two-channel ImFCS strategy establishes diffusion-based comparison as a general methodology to map the functional footprint of the cortical cytoskeleton on live cell membranes.

biophysics↗

bioRxiv2025

Measuring morphogen transport over multiple spatial scales in live zebrafish embryos

Morphogenesis is controlled by signalling morphogens that form gradients across the embryo. The gradient formation requires that the low molecular weight morphogens slow down their diffusion by at least one order of magnitude. However, the precise slow-down of diffusion across the relevant micrometre scales has not been directly observed. Here, we develop and employ Single-Plane Illumination Microscope based spatial Fluorescence Cross-Correlation Spectroscopy (SPIM-sFCCS) to directly measure the diffusion coefficient of the morphogen Squint in early zebrafish embryos as a function of topography and length scale. We show that Squints diffusion coefficient changes on length scales that are commensurate with the diameter of the intercellular spaces in the embryo and that the slow-down is regulated by receptor binding. The slowdown can be reduced by either the knockdown of Activin receptor 2b, a receptor for Squint, or the overexpression of Lefty2, an inhibitor of Squint. Based on our results and supporting simulations, we propose an interstitial space-dependent transient receptor binding and diffusing mechanism to explain this slowdown, which is crucial for gradient formation and embryonic patterning.

developmental biology↗

bioRxiv2024

EGFR does not directly interact with cortical actin: A SRRF'n'TIRF Study

The epidermal growth factor receptor (EGFR) governs pivotal signaling pathways in cell proliferation and survival, with mutations implicated in numerous cancers. The organization of EGFR on the plasma membrane (PM) is influenced by the lipids and the cortical actin (CA) cytoskeleton. Despite the presence of a putative actin-binding domain (ABD) spanning 13 residues, a direct interaction between EGFR and CA has not been definitively established. While disrupting the cytoskeleton can impact EGFR behavior, suggesting a connection, the influence of the static actin cytoskeleton has been found to be indirect. Here, we investigate the potential interaction between EGFR and CA, as well as the extent to which CA regulates EGFRs distribution on the PM using SRRFnTIRF, a spatiotemporal super-resolution microscopy technique that provides sub-100 nm resolution and ms-scale dynamics from the same dataset. To label CA, we constructed PMT-mEGFP-F-tractin, which combines an inner leaflet targeting domain PMT, fluorescent probe mEGFP, and the actin-binding protein F-tractin. In addition to EGFR-mEGFP, we included two control constructs: a) an ABD deletion mutant, EGFR{Delta}ABD-mEGFP serving as a negative control, and b) EGFR-mApple-F-tractin, where F-tractin is fused to the C-terminus of EGFR-mApple, serving as the positive control. We find that EGFR-mEGFP and EGFR{Delta}ABD-mEGFP show similar membrane dynamics, implying that EGFR-mEGFP dynamics and organization are independent of CA. EGFR dynamics show CA dependence when F-tractin is anchored to the cytoplasmic tail. Together, our results demonstrate that EGFR does not directly interact with the CA in its resting and activated state. SIGNIFICANCESRRFnTIRF is a spatiotemporal super-resolution microscopy technique that allows for the investigation of plasma membrane-cytoskeleton interactions. We investigate how cortical actin (CA) influences the dynamic behavior and structural organization of EGFR, employing specific probe targeting CA structure and dynamics. Our results suggest that EGFR, whether in its resting or activated state, does not directly bind to or interact with the CA. Any influence of CA on EGFR is indirect through membrane modulating activities of CA.

biophysics↗

bioRxiv2024

Receptor binding and tissue architecture explain the morphogen local-to-global mobility transition

