Name: preparation of mica supported lipid bilayers for high resolution optical microscopy imaging
Uploaded: 2014-06-07T15:00:00.000+00:00
Duration: 7 min 48 s
Description: Watch this Scientific Journal Video about Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging at JoVE.com

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1. Mica e diapositive Preparazione <ol><li> Luogo No. 1 ½ (0,17 mm) coprioggetto in colorazione rack. </li><li> Ultrasuoni per 30 min a 2% di detergente a 60 ° C. </li><li> Lavare 20 volte con acqua deionizzata. </li><li> Estrarre i vetrini con pinze e asciugare con aria compressa o azoto. </li><li> Tagliare foglio di mica in 10 x 10 mm pezzi quadrati utilizzando forbici o lama di rasoio. </li><li> Tagliare ogni pezzo mica in 2-3 foglioline sottili con lama di rasoio. NOTA: Questa fase richiede l&#39;uso di lama affilata. </li></ol> 2. Montaggio Mica e della Camera di montaggio <ol><li> Pulire vetrino microscopico con etanolo. </li><li> Colla foglio di mica tagliati nel passaggio da 1,6 a vetrino con adesivo ottico. Adesivo a bassa viscosità si consiglia di diffondere meglio la goccia e incollare la mica. </li><li> Cure sotto la lampada UV, 10 min. NOTA: Adhesive è curata da luce UV con massimo di assorbimento nell&#39;intervallo di 350 a 380 nm e l&#39;energia raccomandato required per la piena guarigione è di 4,5 J / cm 2. Tuttavia, diverse fonti di luce possono essere utilizzati per questo passo (vedi materiali forniti dal fornitore adesivo). </li><li> Esporre una superficie di mica pulito rimuovendo i primi strati con nastro adesivo. </li><li> Mettere piccola goccia (~ 20 micron) di adesivo ottico sulla superficie di mica. NOTA: In questo passaggio, si consiglia l&#39;adesivo ad alta viscosità con un basso livello di autofluorescenza. La nostra esperienza ha dimostrato che l&#39;utilizzo di combinazione di basso (punto 2.2) e adesivi ad alta viscosità aumenta in modo significativo l&#39;efficacia di mica splitting (fase 2.7). </li><li> Posizionare delicatamente coprioggetto appena pulita sulla goccia di colla, evitando bolle d&#39;aria, lasciare riposare per 1 min. </li><li> Cure sotto la lampada UV, 10 min. NOTA: dopo questa fase, il sandwich di vetrino, mica e coprioggetto può essere conservato per un periodo relativamente lungo di tempo (fino a poche settimane). Procedere alla fase successiva appena prima l&#39;attuale preparazione SLB, per assicurarsi che la superficie di mica è fresco. NOTA: Adesivo è curata da luce UV con il massimo assorbimento nel range di 350 a 380nm e l&#39;energia consigliata necessario per la piena guarigione è 4.5J/cm 2. Tuttavia, diverse fonti di luce possono essere utilizzati per questo passo (vedi materiali forniti dal fornitore adesivo). </li><li> Utilizzando un taglierino, smontare delicatamente il vetrino dal vetrino lato, come mostrato nel video. Nella maggior parte dei casi, uno strato sottile e piatto di mica rimarrà attaccato alla coprioggetto. Mica attaccato al vetrino può essere riutilizzato (ripetere dal punto 2.4). </li><li> Controllare la qualità della superficie a occhio nudo o sotto un microscopio da dissezione, per assicurarsi che strato mica è ancora incollato al coprioggetto e non è stato rimosso completamente durante la divisione nel passaggio 2.8. Fare un piccolo graffio con una pinza o dissezione ago aiuterà a distinguere tra l&#39;adesivo che ha una consistenza diversa da rivelabile mica. </li><li> Rimuovere la guarnizione di gomma da un ml flacone tappo 1.5 e incollare il tappo a testa in giù alla superficie con otticaadesivo o smalto, e la cura con la lampada UV, o lasciare asciugare 10 minuti, rispettivamente. NOTA: Se il campione è pronto per simultanea ottico (TIRFM o confocale) con imaging AFM, un ml flacone tappo 1.5 potrebbe essere troppo piccolo per montare la testa AFM sul palco microscopio. In tal caso, il tappo può essere sostituito con qualsiasi plastica O-ring con un diametro più grande, adatto per il montaggio testa AFM. Nel caso in cui l&#39;esperimento richiede la rimozione del tappo senza danneggiare la superficie di mica, grasso al silicone può essere usata al posto della colla o smalto. </li></ol> 3. Supportato doppio strato lipidico (SLB) Formazione <ol><li> Posizionare soluzione liposomi preparata nella camera con la superficie. Il volume minimo richiesto per la formazione SLB è ~ 30 ml. NOTA: Per maggiori dettagli sulla liposomi e la formazione SLB, fare riferimento a protocolli pubblicati, come ad esempio il sacchetto et al, 2014 15.. </li><li> Procedere con la formazione SLB utilizzando il protocollo desiderato. Durante l&#39;incubazione el&#39;imaging, la camera può essere collocato in un calore-blocco o fase di microscopio riscaldata per mantenere la temperatura necessaria per mantenere i lipidi in uso sopra della loro temperatura di fusione. </li></ol>

