Gli autori ringraziano il Land Baden-Württemberg e l&#39;Unione-Europäischer Europäische Fonds für die Entwicklung regionale - per il finanziamento ZAFH PHOTON-n, il Bundesministerium für Bildung und Forschung (BMBF) per assegno di ricerca finanziamento n. 1792C08 così come il Baden-Württemberg-Stiftung GmbH per il finanziamento del progetto &quot;Aurami&quot;.

1. Semina ed incubazione di cellule <ol><li> Seme singole cellule, per es U373-MG o U-251-MG cellule di glioblastoma ad una densità tipica di 100 cellule / mm 2 su un vetrino e farle crescere per 4872 ore in terreno di coltura (per es RPMI 1640 integrato con 10% siero di vitello fetale e antibiotici) utilizzando un incubatore a 37 ° C e 5% di CO 2. </li><li> Incubare le cellule con un marcatore di membrana, per esempio 6-dodecanoile dimetilammino-2-naftalene (laurdan) applicata per 60 minuti ad una concentrazione di 8 mM (in mezzo di coltura) 4, o un marcatore citoplasma, ad esempio calceina acetomethlyester applicato per 40 min a concentrazione di 5 pM 3, o una membrana associata fotosensibilizzatore, ad esempio protoporfirina IX indotta upon 4 ore di incubazione con il suo precursore acido 5-aminolevulinico (5-ALA; 1 mM) 4,5. </li><li> Lavare le cellule con mezzo di coltura o tampone fosfato salino (PBS) prima di microscopia a fluorescenza. &lt;/ Li&gt; </ol> 2. VA-TIRFM <ol><li> Utilizzare un microscopio verticale, ad esempio Zeiss Axioplan, e sostituire il suo condensatore da un dispositivo di illuminazione, come descritto in rif. 3 con una emisferica o semicilindrica prisma di vetro accoppiato otticamente alla slitta oggetto (vedi Figura 1). </li><li> Utilizzano un fascio laser collimato parallelo che dopo l&#39;imaging dal prisma di vetro e altri componenti ottici (ad es specchio concavo) risultati anche in un fascio parallelo sulla superficie del campione (raggio pupilla telecentrico). Fare attenzione che il vettore campo elettrico è perpendicolare alla polarizzata piano di incidenza. </li><li> Utilizzare uno specchio orientabile situato all&#39;interno del condensatore che è ripreso nel piano campione e illumina il campione con un angolo di incidenza variabile (raggio oggetto). Lo specchio regolabile può essere azionato da un motore passo passo, dove ogni posizione corrisponde un angolo ben definito di incidenza, come determinato dalle misurazioni goniometriche 3 </sup&gt;. </li><li> Calibrare la posizione angolare dello specchio regolabile utilizzando l&#39;angolo critico Θ = 61,3 ° C per un bicchiere di acqua transizione come valore fisso. Θ c possono essere visualizzati al variare della Θ quando un colorante fluorescente (ad esempio, 1 mM flavina mononucleotide, FMN) è disciolto in acqua. La posizione dello specchio con Θ = Θ c è memorizzato nel programma di posizionamento specchio. </li><li> Impostare i parametri della telecamera (retro illuminato Andor Ixon EMCCD, DV887DC, ANDOR Tecnologia, Belfast, Regno Unito 6), tra cui la temperatura, il guadagno, il tempo di registrazione e la velocità di spostamento. </li><li> Utilizzare una lente obiettivo di ingrandimento adeguato e preferenzialmente moderata apertura numerica (per esempio 40 × / 0,75 o 63 × / 0,90 immersione in acqua) e localizzare la slitta oggetto nel microscopio. Usare olio di immersione per assicurarne il contatto con ottica del campione con il prisma di vetro. Registrare immagini di fluorescenza di campioni identici al variare dellaangolo di illuminazione (Θ ≥ Θ Θ c con c corrispondente a 64,5 ° per una cella di vetro interfaccia). Una gamma angolare di 66 ° -74 ° con passi di 0,5 ° o 1 ° è raccomandato. Utilizzare un adeguato passaggio lungo o filtro passa banda per il rilevamento di fluorescenza. </li></ol> 3. Analisi dei dati <ol><li> Leggere le immagini (ad esempio in formato ASCII) registrate a vari angoli Θ, nonché le impostazioni della fotocamera per discriminazione (sfondo), la lunghezza d&#39;onda di eccitazione e gli indici di rifrazione del vetro (n 1 = 1,52) e cellule (n 2 = 1,37) nel programma di valutazione (LabView, nanocellula). Per ogni set di parametri della profondità di penetrazione d (Θ) e la trasmissione fattore T (Θ) sono calcolati automaticamente. </li><li> Selezionare le immagini utilizzate per la valutazione della cellula-substrato distanze Δ secondo l&#39;equazione 5 (marker di membrana) o equazione 4 (marcatore