Morphogens are intercellular signaling molecules providing spatial information to cells in developing tissues to coordinate cell fate decisions. The spatial information is encoded within long-ranged concentration gradients of the morphogen. Direct measurement of morphogen dynamics in a range of systems suggests that local and global diffusion coefficients can differ by orders of magnitude. Further, local diffusivity can be large, which would potentially abolish any concentration gradient rapidly. Such observations have led to alternative transport models being proposed, including transcytosis and cytonemes. Here, we show that accounting for tissue architecture combined with receptor binding is sufficient to hinder the diffusive dynamics of morphogens, leading to an order of magnitude decrease in the effective diffusion coefficient from local to global scales. In particular, we built a realistic in silico architecture of the extracellular spaces of the zebrafish brain using light and electron microscopy data. Simulations on realistic architectures demonstrate that tortuosity and receptor binding within these spaces are sufficient to reproduce experimentally measured morphogen dynamics. Importantly, this work demonstrates that hindered diffusion is a viable mechanism for gradient formation, without requiring additional regulatory control. SIGNIFICANCEMeasurements of morphogen diffusivity vary significantly depending on experimental approach. Such differences have been used to argue against diffusion as a viable mechanism of morphogen gradient formation. Here, we demonstrate that accounting for the local tissue architecture in concert with including receptor binding is sufficient to explain a range of biological observations. This demonstrates that (hindered) diffusion-driven transport is a viable mechanism of gradient formation.

biophysics↗

bioRxiv2024

Toxin-glycolipid interactions measured by imaging surface plasmon resonance on artificial membranes predicts diffusion behavior and lipid dependence of binding to cells

Membrane-protein interactions mediate cellular invasion by toxins, and are thought to involve organized plasma membrane lipid domains, often containing glycolipids, other sphingolipids, and/or cholesterol. Here, we characterize an isolated glycolipid-interacting domain of the tetanus toxin heavy chain (Hc) as a fluorescently labelled peptide, TeNT46, and describe its membrane dynamics and binding characteristics on artificial bilayers and cellular membranes. We show that this novel ganglioside-interacting probe TeNT46 retains the glycolipid binding preferences of the parent toxin, using imaging-SPR (iSPR) on a micro-patterned hybrid bilayer surface. On live cell membranes, using fluorescence correlation spectroscopic (FCS) diffusion measurements to compare TeNT46 to the well-studied GM1-binding toxin CTxB, we find that both probes display ordered domain-binding characteristics, but distinct cholesterol and sphingolipid dependencies. Strikingly, the contrasting lipid requirements of TeNT46 from those of CTxB in cells are predicted by their iSPR binding preferences on hybrid synthetic membranes. Based on the combined findings from iSPR and FCS, we propose a model for toxin-membrane interaction whereby a unique lipid constellation determines optimum binding for each probe independently of lateral confinement, which is more generally imposed by cholesterol. Our resulting understanding of the specific lipid requirements of these toxin targets and their dynamics in cell membranes could be important for the future design of preventive membrane-based nano-decoys and cell-delivery tools.

cell biology↗

bioRxiv2023

Midkine and Ptprz1b act upstream of Wnt planar cell polarity to establish a midline in the developing zebrafish hindbrain

A midline in the developing central nervous system (CNS) is essential for the symmetric distribution of neural progenitors that later establish functional, bilaterally symmetric neural circuits. In the zebrafish hindbrain, a midline forms early during neurulation and requires a coordinated interplay of cell convergence and midline-crossing cell divisions (C-divisions). These two processes are controlled by the Wnt/planar cell polarity (PCP) pathway. However, upstream cues that control the timely production of PCP components remain unknown. Midkine (Mdk) and pleiotrophin (Ptn) are structurally related heparin-binding growth factors that are dynamically expressed in the developing zebrafish hindbrain. We used proximity ligation assays (PLAs) and fluorescence cross correlation spectroscopy (FCCS) in vivo to show that two zebrafish Mdks, Mdka and Mdkb, as well as Ptn interact with protein tyrosine phosphatase receptors type Z1, Ptprz1a and Ptprz1b, with distinct affinities. Ligand binding triggered Ptprz1b internalization and thereby determined the availability of signaling receptor on cell membranes. In zebrafish mdka, ptn and ptprz1b mutants, cell migration and convergence were significantly impaired during hindbrain neurulation. Impaired convergence led to misplaced C-divisions, defective cell polarity and consequently duplicated midlines. These duplications were rescued by overexpression of Drosophila Prickle, a key component of the Wnt/PCP pathway. Here, we provide evidence that zygotic Mdka controls the distribution of maternally provided Ptprz1b, which in turn is needed for transcription of zebrafish prickle1b. Our findings thus reveal a role for Mdka and Ptprz1b upstream of Wnt/PCP to coordinate neural plate convergence, neural progenitor positioning and midline formation.

developmental biology↗

bioRxiv2023

Deep learning reduces data requirements and allows real-time measurements in Imaging Fluorescence Correlation Spectroscopy

