Questo lavoro è stato sostenuto da NIH di Grant EY 14990.

Parte 1: Dissection e cella di isolamento bipolare <ol><li> Preparare le soluzioni di cui alla tabella 2; Il pH delle suonerie &#39;(esterni) le soluzioni devono essere regolato a 7,4 con NaOH e il pH della soluzione interna deve essere regolato a 7,2 con CsOH. Proteggere la soluzione dalla luce interna con foglio di alluminio e conservarlo a 4 ° C fino all&#39;utilizzo; </li><li> Dark-adattare un pesce rosso per almeno 30 minuti prima della dissezione; </li><li> Mentre l&#39;animale scuro si adatta, preparare 5 ml di ialuronidasi (ialuronidasi tipo V, 1100 unità / mL in basso Ca 2 + Ringer, Sigma, St. Louis, MO) e 10 mL di L-cisteina (0,5 mg / mL in basso Ca 2 + Ringer) soluzioni e pesare la papaina (polvere liofilizzata, 40 unità / ml; Sigma, St. Louis, MO) per 5 ml di soluzione digestione; </li><li> Euthanize il pesce rosso per decapitazione veloce con le forbici chirurgiche e distruggere il cervello e il midollo spinale con una lama di bisturi # 11; </li><li> Rimuovere gli occhi distruggendo i muscoli extraoculari con l&#39;aiuto di una pinza # 7 curve Dumont e il taglio del nervo ottico con le forbici iris; </li><li> Mettere una lampadina occhio su un pezzo di carta da filtro e forare il limbo sclerale con la punta della lama di un bisturi # 11; </li><li> Introdurre la lama di un paio di forbici Vännäs all&#39;interno del tutto forato e tagliato l&#39;intero segmento anteriore lontano; </li><li> Posizionare un piccolo pezzo di carta da filtro in cima alla coppa rimanenti ottica ed esercitare una certa pressione al fine di avere la carta bagnare con vitreo; </li><li> Sollevare la carta da filtro con la retina ad esso e tagliare il nervo ottico con un par di forbici Vännäs; </li><li> Trasferire il filtro contenente la retina in un piatto di plastica 35 millimetri con soluzione cultura ialuronidasi e staccare la retina dalla carta da filtro con l&#39;aiuto di pinzette Dumont # 7; </li><li> Tagliare la retina in 4-6 pezzi con metà di un industriale in acciaio al carbonio singolo taglio della lama e lasciare riposare nella soluzione ialuronidasi per 20 minuti; </li><li> In attesa di ialuronidasi abbiano effetto, aggiungere 5 ml di L-cisteina soluzione alla papaina e lasciare riposare fino a quando il liquido diventa trasparente (circa 5-10 minuti); </li><li> Lavate i pezzi di retina 3x a basso Ca 2 + Ringer e farli sedere nella soluzione di papaina per 30-35 minuti; </li><li> Lavate i pezzi di retina 3x a basso Ca 2 + Ringer e conservarli fino al momento dell&#39;uso a 4 ° C in un piatto da 35 millimetri cultura di plastica contenente basso Ca 2 + di Ringer; </li><li> Dissociare le cellule, mettere un pezzo di retina in una provetta contenente 500 ml di basso Ca 2 + Ringer e lentamente triturare la retina pipettando su e giù con una dissociazione pipetta di vetro, con attenzione per non produrre eventuali bolle d&#39;aria. Pipette dissociazione sono fabbricati con il riscaldamento fino alla punta di una pipetta Pasteur di vetro con un becco Bunsen e piegando leggermente con l&#39;aiuto di pinze anatomiche; </li><li> Piastra le cellule isolate aggiungendo una goccia di sospensione della retina ad un fatto in casa da camera di registrazione precedentemente riempiti con 2 ml di basso Ca 2 + Ringer. La camera è costituito dalla metà inferiore di un 35 mm, piatto di plastica con la cultura nel suo complesso circolare nel mezzo e un