citoplasma), le immagini individuali con disomogeneailluminazione (e rivelatrice, g. strisce) possono essere esclusi. </li><li> Calcola le immagini di cellule-substrato topologia e selezionare un codice di colore appropriato per la visualizzazione. Durante profili di valutazione dei singoli pixel possono essere visualizzati, come illustrato nella Figura 2. Questi profili possono essere utilizzati come indicatore per la qualità della calzata. </li><li> Memorizzare il protocollo di valutazione. </li></ol> 4. Risultati rappresentativi Esperimenti rappresentativi sono eseguite con tecniche di ingegneria genetica cellule di glioblastoma U251-MG gentilmente fornite dal Prof. Jan Mollenhauer, Dipartimento di Oncologia Molecolare, University of South Denmark, Odense. In due subcloni di tali cellule U251 geni oncosoppressori TP53 o PTEN sono sovra-espressi utilizzando un Tet-On sistema inducibile di espressione, in modo tale che queste cellule presentano un potenziale oncogeno ridotta e può essere considerato meno maligna. Altamente maligni U251-MG cellule senza gene soppressore vengono utilizzati come controlli, e U251-Cellule di tipo MG selvatici in carica per un ulteriore confronto. Un diodo laser che emette una serie quasi continua di impulsi picosecondi (LDH400 con conducente PDL 800-B, Picoquant, Berlin, Germany, lunghezza d&#39;onda: 391 nm; impulso di energia: 12 pJ; tasso di ripetizione: 40 MHz) è utilizzato come una sorgente di eccitazione. Nella Figura 3 immagini TIRFM di U251-MG cellule di glioblastoma con un attivato TP53 gene soppressore del tumore e incubate con laurdan (8 uM, 60 min), sono raffigurati per gli angoli di incidenza di 66,0 °, 69,0 ° e 73,0 ° corrispondenti a profondità di penetrazione del campo elettromagnetico evanescente di circa 140 nm, 75 nm e 60 nm, rispettivamente. Con profondità decrescente fluorescenza penetrazione diminuisce di intensità (vedi tabelle) e proviene da altri siti superficiali delle cellule (ad esempio contatti focali sui bordi). Cell-substrato distanze (visualizzato in 3d figura da un codice colore che va 0-600 nm) fortemente variare nel corso del le celle comprese tra illy pochi nanometri (bianco-giallo) e 250-300 nm (rosso scuro), vale a dire i contatti focali e grandi cellule substrato distanze sono nelle immediate vicinanze. Lo stesso vale per U251-MG cellule di glioblastoma con un gene PTEN attivato soppressore (Figura 4d), ​​mentre la cellula-substrato distanze sono piuttosto costanti per U251-MG di controllo e cellule wild type con i valori tipici di 80-100 nm (giallo; Figura 4b ) e 150-200 nm (rosso-arancio; la figura 4a). <img alt="Figura 1" src="/files/ftp_upload/4133/4133fig1.jpg" /> Figura 1. Dispositivo di illuminazione per VA-TIRFM in un microscopio verticale. Uno specchio orientabile comandata da un motore passo permette una risoluzione angolare di ± 0,25 °. <img alt="Figura 2" src="/files/ftp_upload/4133/4133fig2.jpg" /> Figura 2. Profilo di valutazione per un pixel (schematica). Quando ln [I F / T (Θ)] viene tracciata in funzione di 1 / d (Θ), rappresenta la pendenza della cellula-substrato distanza Δ. Tale pendenza è comunemente determinata con una precisione di circa ± 10%. Un valore - derivante dalla illuminazione disomogenea - è stata segnata ed esclusi dalla valutazione. <img alt="Figura 3" src="/files/ftp_upload/4133/4133fig3.jpg" /> Figura 3 immagini TIRFM di U251-MG cellule di glioblastoma con attivata TP53 gene soppressore su incubazione con laurdan (8 uM, 60 min). Registrato a vari angoli di illuminazione (ac); cellula-substrato distanze nell&#39;intervallo 0-600 nm mostrato da un codice colore (d); lunghezza d&#39;onda di eccitazione: 391 nm, la rilevazione: ≥ 420 nm, dimensioni immagine: 210 micron. × 210 micron <a href="http://www.jove.com/files/ftp_upload/4133/4133fig3large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . Figura 4 Cell-substrato distanze U251-MG cellule glioblatoma nell&#39;intervallo 0-600 nm indicati da un codice colore,. (A) di tipo selvatico (b) i controlli, (c) cellule con attivata gene soppressore TP53, (d) cellule con attivato PTEN gene soppressore; lunghezza d&#39;onda di eccitazione: 391 nm, la rilevazione: ≥ 420 nm, dimensioni immagine: 210 micron. × 210 micron <a href="http://www.jove.com/files/ftp_upload/4133/4133fig4large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> .