Imaging Fluorescence Correlation Spectroscopy (Imaging FCS) is a powerful tool to extract information on molecular mobilities, actions and interactions in live cells, tissues and organisms. Nevertheless, several limitations restrict its applicability. First, FCS is data hungry, requiring 50,000 frames at 1 ms time resolution to obtain accurate parameter estimates. Second, the data size makes evaluation slow. Thirdly, as FCS evaluation is model-dependent, data evaluation is significantly slowed unless analytic models are available. Here we introduce two convolutional neural networks (CNNs) - FCSNet and Im-FCSNet - for correlation and intensity trace analysis, respectively. FCSNet robustly predicts parameters in 2D and 3D live samples. ImFCSNet reduces the amount of data required for accurate parameter retrieval by at least one order of magnitude and makes correct estimates even in moderately defocused samples. Both CNNs are trained on simulated data, are model-agnostic, and allow autonomous, real-time evaluation of Imaging FCS measurements.

biophysics↗

bioRxiv2022

Long-ranged formation of the Bicoid gradient requires multiple dynamic modes that spatially vary across the embryo

Morphogen gradients provide essential positional information to gene networks through their spatially heterogeneous distribution. Yet, how morphogen gradients form is still hotly contested, with multiple models proposed for different systems. Here, we focus on the transcription factor Bicoid (Bcd), a morphogen that forms an exponential gradient across the anterior-posterior (AP) axis of the early Drosophila embryo. We utilise fluorescence correlation spectroscopy (FCS) and perturbations to Bcd, to dissect Bcd dynamics at multiple spatial and temporal locations. In both the cytoplasm and nucleus, we find two dynamic modes for Bicoid diffusion dynamics, consisting of fast and slow populations of Bcd. Surprisingly, there are spatial differences in Bcd diffusivity along the AP-axis, with Bcd diffusing more rapidly in the posterior. We establish that such spatially varying differences in the Bcd dynamics are sufficient to explain how Bcd can have a steep exponential gradient in the anterior half of the embryo and yet still have an observable fraction of Bcd near the posterior pole. We subsequently investigated which binding elements of Bcd are playing a role in its dynamics. In the nucleus, we demonstrate that the slower mode of Bcd transport is due to Bcd DNA binding. Addition of the Bcd homeodomain to eGFP::NLS can qualitatively replicate the observed Bcd concentration profile, suggesting this domain is the primary region regulating Bcd dynamics. This study provides a detailed analysis of morphogen dynamics at different spatial and temporal locations, revealing multiple modes of transport. These results explain how a long-ranged gradient can form while retaining a steep profile through much of its range.

developmental biology↗

bioRxiv2022

The dependence of EGFR oligomerization on environment and structure: A camera-based N&B study

Number and Brightness analysis (N&B) is a fluorescence spectroscopy technique to quantify protein oligomerization. Accurate results, however, rely on a good knowledge of non-fluorescent states of the fluorescent labels, especially of fluorescent proteins (FP), which are widely used in biology. FPs have been characterized for confocal but not camera-based N&B, which allows in principle faster measurements over larger areas. Here, we calibrate camera-based N&B implemented on a total internal reflection fluorescence (TIRF) microscope for various fluorescent proteins by determining their propensity to be fluorescent. We then apply camera-based N&B in live CHO-K1 cells to determine the oligomerization state of the epidermal growth factor receptor (EGFR), a transmembrane receptor tyrosine kinase that is a crucial regulator of cell proliferation and survival with implications in many cancers. EGFR oligomerization in resting cells and its regulation by the plasma membrane microenvironment is still under debate. Therefore, we investigate the effects of extrinsic factors, including membrane organization, cytoskeletal structure, and ligand stimulation, and intrinsic factors, including mutations in various EGFR domains, on the receptors oligomerization. Our results demonstrate that EGFR oligomerization increases with removal of cholesterol or sphingolipids, or the disruption of GM3-EGFR interactions, indicating raft association. However, oligomerization was not significantly influenced by the cytoskeleton. Mutations in either I706/V948 residues or E685/E687/E690 residues in the kinase and juxtamembrane domains, respectively, led to a decrease in oligomerization, indicating their necessity for EGFR dimerization. Finally, EGFR phosphorylation is oligomerization-dependent involving the extracellular domain (550-580 residues). Coupled with biochemical investigations, camera-based N&B indicates that EGFR oligomerization and phosphorylation is the outcome of several molecular interactions involving the lipid content and structure of the cell membrane and multiple residues in the kinase, juxtamembrane, and extracellular domains. STATEMENT OF SIGNIFICANCENumber and brightness (N&B) analysis is a powerful tool to determine protein association but is mostly conducted in confocal microscopes. This work determines the brightness and fluorescence probability of a range of fluorescent proteins for camera-based N&B on a total internal reflection microscope, demonstrating that with proper calibration different fluorescent proteins provide the same answers on oligomerization within the margins of error. This camera-based approach allows measuring N&B values across whole cell basal membranes up to an area of ~1,000 m2 simultaneously. N&B is then used in combination with biochemical assays to investigate the oligomerization and activation of the epidermal growth factor receptor (EGFR), a prototypical receptor tyrosine kinase with importance in cell signalling, division and survival and implicated in various cancers. The results indicate that EGFR oligomerization and activation is governed by an interplay between membrane structure and composition and key amino acid residues of EGFR that span the extracellular to the intracellular domains.

