STED-FLCS - an advanced tool to reveal spatio-temporal heterogeneity of molecular membrane dynamics (original) (raw)
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Nature Communications, 2014
The interaction of lipids and proteins plays an important role in plasma membrane bioactivity, and much can be learned from their diffusion characteristics. Here we present the combination of super-resolution STED microscopy with scanning fluorescence correlation spectroscopy (scanning STED-FCS, sSTED-FCS) to characterize the spatial and temporal heterogeneity of lipid interactions. sSTED-FCS reveals transient molecular interaction hotspots for a fluorescent sphingolipid analogue. The interaction sites are smaller than 80 nm in diameter and lipids are transiently trapped for several milliseconds in these areas. In comparison, newly developed fluorescent phospholipid and cholesterol analogues with improved phase-partitioning properties show more homogenous diffusion, independent of the preference for liquid-ordered or disordered membrane environments. Our results do not support the presence of nanodomains based on lipid-phase separation in the basal membrane of our cultured nonstimulated cells, and show that alternative interactions are responsible for the strong local trapping of our sphingolipid analogue.
Journal of physics D: Applied physics, 2018
Observation techniques with high spatial and temporal resolution, such as single-particle tracking based on interferometric scattering (iSCAT) microscopy, and fluorescence correlation spectroscopy applied on a super-resolution STED microscope (STED-FCS), have revealed new insights of the molecular organization of membranes. While delivering complementary information, there are still distinct differences between these techniques, most prominently the use of fluorescent dye tagged probes for STED-FCS and a need for larger scattering gold nanoparticle tags for iSCAT. In this work, we have used lipid analogues tagged with a hybrid fluorescent tag-gold nanoparticle construct, to directly compare the results from STED-FCS and iSCAT measurements of phospholipid diffusion on a homogeneous supported lipid bilayer (SLB). These comparative measurements showed that while the mode of diffusion remained free, at least at the spatial (>40 nm) and temporal (50 ⩽ t ⩽ 100 ms) scales probed, th...
2019
ABSTRACTA 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 analo...
2021
The primary cilium is a specialized plasma membrane protrusion with important receptors for signalling pathways. In polarized epithelial cells, the primary cilium assembles after the midbody remnant (MBR) encounters the centrosome at the apical surface. The membrane surrounding the MBR, namely remnant associated membrane patch (RAMP) once situated next to the centrosome, releases some of its lipid components to form a centrosome-associated membrane patch (CAMP) from which the ciliary membrane stems. The RAMP undergoes a spatiotemporal membrane refinement during the formation of the CAMP, which becomes highly enriched in condensed membranes with low lateral mobility. To better understand this process, we have developed a correlative imaging approach that yields quantitative information about the lipid lateral packing, its mobility and collective assembly at the plasma membrane at different spatial scales over time. Our work paves the way towards a quantitative understanding of lipid ...
Membranes, 2015
Diffusion in lipid membranes is an essential component of many cellular process and fluorescence a method of choice to study membrane dynamics. The goal of this work was to directly compare two common fluorescence methods, line-scanning fluorescence correlation spectroscopy and single-particle tracking, to observe the diffusion of a fluorescent lipophilic dye, DiD, in a complex five-component mitochondria-like solid-supported lipid bilayer. We measured diffusion coefficients of DFCS ~ 3 um2 * s-1 and DSPT ~ 2 um2 * s-1, respectively. These comparable, yet statistically different values are used to highlight the main message of the paper, namely that the two considered methods give access to distinctly different dynamic ranges: D sup or approximatively 1um2 * s-1 for FCS and D inf or approximatively 5 um2 s-1 for SPT (with standard imaging conditions). In the context of membrane diffusion, this means that FCS allows studying lipid diffusion in fluid membranes, as well as the diffusio...
Most STED measurements were performed on a custom-built STED microscope setup, as described previously 1, 2 . Briefly, the setup had a 532 nm (PicoTA, P icoQuant, Berlin, Germany; for excitation of NV centres) or a 635 nm pulsed diode laser (LDH-D-C-635, PicoQuant, Berlin, Germany; for excitation of lipid analogues) and a Ti:Sapphire laser tuned to 740-770 nm and operating in continuous-wave mode for STED (Mira900, Coherent, Santa Clara, CA). The fluorescence was detected with a single-photon avalanche photodiode (APD) (id100-MMF50, ID Quantique, Carouge, Switzerland) for imaging or split by a 50:50 beam splitter and detected by two APDs (SPCMAQR-13-FC, Perkin Elmer Optoelectronics, Fremont, CA) for FCS. The detector signals were acquired by a time-correlated single-photon counting (TCSPC) board (SPC-730 or DPC-230, Becker & Hickl, Berlin, Germany) for post-acquisition, timegating, lifetime analysis and calculation of the correlation functions. Scanning of the sample was realized using a three-axis piezo table (NanoMax, Thorlabs GmbH Europe, Dachau, Germany). The focal STED doughnut was created by placing a polymer phase plate (RPC Photonics, Rochester, NY) in the circular polarized STED beam applying a helical phase ramp from 0 to 2π into the back aperture of a 1.4 NA objective lens (HCX PL APO, 100×/1.40, oil, Leica, Wetzlar, Germany). Excitation and STED beams were overlapped and separated from the fluorescence by two dichroic mirrors (AHF Analysentechnik, Tuebingen, Germany). The fluorescence was detected by the same objective lens, cleaned up with appropriate bandpass and notch filters and collected through multimode fibers for confocal detection. The data of figure S9 was recorded on a commercial Leica TCS SP8 STED (Leica. Microsystems, Leica Microsystems, Mannheim, Germany) configured for single-molecule-detection experiments. The system is equipped with a 592 nm continuous-wave laser (VFL-P_2000 592, MBP Communications, Canada) for STED and a 80 MHz pulsed white-light laser (SuperK, NKT Photonics A/S, Denmark) tuned to 488 nm for excitation. The fluorescence signal is collected by two single-photon-avalanche-diodes (SPADs) and processed for STED-FLCS by a TCSPC card (PicoHarp 300, PicoQuant, Berlin, Germany).