coprioggetto circolare in vetro di 1,78 indice di rifrazione (PlanOptik, Germania) incollati al fondo con un elastomero di silicone (Sylgard 184; Dow Corning , Midland, MI). </li></ol> Parte 2: cella di carico bipolare e Wash Out TIRF immagini delle vescicole sinaptiche è meglio effettuato con un obiettivo di tipo TIRFM microscopio con un obiettivo molto alto NA e una macchina fotografica sensibile. Per i nostri esperimenti, abbiamo scelto di usare un obiettivo NA 1,65 (Apo x100 O HR, NA 1.65, Olympus, Giappone) con un EMCCD (Cascade 512B, Roper Scientific, Tucson, AZ). L&#39;uso del obiettivo molto alto NA richiede l&#39;uso di coprioggetto di vetro ad alta rifrazione e fluido ad immersione (di-iodomethane con lo zolfo). Nelle nostre condizioni, luce di eccitazione è limitato a un campo diminuisce in modo esponenziale con una lunghezza costante di circa 50 nm. <ol><li> Aggiungere una goccia di liquido ad alto indice di rifrazione (serie M, indice di rifrazione = 1,7800, Cargille Labs, Cedar Grove, NJ) con l&#39;obiettivo microscopio; </li><li> Posizionare la camera di registrazione con attenzione sulla parte superiore del microscopio e l&#39;obiettivo con cura il montaggio dell&#39;elettrodo di massa e l&#39;uscita del tubo superfusione alla camera; </li><li> Lasciate camera di sedersi sul microscopio per 10-20 minuti per consentire alle cellule di affondare e di aderire al fondo; </li><li> Nel frattempo, preparare 5 ml di una Trolox 1mM ® ((±)- 6-idrossi-2 ,5,7,8-tetramethylchromane-2-carbossilico, Sigma, St. Louis, MO) una soluzione di alta K + Ringer . Sonicare fino a completa dissoluzione; </li><li> Preparare 15 mL di ADVASEP-7 soluzione di lavaggio: 1 mM ADVASEP-7 (Sigma, St. Louis, MO) a basso Ca 2 + Ringer. Si noti che ADVASEP-7 uso è facoltativo e può essere omesso se lo si desidera; </li><li> Spurgare il li superfusionesoluzione NES e aggiungere ADVASEP-7 di lavaggio, basso Ca 2 + suoneria e il controllo della suoneria al sistema superfusione; </li><li> Tirare carico pipette da pareti sottili in vetro borosilicato (Kwik-Fil ® TW150-3; WPI, Sarasota, FL). Puffer resistenze pipetta sono nella gamma 1,5-2,5 MΩ; </li><li> Preparare la FM1-43 ® (N-(3-triethylammoniumpropyl) -4 - (4 - (dibutylamino) styryl) piridinio dibromuro &quot;confezione speciale&quot;; Invitrogen, Carlsbad, CA) soluzioni. In primo luogo, fare un 1 mM magazzino con l&#39;aggiunta di 160 microlitri di acqua distillata per un flaconcino (100 mg) di FM1-43 ®. Questo stock può essere conservato a 4 ° C per un massimo di una settimana. Poi, aggiungere 5 ml di FM1-43 ® a 1 ml di alta K + suoneria + di 1 mM Trolox ®. Proteggere la soluzione dalla luce con un foglio di alluminio e conservarlo a 4 ° C fino all&#39;utilizzo; </li><li> Girare la brillante luce campo ottico e la ricerca delle cellule bipolari intatto. Leggermente toccare il microscopio per fare in modo che i neuroni sono saldamente attaccato al fondo della camera; </li><li> Posizionare la penna superfusione vicino alla cella di interesse e profumato continua la preparazione con un basso Ca 2 + Ringer; </li><li> Spegnere le luci della stanza e aggiungere un filtro passa lunghe rosso (cioè RG630, Schott, Germania) per il percorso ottico per ridurre al minimo l&#39;eccitazione del colorante FM; </li><li> Riempire una pipetta di carico con 10 microlitri della soluzione colorante FM, montare la pipetta nel micromanipolatore e abbassare la pipetta sulla