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Nessun conflitto di interessi dichiarati.

Un metodo è descritto per cella di misura-substrato distanze con precisione nanometrica. Attualmente, i metodi di super-risoluzione microscopia, ad esempio sulla base di illuminazione strutturata 7 o 8-10 singoli rilevazione molecola nonché deplezione emissione stimolata (STED) microscopia 11 sono di notevole interesse. Nessuna di queste tecniche, tuttavia, consente una risoluzione assiale inferiore a 50 nm. Inoltre, irradianza piuttosto elevato di 50.100 W / cm 2 ?...

Quando un fascio di luce che si propaga attraverso un mezzo di indice di rifrazione n 1 incontra un&#39;interfaccia ad un secondo mezzo di rifrazione n 2 &lt;n 1, riflessione interna totale avviene a tutti gli angoli di incidenza Θ, che sono maggiore dell&#39;angolo critico Θ = c arcsin (n 2 / n 1). Nonostante sia totalmente riflesso del fascio di luce incidente evoca un campo evanescente elettromagnetico che penetra nel secondo mezzo e decade esponenzialmente con la distanza z perpendicolare dall&#39;interfaccia secondo I (z) = I 0 ez / d (Θ). I (z) corrisponde all&#39;intensità del campo elettromagnetico e d (Θ) alla profondità di penetrazione alla lunghezza d&#39;onda λ, come indicato da d (Θ) = (λ/4π) (n 1 2 sin 2 Θ - n 2 2) -1 / 2 Come riportato in letteratura 1,2, I 0 </sub&gt; corrisponde all&#39;intensità della luce incidente I e moltiplicato per il coefficiente di trasmissione T (Θ) e il rapporto n 2 / n 1. Se il vettore di campo elettrico di questo fascio di luce polarizzata è perpendicolare al piano di incidenza (cioè il piano attraversato dal incidente e il raggio riflesso), questo fattore di trasmissione è data dalla T (Θ) = 4 cos 2 Θ / [1 - (2 n / NL 1) 2] Per l&#39;intensità di fluorescenza rilevata in misurazioni TIRFM, assorbimento di luce deve essere integrati su tutti gli strati del campione e moltiplicato per il rendimento η quantica di fluorescenza del colorante rilevante e con l&#39;angolo solido di rilevamento Ω. Come riportato in precedenza 3, questa intensità può essere descritto da I F (Θ) = AT (Θ) OC (z) e-z / d (Θ) dz se tutti i fattori che sono indipendenti f<html> rom l&#39;angolo di incidenza e Θ coordinata z sono inclusi nella equazione sperimentale A. costante 3 può essere risolto analiticamente, se la concentrazione c (z) di fluorofori è considerato essere quasi costante - O affatto coordinate z ≥ Δ, ad esempio all&#39;interno del citoplasma delle cellule aventi una distanza Δ dall&#39;interfaccia. In questo caso, l&#39;integrale deve essere calcolata da z = Δ per z = ∞ conseguente I F = A c T (Θ) d (Θ) e-Δ / d (Θ) - O tra z = Δ - t / 2 e z = + Δ t / 2, ossia in un sottile strato di spessore t, ad esempio una membrana cellulare a una distanza Δ dall&#39;interfaccia. In questo caso, l&#39;intensità di fluorescenza può essere calcolato come I F = A c T (Θ) te-Δ / d (Θ), se t è piccolo in confronto con Δ. ent &quot;&gt; dalle equazioni (4) e (5) cellula-substrato distanze Δ può essere calcolato, se le immagini di fluorescenza I F sono registrati per angoli variabili Θ, e se ln (I F / T d) (Eq. 4) o ln (I F / T) (Eq. 5) viene valutato come una funzione di 1 / d per questi angoli. Per la membrana marcatore laurdan impiegate in questo documento (vedi sotto), la valutazione è stata eseguita secondo l&#39;Eq. 5. Come riportato in precedenza 3, l&#39;acquisizione delle immagini e la valutazione richiede - Distribuzione omogenea della luce di eccitazione sul campione, - Validità di un 2-strato del modello (con gli indici di rifrazione n 1 e n 2 per il substrato e il citoplasma, rispettivamente). Ciò vale, se la membrana plasmatica è molto sottile, e se il livello del mezzo extracellulare (tra la cella e il substrato) è piccolo rispetto alla lunghezza d&#39;onda della luce incidente. Inoltre, un&#39;apertura numerica moderata(Tipicamente A ≤ 0,90) della lente di obiettivo microscopio è vantaggioso per evitare deviazioni di luminosità dovuta alla anisotropia della radiazione nel campo vicino dell&#39;interfaccia dielettrico.