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	<li>Giocondi, M. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+topography+of+membrane+domains.">Surface topography of membrane domains.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 1798, 703-718 (2010).</li><li>Garcia-Saez, A. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+scanning+probe+and+total+internal+reflection+fluorescence+microscopy.">Combined scanning probe and total internal reflection fluorescence microscopy.</a> Methods. 46, 2-10 (2008).</li><li>Shaw, J. E., Slade, A., Yip, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+in+situ+total+internal+reflectance+fluorescence/atomic+force+microscopy+studies+of+DPPC/dPOPC+microdomains+in+supported+planar+lipid+bilayers.">Simultaneous in situ total internal reflectance fluorescence/atomic force microscopy studies of DPPC/dPOPC microdomains in supported planar lipid bilayers.</a> J Am Chem Soc. 125, 11838-11839 (2003).</li><li>Skaug, M. J., Faller, R., Longo, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlating+anomalous+diffusion+with+lipid+bilayer+membrane+structure+using+single+molecule+tracking+and+atomic+force+microscopy.">Correlating anomalous diffusion with lipid bilayer membrane structure using single molecule tracking and atomic force microscopy.</a> J Chem Phys. 134, (2011).</li><li>Bag, N., Yap, D. H., Wohland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+dependence+of+diffusion+in+model+and+live+cell+membranes+characterized+by+imaging+fluorescence+correlation+spectroscopy.">Temperature dependence of diffusion in model and live cell membranes characterized by imaging fluorescence correlation spectroscopy.</a> Biochimica et Biophysica Acta. , (2013).</li><li>Sbalzarini, I. F., Koumoutsakos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feature+point+tracking+and+trajectory+analysis+for+video+imaging+in+cell+biology.">Feature point tracking and trajectory analysis for video imaging in cell biology.</a> J Struct Biol. 151, 182-195 (2005).</li><li>Linkert, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metadata+matters:+access+to+image+data+in+the+real+world.">Metadata matters: access to image data in the real world.</a> J Cell Biol. 189, 777-782 (2010).</li><li>Schutz, G. 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Questo protocollo descrive un metodo per preparare superfici mica lisce e sottili per deposizione bistrato lipidico e imaging ad alta risoluzione. La tecnica richiede abilità manuale minimo, limitato soprattutto alla attenta smontaggio del sandwich vetro-mica-vetro (passo 2.8), che è essenziale per ottenere una superficie di mica alta qualità. Controllo della mica appena spaccati è sempre necessaria, a questo punto, poiché è possibile per la mica staccare dall&#39;adesivo ottica senza fende, lasciando aree esposte...