preparazione senza sovrapressione fino a quando non è sullo stesso piano focale, come la cellula bipolare che si desidera caricare. Assicurarsi di avere almeno due portaelettrodi: quello usato per la tintura FM non può essere utilizzato per la patch di bloccaggio, altrimenti potrebbero contaminare la soluzione intracellulare; </li><li> Posizionare l&#39;apertura palla a una distanza di circa 10 micron dal terminale dell&#39;assone, spegnere il sistema superfusione off e sfoglia la soluzione colorante per 10 secondi, girando la sovrapressione pipetta sulla; </li><li> Girare la sovrapressione off e, senza muovere la pipetta, attendere per 30 secondi; </li><li> Girare il superfusione su e fare il bagno della camera in ADVASEP-7 soluzione per 5 minuti. Nel frattempo, togliere la pipetta palla dal bagno; </li><li> Dopo 5 minuti, passare al basso Ca 2 + suoneria e profumato della camera per 25-30 minuti per permettere la rimozione del colorante in eccesso. </li></ol> Parte 3: Patch di serraggio e TIRFM Imaging <ol><li> Mentre la preparazione sta lavando fuori, tirare pipette patch da pareti spesse in vetro borosilicato (B150-86-10; Instrument Company Sutter, Novato, CA). Patch resistenze pipetta sono MΩ 8-10 gamma; </li><li> Dopo wash out è completa, posizionare il terminale assone al centro del campo di vista; </li><li> Riempire una pipetta di patch con 7μL di soluzione interna, toccare la pipetta per liberarsi di bolle d&#39;aria, cappotto la pipetta con fuso impronte dentarie (Sticky Cera; Kerr Corporation, Orange, CA) e montarlo nel micromanipolatore; </li><li> Girare la sovrapressione pipetta sulla e abbassare la pipetta lentamente sulla preparazione. Controllare la resistenza pipetta in dell&#39;amplificatore e offset pipetta corretta e capacità con i controlli amplificatore rispettive; </li><li> Accendere il superfusione per controllare la suoneria. Per creare un gigaseal tra la pipetta e la cellula, un po &#39;toccare la punta dell&#39;elettrodo contro il corpo cellulare e girare la sovrapressione pipetta off mentre delicatamente applicando una pressione negativa per l&#39;elettrodo; </li><li> Mentre sigillato, scegliere &quot;a cellula intera&quot; modalità dell&#39;amplificatore e impostare la cella di detenzione potenziale a -60 mV; </li><li> Scegli una regione di interesse con il software di imaging che comprende tutto il terminal assone, spegnere la luce campo e fuori, con una breve esposizione (30 ms) il terminale al laser 488 nm, trovare il giusto piano focale per TIRF per immagini; </li><li> Pausa nella cella utilizzando il comando &quot;zap&quot; dell&#39;amplificatore, mentre applicando una pressione leggermente negativa per la pipetta; </li><li> Corretto per capacità delle cellule e la resistenza serie, e quindi applicare il protocollo di tensione di interesse, mentre l&#39;imaging del movimento delle vescicole sinaptiche. E &#39;in questa fase importante avere il protocollo di tensione sincronizzato con il frame rate della telecamera. Nel nostro caso, ogni fotogramma è di 30 ms lunga, quindi i cambiamenti di tensione si verificano in multipli di questo valore (cioè ogni 300 ms o 10 fotogrammi); </li><li> Attendere almeno 40 secondi tra le prove per consentire il recupero; </li><li> Per controllare la posizione dei nastri sinaptici, scattare foto mentre si accende il laser a 561 nm. </li></ol><img src="/files/ftp_upload/1305/fig1.jpg" alt="figura 1" /> Figura 1: Il setup