<table border="1"><tbody><tr><td> Nome </td><td> Azienda </td><td> Tipo </td><td> Commenti </td></tr><tr><td> Incubatrice </td><td> Nunc </td><td> Nunc Cellstar QM1300SVBA </td><td></td></tr><tr><td> A flusso laminare </td><td> Holten sicuro </td><td> S2010 1.2 IT GG 1LN </td><td></td></tr><tr><td> DMEM + 10% FCS + 1% di penicillina / streptomicina </td><td> Biochrom </td><td></td><td> Coltivazione media </td></tr><tr><td> Vetro oggetto diapositive </td><td> Marienfeld </td><td> Pure vetro bianco </td><td> Pulizia Procedura speciale utilizzato </td></tr><tr><td> 6-dodecanoile dimetilammino-2-naftalene (laurdan) </td><td> Molecular Probes </td><td></td><td> Marcatore fluorescente (soluzione stock: 2 mM in etanolo) </td></tr><tr><td> Microscopio </td><td> Carl Zeiss </td><td> Axioplan 1 </td&gt; <td></td></tr><tr><td> Diodo laser </td><td> PicoQuant </td><td> LDH 400 con autista PDL 800-B </td><td> Lunghezza d&#39;onda: 391 nm </td></tr><tr><td> Singola modalità del sistema di fibra </td><td> Punto di Origine </td><td> kineFlex </td><td> Usato con ottiche collimanti </td></tr><tr><td> EMCCD fotocamera </td><td> Andor </td><td> DV887DC </td><td> Fotocamera posteriore illuminato </td></tr></tbody></table>

nanotopology of cell adhesion upon variable-angle total internal reflection fluorescence microscopy (va-tirfm)

Topologia superficiale, ad esempio di cellule che crescono su un substrato, è determinata con precisione nanometrica Variable-Angle Microscopy Total Internal Reflection Fluorescence (VA-TIRFM). Cellule sono coltivate su vetrini trasparenti ed incubate con un marcatore fluorescente omogeneamente distribuiti nella loro membrana plasmatica. L&#39;illuminazione avviene mediante un raggio laser parallelo sotto angoli variabili di riflessione interna totale (TIR) ​​con differenti profondità di penetrazione del campo elettromagnetico evanescente. Registrazione di immagini di fluorescenza upon irradiazione a circa 10 diverse angolazioni permette di calcolare cellula-substrato distanze con una precisione di pochi nanometri. Differenze di adesione tra varie linee cellulari, cellule tumorali e cellule es meno maligne, sono così determinato. Inoltre, eventuali modifiche di adesione cellulare su di trattamento chimico o fotodinamica può essere esaminato. In confronto con altri metodi di super-risoluzione microscopia luce di esposizione rimanedanni molto piccola, e non di cellule viventi ci si aspetta.

Watch this Scientific Journal Video about Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM at JoVE.com

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM

Topologia di adesione cellulare su un substrato viene misurata con precisione nanometrica variabile-angolo microscopia a fluorescenza di riflessione interna totale (VA-TIRFM).

Nanotopology dell&#39;adesione cellulare su angolo di inclinazione variabile Microscopia Total Internal Reflection Fluorescence (VA-TIRFM)

Surface topology, e.g. of cells growing on a substrate, is determined with nanometer precision by Variable-Angle Total Internal Reflection Fluorescence ...

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Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

10.1K Views.  Institut für Angewandte Forschung. Topologia di adesione cellulare su un substrato viene misurata con precisione nanometrica variabile-angolo microscopia a fluorescenza di riflessione interna totale (VA-TIRFM).

Video: Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

Visualizzazione di Cortex Organizzazione e Dynamics in microrganismi, utilizzando Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

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Bioingegneria

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Creazione di sfumature adesivi e solubile per la migrazione di imaging cellulare con microscopia a fluorescenza

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

Perlina di aggregazione saggi per la caratterizzazione di molecole di adesione cellulare putativo

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3D Orbital Inseguimento in una Modified microscopio a due fotoni: un&#39;applicazione per il monitoraggio di intracellulare vescicole

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

Da Veloce imaging di fluorescenza di Molecular legge Diffusione su membrane cellulare dal vivo in un microscopio commerciale

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Utilizzando impedenza Cell-substrato e imaging cellulare dal vivo per misurare i cambiamenti in tempo reale in Cellular Adesione e De-adesione indotta da Matrix Modifica

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

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 ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Wet Chimica e Peptide immobilizzazione su politetrafluoroetilene per Miglioramento di adesione cellulare

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

Divisione delle cellule dei mammiferi in matrici 3D tramite microscopia confocale quantitativa di riflessione

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

Visualizzazione di formazione di aderenze in cellule per mezzo di avanzati filatura microscopia di fluorescenza di riflessione interna totale disco

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...