L&#39;obiettivo generale del presente protocollo è quello di mostrare un metodo per preparare le superfici mica per imaging ad alta risoluzione di mica sostenuta lipidi bistrati (SLB) utilizzando la microscopia ottica riflessione interna totale in fluorescenza (TIRFM) o la microscopia confocale, che potrebbe anche essere combinato con forza atomica Microscopy (AFM). SLB sono un modello ampiamente utilizzato per numerosi studi di raggruppamento lipidi, separazione di fase, la dinamica di componenti a doppio strato o loro interazioni con peptidi, proteine ​​o altri composti 1-5. Substrati differenti potrebbero essere utilizzati per la formazione SLB (cioè vetro, mica, biossido di silicio, polimeri) a seconda della natura dello studio 4,6-8. Gli studi di membrana tipiche si basano su tecniche di imaging basata microscopia-, come TIRFM e AFM. Così, per l&#39;imaging TIRFM, una superficie di vetro è una tipica scelta perché il vetro è trasparente. Preparazione del vetro è relativamente facile, e la qualità dei risultati è principalmentedeterminato dalla accurata pulizia prima della deposizione delle vescicole lipidiche superficie. AFM grazie alla sua elevata risoluzione assiale richiede superfici di mica. Mica è un minerale silicato, con quasi perfetta scissione basale. Così, la mica appena spaccati è atomicamente piatta, che permette l&#39;osservazione di membrana dislivelli anche a scala sub-nanometrica 9. Studi di diffusione con metodi quali la spettroscopia di correlazione di fluorescenza (FCS), singola molecola di inseguimento (SMT), e recupero di fluorescenza dopo photobleaching (FRAP) hanno mostrato tuttavia che la dinamica della membrana lipidica dipende fortemente dal tipo di superficie su cui sono depositati, per cui vetro e mica può dare risultati ampiamente variabili 10,11. Queste differenze sono non solo i coefficienti di diffusione delle sonde di membrana, ma anche l&#39;individuazione di popolazioni separate di particelle diffondenti con tassi differenti, e possibilmente la commutazione tra i diversi stati. Così,il confronto diretto dei risultati ottenuti utilizzando tecniche TIRFM e AFM è spesso problematico, a meno che la stessa superficie (in questo caso mica) viene utilizzato. Anche se ci sono alcuni studi in cui TIRFM e imaging AFM doppio strato è stata condotta sulla stessa superficie mica 12,13, mica è usato raramente per la microscopia ottica, soprattutto a causa di problemi di gestione. Preparazione Mica richiede il taglio a mano in foglioline sottili, che sono incollati ad coprioggetto con adesivo ottico 12. Questo metodo richiede tuttavia una certa pratica per ottenere risultati soddisfacenti. Inoltre, le superfici ottenute sono spesso ondulati e di spessore, che li rende difficili da utilizzare con bassa distanza di lavoro, alti lenti apertura numerica. Mica superfici preparate come descritto in questo protocollo sono molto sottili (~ 220 micron, compreso lo spessore vetrino di 170 micron) ed estremamente piatta, evitando di &quot;ondulazione&quot;, che è fondamentale per il successo di imaging ad alta risoluzione. Possono essere utilizzatiper TIRFM o confocale configurazioni. Inoltre, gli stessi campioni possono essere trasferiti AFM, e anche ripreso contemporaneamente TIRFM / confocale e AFM. La combinazione di queste due tecniche consente di correlazione diretta del comportamento diffusione con struttura a membrana a doppio strato 14. Poiché le superfici mica sono appena spaccati, sono pulite e non richiedono molto tempo, scarsamente riproducibile, e le procedure di pulizia potenzialmente pericolosi (protocolli di pulizia di vetro di solito includono sostanze chimiche come soluzione Piranha, acido solforico, idrossido di sodio / potassio). Montaggio di una piccola camera, anche descritto nel protocollo, riduce il volume di vescicole necessari per la formazione bistrato efficace e meno di 50 microlitri. Infine, l&#39;intero processo di assemblaggio superficie non è molto tempo (preparazione richiede meno di 30 minuti), e non richiede un elevato grado di abilità manuale, così come convenzionale scissione mica e incollaggio.