biophysics↗

bioRxiv2022

Microscope Alignment Using Real-Time Imaging FCS

AO_SCPLOWBSTRACTC_SCPLOWModern EMCCD and sCMOS cameras read out fluorescence data with single-molecule sensitivity at a rate of thousands of frames per second. Exploiting these capabilities in full requires data evaluation in real-time. The direct camera-read-out tool presented here allows access to the data while the camera is recording. This provides simplified and accurate alignment procedures for Total Internal Reflection and Light Sheet Fluorescence Microscopy (TIRFM, LSFM), and simplifies and accelerates fluorescence experiments. The tool handles a range of widely used EMCCD and sCMOS cameras and uses Imaging Fluorescence Correlation Spectroscopy (Imaging FCS) for its evaluation. It is easily extendable to other camera models and other techniques and is a base for automated TIRMF and LSFM data acquisition. SignificanceWe developed a direct camera readout (DCR) software tool that allows access to camera data during acquisition and provides real-time Imaging Fluorescence Correlation Spectroscopy (Imaging FCS) analysis. DCR displays live feedback and due to the sensitivity of correlation analysis provides a sensitive tool for microscope alignment using simple solutions of fluorescent particles. DCR is adaptable to different cameras and evaluation strategies, reduces alignment time, accelerates experiments, and can be used for automation.

biophysics↗

bioRxiv2022

GM1a functions as a coreceptor/ attachment factor for Dengue virus during infection in mammalian systems

Dengue virus (DENV) is a flavivirus causing an estimated 390 million infections per year around the world. Despite the immense global health and economic impact of this virus, its true receptor(s) for internalization into live cells has not yet been identified, and no successful antivirals or treatments have been isolated to this date. This study aims to improve our understanding of virus entry routs by exploring the sialic acid-based cell surface molecule GM1a and its role in DENV infection. The interaction of the virus with GM1a was studied using fluorescence correlation spectroscopy (FCS), fluorescence cross correlation spectroscopy (FCCS), imaging FCS (ImFCS) and amide hydrogen/deuterium exchange mass spectrometry (HDXMS), and the effect on infectivity and movement of the virus during infection was explored using plaque assays and fluorescence-based imaging and single particle tracking (SPT). GM1a was deemed to interact with DENV at domain I (DI) and domain II (DII) of the E protein of the protein coat at quaternary contacts of a fully assembled virus, leading to a ten-fold increase and seven-fold increase in infectivity for DENV1 and DENV2 in mammalian cell systems respectively. The interaction of virus with GM1a triggers a speeding up of virus movement on live cell surfaces, possibly resulting from a reduction in rigidity of cellular rafts during infection, and functions as a coreceptor/ attachment factor for DENV during infection in mammalian systems. Author SummaryDengue virus (DENV) is a flavivirus causing an estimated 390 million infections per year around the world. Despite the immense global health and economic impact of this virus, no successful antivirals or treatments have been isolated to this date. This may be due to the incomplete understanding of the virus infection mechanism, including a lack of an identified true receptor and entry related attachment factors or co-receptors responsible for internalization of the virus. This work focuses on the early infection stage of DENV1 and DENV2 strains, to identify how the virus moves on cell surfaces in its search for its receptors, and identifies the critical role of the sialic acid ganglioside GM1a during internalization of the virus.