Detection of lipid domains in model and cell membranes by fluorescence lifetime imaging microscopy
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2010
The presence of lipid domains in cellular membranes and their characteristic features are still an issue of dividing discussion. Several recent studies implicate lipid domains in plasma membranes of mammalian cells as short lived and in the submicron range. Measuring the fluorescence lifetime of appropriate lipid analogues is a proper approach to detect domains with such properties. Here, the sensitivity of the fluorescence lifetime of1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phospholipid (C6-NBD-phospholipid) analogues has been employed to characterize lipid domains in giant unilamellar vesicles (GUVs) and the plasma membrane of mammalian cells by fluorescence lifetime imaging (FLIM). Fluorescence decay of C6-NBD-phosphatidylcholine is characterized by a short and long lifetime. For GUVs forming microscopically visible lipid domains the longer lifetime in the liquid disordered (ld) and the liquid ordered (lo) phase was clearly distinct, being ϳ7 ns and 11 ns, respectively. Lifetimes were not sensitive to variation of cholesterol concentration of domainforming GUVs indicating that the lipid composition and physical properties of those lipid domains are well defined entities. Even the existence of submicroscopic domains can be detected by FLIM as demonstrated for GUVs of palmitoyloleoyl phosphatidylcholine/N-palmitoyl-D-sphingomyelin/cholesterol mixtures. A broad distribution of the long lifetime was found for C6-NBD-phosphatidylcholine inserted in the plasma membrane of HepG2 and HeLa cells centered around 11 ns. FLIM studies on lipid domains forming giant vesicles derived from the plasma membrane of HeLa cells may suggest that a variety of submicroscopic lipid domains exists in the plasma membrane of intact cells.
Chemistry and Physics of Lipids, 2009
Membrane microdomains and their involvement in cellular processes are part of the current paradigm of biomembranes. However, a better characterization of domains, namely lipid rafts, is needed. In this review, it is shown how the use of time-resolved fluorescence, with the adequate parameters and probes, helps elucidating the type, number, fraction, composition and size of lipid phases and domains in multicomponent model systems. The determination of phase diagrams for lipid mixtures containing sphingolipids and/or cholesterol is exemplified. The use of fluorescence quenching and Förster resonance energy transfer (FRET) are also illustrated. Strategies for studying protein-induced domains are presented. The advantages of using single point microscopic decays and fluorescence lifetime imaging microscopy (FLIM) in systems with three-phase coexistence are explained. Finally, the introduction of FLIM allows studies in live cell membranes, and the nature of the microdomains observed is readily elucidated due to the information retrieved from fluorescence lifetimes.
Biophysical Journal, 2011
Details about molecular membrane dynamics in living cells, such as lipid-protein interactions, are often hidden from the observer because of the limited spatial resolution of conventional far-field optical microscopy. The superior spatial resolution of stimulated emission depletion (STED) nanoscopy can provide new insights into this process. The application of fluorescence correlation spectroscopy (FCS) in focal spots continuously tuned down to 30 nm in diameter distinguishes between free and anomalous molecular diffusion due to, for example, transient binding of lipids to other membrane constituents, such as lipids and proteins. We compared STED-FCS data recorded on various fluorescent lipid analogs in the plasma membrane of living mammalian cells. Our results demonstrate details about the observed transient formation of molecular complexes. The diffusion characteristics of phosphoglycerolipids without hydroxyl-containing headgroups revealed weak interactions. The strongest interactions were observed with sphingolipid analogs, which showed cholesterol-assisted and cytoskeletondependent binding. The hydroxyl-containing headgroup of gangliosides, galactosylceramide, and phosphoinositol assisted binding, but in a much less cholesterol-and cytoskeleton-dependent manner. The observed anomalous diffusion indicates lipid-specific transient hydrogen bonding to other membrane molecules, such as proteins, and points to a distinct connectivity of the various lipids to other membrane constituents. This strong interaction is different from that responsible for forming cholesterol-dependent, liquid-ordered domains in model membranes.