<table><tbody><tr><td>Bath Sonicator</td><td>Fisher Scientific</td><td>FB15051</td><td></td></tr><tr><td>Coverslips 24 x 50 mm - No H1.5</td><td>Marienfeld</td><td>102222</td><td></td></tr><tr><td>DOPC</td><td>Avanti Polar Lipids</td><td>850357</td><td></td></tr><tr><td>Hellmanex III (detergent)</td><td>Hellma Analytics</td><td>320.003</td><td></td></tr><tr><td>Mica V-1 Grade</td><td>SPI Suppliers</td><td>1872-CA</td><td></td></tr><tr><td>Optical Adhesive (high viscosity)</td><td>Norland Products</td><td>NOA63</td><td></td></tr><tr><td>Optical Adhesive (low viscosity)</td><td>Norland Products</td><td>NOA60</td><td></td></tr><tr><td>Sphingomyelin-ATTO647N</td><td>AttoTec</td><td>AD 647N-171</td><td></td></tr><tr><td>UV lamp</td><td>Synoptics Ltd.</td><td>GelVue GVM20</td><td>The lamp was set to 100% power</td></tr></tbody></table>

preparation of mica supported lipid bilayers for high resolution optical microscopy imaging

Doppi strati lipidici supportati (SLB) sono ampiamente usati come modello per lo studio delle proprietà di membrana (separazione di fase, di clustering, dinamica) e la sua interazione con altri composti, come i farmaci o peptidi. Tuttavia caratteristiche SLB variano a seconda del supporto utilizzato. Tecniche utilizzate comunemente per SLB l&#39;imaging e le misure sono single microscopia a fluorescenza molecola, FCS e microscopia a forza atomica (AFM). Poiché la maggior parte degli studi di imaging ottico sono effettuate su un supporto di vetro, mentre AFM richiede una superficie estremamente piatta (generalmente mica), i risultati di queste tecniche non sono direttamente confrontabili, in quanto le proprietà di carica e di funzionamento di questi materiali influenzano fortemente diffusione. Purtroppo, l&#39;alto livello di destrezza manuale richiesto per il taglio e incollaggio fette sottili di mica al vetrino presenta un ostacolo di utilizzare sistematicamente mica per la preparazione SLB. Sebbene questo sarebbe il metodo di scelta, come mica preparatosuperfici spesso finiscono per essere irregolare (ondulato) e difficile da immagine, soprattutto con la piccola distanza di lavoro, alti lenti apertura numerica. Qui vi presentiamo un metodo semplice e riproducibile per la preparazione di sottili superfici piane mica per la deposizione lipidica delle vescicole e preparazione SLB. Inoltre, la nostra camera di misura richiede solo piccole quantità di vescicole per la formazione SLB. I risultati procedura generale nella produzione efficiente, semplice ed economico di superfici doppio strato lipidico alta qualità che sono direttamente paragonabili a quelli utilizzati negli studi AFM.

Il comportamento diffusione di sonde fluorescenti lipidici in SLB è diverso a seconda del substrato. TIRFM combinata con la tecnica SMT è un metodo utile per la visualizzazione movimenti di particelle ed estraendo i loro coefficienti di diffusione. Segnali singola molecola di una sonda sfingomielina-ATTO647N diffusione in un DOPC (1,2-dioleoyl-sn-glycero-3-fosfocolina) doppio strato supportato su vetro e mica sono mostrate nella figura animata allegata. La superficie mica è stato preparato secondo il protoco...

Watch this Scientific Journal Video about Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging at JoVE.com

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Vi presentiamo un metodo di preparazione mica supportato doppi strati lipidici per microscopia ad alta risoluzione. Mica è trasparente e piatto su scala atomica, ma raramente usata nell&#39;imaging a causa della difficoltà di manipolazione; nostra preparazione comporta anche deposizione del foglio di mica, e riduce il materiale utilizzato nella preparazione bistrato.