biophysics↗

bioRxiv2021

Heptanol-mediated phase separation determines phase preference of molecules in live cell membranes

Plasma membranes contain diverse nanoscale assemblies of lipid and protein domains. Specific localization of lipids and proteins in these domains is often essential for membrane function and integrity. Due to the nanoscale size and dynamic nature of membrane domains, identification of molecules residing in domains either is not possible with modern imaging techniques or requires advanced methods with high spatiotemporal resolution. Such methods need expensive equipment making these approaches inaccessible and thus difficult to implement at large scale. Here, we present a novel membrane fluidizer-induced clustering (MFIC) approach to identify the phase-preference of molecules in intact cell membranes. Experiments in phase-separated bilayers and live cells on molecules with known phase preference demonstrate that heptanol hyperfluidizes the membrane and stabilizes phase separation in cell membranes. The domain stabilization results in a transition of nano-to micron-sized clusters of associated molecules and allows identification of molecules localized in domains using routine microscopy techniques. This assay can be carried out on both genetically and extrinsically labelled molecules in live cell membranes, does not require any invasive sample preparation and can be carried out in 10-15 minutes. This inexpensive and easy to implement assay can be conducted at large-scale and will allow easy identification of molecules partitioning into domains.

biophysics↗

bioRxiv2021

Dengue virus 2 capsid protein chaperones strand displacement without altering the capsid-coding region hairpin element's structural functionality

By virtue of its chaperone activity, the capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements. However, the role of DENV2C during the interaction of RNA elements involved in stabilizing the 5-3 panhandle structure of DENV RNA is still unclear. Therefore, we determined how DENV2C affects structural functionality of the capsid-coding region hairpin element (cHP) during RNA rearrangement of the 9-nt conserved sequence (5CS) to its complementary 3CS counterpart. The cHP element has two distinct functions: a role in translation start codon selection and a role in RNA synthesis. Our results showed that the cHP hairpin impedes annealing between the 5CS and the 3CS elements. Although DENV2C does not modulate structural functionality of the cHP hairpin, it accelerates annealing and specifically promotes strand displacement of 3CS during 5-3 panhandle formation. Furthermore, DENV2C exerts its chaperone activity by favoring one of the active conformations of the cHP element. Based on our results, we propose mechanisms for annealing and strand displacement involving the cHP element. Thus, our results provide mechanistic insights on how DENV2C regulates RNA synthesis by modulating essential RNA elements in the capsid-coding region, that in turn allow for DENV replication.

molecular biology↗

bioRxiv2020

Dengue virus strain 2 capsid protein switches the annealing pathway and reduces the intrinsic dynamics of the conserved 5' untranslated region

The capsid protein of Dengue Virus strain 2 (DENV2C) is a structural protein with RNA chaperone activity that promotes multiple nucleic acid structural rearrangements, critical for transcription of the single-stranded positive-sense DENV2 genomic RNA. Annealing of the conserved 5 untranslated region (5UTR) to either its complementary sequence or to the 3 untranslated region (3UTR) occurs during (+)/(-) ds-RNA formation and (+) RNA circularization, respectively, both essential steps during DENV RNA replication. We investigated the effect of DENV2C on the annealing mechanism of two hairpin structures from the 5UTR region (21-nt upstream AUG region (5UAR) and 23-nt capsid-coding hairpin (5cHP)) to their complementary sequences during (+)/(-) ds-RNA formation and (+) RNA circularization. Using fluorescence spectroscopy, DENV2C was found to switch annealing reactions nucleated mainly through kissing-loop intermediates to stem-stem interactions during (+)/(-) ds-RNA formation while it promotes annealing mainly through kissing-loop interactions during the (+) RNA circularization. Using FRET-FCS and trFRET, we determined that DENV2C exerts RNA chaperone activities by modulating intrinsic dynamics and by reducing the kinetically trapped unfavorable conformations of the 5UTR sequence. Thus, DENV2C is likely to facilitate genome folding into functional conformations required for replication, playing a role in modulating (+)/(-) ds-RNA formation and (+) RNA circularization.

molecular biology↗

bioRxiv2020

Wnt3 distribution in the zebrafish brain is determined by expression, diffusion and multiple molecular interactions

Wnt3 proteins are lipidated and glycosylated, secreted signaling molecules that play an important role in zebrafish neural patterning and brain development. However, the transport mechanism of lipid-modified Wnts through the hydrophilic extracellular environment for long-range action remains unresolved. Here, we determine how Wnt3 accomplishes long-range distribution in the zebrafish brain. First, we characterize the Wnt3-producing source and Wnt3-receiving target regions. Subsequently, we analyze Wnt3 mobility at different length scales by fluorescence correlation spectroscopy and fluorescence recovery after photo-bleaching. We demonstrate that Wnt3 spreads extracellularly and interacts with heparan sulfate proteoglycans (HSPG). We then determine the binding affinity of Wnt3 to its receptor, Frizzled1 (Fzd1), using fluorescence cross-correlation spectroscopy, and show that the co-receptor, low-density lipoprotein receptor-related protein 5 (Lrp5), is required for Wnt3-Fzd1 interaction. Our results are consistent with the extracellular distribution of Wnt3 by a diffusive mechanism that is modified by tissue morphology, interactions with HSPG and Lrp5-mediated receptor binding, to regulate zebrafish brain development.

developmental biology↗

bioRxiv2020

SRRF 'n' TIRF - FCS: Simultaneous spatiotemporal super-resolution microscopy

Super-resolution microscopy and single molecule fluorescence spectroscopy require mutually exclusive experimental strategies optimizing either time or spatial resolution. To achieve both, we implement a GPU-supported, camera-based measurement strategy that highly resolves spatial structures (~60 nm), temporal dynamics ([≤] 2 ms), and molecular brightness from the exact same data set. We demonstrate the applicability and advantages of multi-parametric measurements to monitor the super-resolved structure and dynamics of two different biomolecules, the actin binding polypeptide LifeAct, and the epidermal growth factor receptor (EGFR). Simultaneous super-resolution of spatial and temporal details leads to an improved precision in estimating the diffusion coefficient of LifeAct in dependence of the cellular actin network. Multi-parametric analysis suggests that the domain partitioning of EGFR is primarily determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-EGFR interactions but is largely independent of EGFR-actin interactions. These results demonstrate that pixel-wise cross-correlation of parameters obtained from different techniques on the same data set enables robust physicochemical parameter estimation and provides new biological knowledge that cannot be obtained from sequential measurements.

biophysics↗

bioRxiv2019

Analysing plasma membrane asymmetry of lipid organisation by fluorescence lifetime and correlation spectroscopy

A fundamental feature of a eukaryotic cell membrane is the asymmetric arrangement of lipids in the two leaflets. A cell invests significant energy to maintain this asymmetry and utilizes it to regulate important biological processes such as apoptosis and vesiculation. Here, we employ fluorescence lifetime imaging microscopy (FLIM) and imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) to differentiate the dynamics and organization of the exofacial and cytoplasmic leaflet of live mammalian cells. We characterize the biophysical properties of fluorescent analogues of phosphatidylcholine (PC), sphingomyelin (SM) and phosphatidylserine (PS) in two mammalian cell membranes. Due to their specific transverse membrane distribution, these probes allow leaflet specific investigation of the plasma membrane. We compare the results with regard to the different temporal and spatial resolution of the methods. Fluorescence lifetimes of fluorescent lipid analogues were found to be in a characteristic range for the liquid ordered phase in the outer leaflet and liquid disordered phase in the inner leaflet. The observation of a more fluid inner leaflet is supported by free diffusion in the inner leaflet with high average diffusion coefficients. The liquid ordered phase in the outer leaflet is accompanied by slower diffusion and diffusion with intermittent transient trapping. Our results show that the combination of FLIM and ITIR-FCS with specific fluorescent lipid analogues provides a powerful tool to investigate lateral and trans-bilayer characteristics of plasma membrane in live cells.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC=\"FIGDIR/small/776831v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (24K):\norg.highwire.dtl.DTLVardef@1cb68f1org.highwire.dtl.DTLVardef@156a92forg.highwire.dtl.DTLVardef@7d84bdorg.highwire.dtl.DTLVardef@95bfa0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