Preparazione di Mica supportati doppi strati lipidici per alta risoluzione microscopia ottica Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

preparation-mica-supported-lipid-bilayers-for-high-resolution-optical

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

13.3K Views.  Nanyang Technological University.  Vi presentiamo un metodo di preparazione mica supportato doppi strati lipidici per microscopia ad alta risoluzione. Mica è trasparente e piatto su scala atomica, ma raramente usata nell'imaging a causa della difficoltà di manipolazione; nostra preparazione comporta anche deposizione del foglio di mica, e riduce il materiale utilizzato nella preparazione bistrato. 

Video: Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions between lymphocytes and antigen presenting cells, we use Ni2+-chelating lipids to anchor recombinant cell adhesion and MHC proteins to the upper leaflet of a bilayer with poly-histidine tags. Planar bilayers are generated by preparing lipid, treating the glass surfaces where the bilayer will form, and then forming the bilayer in a specialized chamber containing a flow-cell where the lymphocytes will be added.  Then, bilayers are charged with Ni and his-tagged recombinant proteins are added.  Finally,  lymphocytes are injected into the flow cell and TIRF microscopy can be used to image synapse formation and the mechanisms that control T cell locomotion, sites of receptor sorting,  and sites of receptor degradation.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.

Supportato bilayers Planar per la formazione degli Studi di sinapsi immunologica e Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions ...

Ricerca

Biologia

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Formazione di microarray di membrana con un metodo di assemblaggio con sede a seccatoio

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Bioingegneria

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Atomic Force Microscopy Imaging e Spettroscopia Forza supportati doppi strati lipidici

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

Biomembrane Fabrication dal solvente assistita doppio strato lipidico (SALB) Metodo

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

Singola molecola microscopia a fluorescenza su Planar bilayers supportati

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

Super-Resolution Imaging di cellule Natural Killer immunologica Sinapsi su un vetro supportato Planar doppio strato lipidico

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Immunologia e infezioni

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Diffusione e assemblaggio di singole molecole su membrane lipidiche affollate di polimeri

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

Biochimica

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

Assemblaggio di modelli di doppio strato lipidico supportati e sospesi per lo studio delle interazioni molecolari

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

Reconstitution of Membrane-Tethered Minimal Actin Cortices on Supported Lipid Bilayers

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane with the underlying actin cortex. In the past decades, reconstituted, minimal systems based on supported lipid bilayers in combination with actin filament networks have proven to be very instrumental in unraveling basic mechanisms and consequences of membrane-tethered actin networks, as well as in studying the functions of individual membrane-associated proteins. Here, we describe how to reconstitute such active composite systems in vitro that consist of fluid supported lipid bilayers coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors that can be readily observed via total internal reflection fluorescence microscopy. An open-chamber design allows one to assemble the system in a step-by-step manner and to systematically control many parameters such as linker protein concentration, actin concentration, actin filament length, actin/myosin ratio, as well as ATP levels. Finally, we discuss how to control the quality of the system, how to detect and troubleshoot commonly occurring problems, and some limitations of this system in comparison with the living cell surface.

This protocol describes the formation of supported lipid bilayers and the addition of cytoskeletal filaments and motor proteins to study the dynamics of reconstituted, membrane-tethered cytoskeletal networks using fluorescence microscopy.

Ricostituzione di cortecce di actina minima legate alla membrana su doppi strati lipidici supportati

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane ...

Planar Supported Lipid Bilayer Assay: A Microscopy-Based In Vitro Technique to Investigate Septin Assembly on Biological Membrane Mimics

Source: Curtis, B. N. et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/64090">Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions.</a> J. Vis. Exp. (2022)
In this video, we demonstrate a procedure to investigate septin organization on a glass-supported lipid bilayer in vitro using total internal reflection fluorescence microscopy.

Saggio a doppio strato lipidico supportato planare: una tecnica in vitro basata sulla microscopia per studiare l'assemblaggio della septina su imitazioni della membrana biologica

Source: Curtis, B. N. et al. Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions. J. Vis. Exp. (2022)
In this ...