sperimentale. Un laser a 488 nm (blu) è focalizzata alla periferia del piano focale posteriore dell&#39;obiettivo e soffre riflessione interna totale quando raggiunge il vetro acquoso interfaccia media. Il campo elettromagnetico generato dal raggio riflesso eccita il fluoroforo caricato nel vescicole sinaptiche più vicino al bottom della camera di vetro, che poi fluorescenti (verde). La luce fluorescente viene poi guidato all&#39;occhio dell&#39;osservatore (raffigurato) o di una telecamera CCD. Il potenziale di membrana delle cellule è controllata ripreso contemporaneamente da loro patch-bloccaggio. Questo approccio permette lo studio del rapporto tra i segnali in arrivo (la tensione di membrana) e l&#39;uscita neuronale (esocitosi). <img src="/files/ftp_upload/1305/fig2.jpg" alt="figura 2" /> Figura 2: I risultati tipici. Sinistra: immagine in campo chiaro di una cellula isolata bipolare pesci rossi. In alto a destra: immagine TIRF delle vescicole sinaptiche nel terminale assone delle cellule bipolari caricato con FM 1-43 ® e ripreso con il nm laser 488 (FM colorante). In basso a destra: l&#39;immagine del terminale stesso dopo aver patchato la cellula e di imaging il terminale assone con il laser a 561 nm. Nastri sinaptici sono etichettati dalla rodamina-based Ribeye di legame peptidico (Rpep_rhod). Tabella 1: Reagenti e attrezzature specifiche. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Nome </td><td> Tipo </td><td> Fabbricante </td><td> Catalogo </td><td> Commento </td></tr><tr><td> - </td><td> Air Tabella </td><td> Newport Corporation </td><td> - </td><td> - </td></tr><tr><td> IX70 </td><td> Microscopio invertito </td><td> Olimpo </td><td> - </td><td> Dotato di una lampada al tungsteno per campo chiaro e una porta laterale per l&#39;apertura TIRF </td></tr><tr><td> TH4-100 </td><td> Alimentazione lampada </td><td> Olimpo </td><td> - </td><td> - </td></tr><tr><td> FF498_581-DI01 </td><td> Dicroico Filtro </td><td> Semrock </td><td> - </td><td> - </td></tr><tr><td> NF01-405_488_568 </td><td> Emissione Filtro </td><td> Semrock </td><td> - </td><td> - </td></tr><tr><td> Apo x100 O HR </td><td> Obiettivo </td><td> Olimpo </td><td> - </td><td> NA 1,65 </td></tr><tr><td> RG630 </td><td> Red Glass Filter </td><td> Schott </td><td> - </td><td> - </td></tr><tr><td> - </td><td> 488 nm laser </td><td> Coerente </td><td> - </td><td> Uso alla minima potenza </td></tr><tr><td> - </td><td> Otturatore </td><td> Uniblitz </td><td> - </td><td> - </td></tr><tr><td> VMM-D1 </td><td> Shutter driver </td><td> Uniblitz </td><td> - </td><td> - </td></tr><tr><td> - </td><td> 561 nm laser </td><td> Melles Griot </td><td> - </td><td> - </td></tr><tr><td> - </td><td> Otturatore </td><td> Uniblitz </td><td> - </td><td> - </td></tr><tr><td> VMM-D3 </td><td> Shutter driver </td><td> Uniblitz </td><td> - </td><td> - </td></tr><tr><td> Perfusione pressione Kit </td><td> Superfusione sistema </td><td> Automatizzare scientifico </td><td> 09-04 </td><td> - </td></tr><tr><td> Perfusione Penna </td><td> Superfusione sistema </td><td> Automatizzare scientifico </td><td> - </td><td> - </td></tr><tr><td> ValveLink 8 </td><td> Superfusione System Controller </td><td> Automatizzare scientifico </td><td> - </td><td> - </td></tr><tr><td> Cascade 512B </td><td> EM CCD </td><td> Roper Scientific </td><td> - </td><td> - </td></tr><tr><td> Metamorph 7,1 </td><td> Imaging Software </td><td> Molecular Devices </td><td> - </td><td> - </td></tr><tr><td> EPC-9 </td><td> Patch Morsetto Amplificatore </td><td> HEKA Elektronik </td><td> - </td><td> - </td></tr><tr><td> Impulso </td><td> Amplificatore Software </td><td> HEKA Elektronik </td><td> - </td><td> - </td></tr><tr><td> MP-285 </td><td> Micromanipolatore </td><td> Sutter strumento </td><td> - </td><td> - </td></tr><tr><td> - </td><td> Porta elettrodo </td><td> HEKA Elektronik </td><td> - </td><td> Per 1,5 mm di diametro in vetro, 2 unità </td></tr><tr><td> Kwik-Fil ® TW150-3 </td><td> Capillare di vetro borosilicato </td><td> WPI </td><td> - </td><td> Senza filamento </td></tr><tr><td> B150-86-10 </td><td> Capillare di vetro borosilicato </td><td> Sutter strumento </td><td> - </td><td> Con filamento </td></tr><tr><td> P-97 </td><td> Microelettrodo Puller </td><td> Sutter strumento </td><td> - </td><td> Dotato di filamento box 3x3 e camera ambientale </td></tr><tr><td> - </td><td> Pressione pompa a vuoto d&#39;aria </td><td> Thomas scientifico </td><td> 7893B05 </td><td> Crea vuoto per rimuovere i liquidi da camera e sovrapressione per pipette </td></tr><tr><td> MatLab R2008a </td><td> Software per l&#39;analisi </td><td> The MathWorks </td><td> - </td><td> - </td></tr><tr><td> 353001 </td><td> Piatti 35 millimetri Cultura Plastica </td><td> Falco </td><td> - </td><td> - </td></tr><tr><td> - </td><td> Alto indice di rifrazione di vetro </td><td> PlanOptik </td><td> - </td><td> Indice di rifrazione 488 nm = 1,78 </td></tr><tr><td> Serie M </td><td> Alto indice di rifrazione liquido </td><td> Cargille Labs </td><td> - </td><td> Indice di rifrazione = 1.78 </td></tr><tr><td> Sylgard 184 </td><td> Silicone elastomero Kit </td><td> Dow Corning </td><td> - </td><td> - </td></tr><tr><td> Glutatione </td><td> Tripeptide </td><td> EMD chimici </td><td></td><td> Libero radical scavenger </td></tr><tr><td> Ialuronidasi </td><td> Enzima </td><td> Sigma </td><td> H6254 </td><td> Tipo V </td></tr><tr><td> L-cisteina </td><td> Amino Acid </td><td> Fluka </td><td> 30090 </td><td> Attiva la papaina </td></tr><tr><td> Papaina </td><td> Enzima </td><td> Fluka </td><td> 76220 </td><td> Dalla Carica papaya </td></tr><tr><td> Trolox ® </td><td> Solubile vitamina E </td><td> Sigma </td><td> 56510 </td><td> Scavenger radicali liberi </td></tr><tr><td> ADVASEP-7 </td><td> Solfonati B-ciclodestrina derivati </td><td> Sigma </td><td> A3723 </td><td> Riduce FM 1-43 ® fluorescenza di fondo </td></tr><tr><td> FM 1-43 ® </td><td> Colorante fluorescente </td><td> Invitrogen </td><td> T35356 </td><td> &quot;Confezione speciale&quot; </td></tr><tr><td> Cera appiccicoso </td><td> Pipetta di rivestimento agente </td><td> Kerr Corporation </td><td> - </td><td> Diminuisce la capacità pipetta </td></tr></tbody></table> Tabella 2: soluzioni fisiologiche utilizzate in questo studio. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Sostanza </td><td> Basso Ca 2 + Ringer </td><td> Controllo di Ringer </td><td> Alta K + Ringer </td><td> Soluzione interna </td></tr><tr><td> NaCl </td><td> 120 mm </td><td> 120 mm </td><td> 97,5 mm </td><td> - </td></tr><tr><td> KCl </td><td> 2,5 mM </td><td> 2,5 mM </td><td> 25 mM </td><td> - </td></tr><tr><td> MgCl 2 </td><td> 1 mM </td><td> 1 mM </td><td> 1 mM </td><td> 4 mM </td></tr><tr><td> CaCl 2 </td><td> 0,5 mM </td><td> 2,5 mM </td><td> 2,5 mM </td><td> - </td></tr><tr><td> HEPES </td><td> 10 mM </td><td> 10 mM </td><td> 10 mM </td><td> 10 mM </td></tr><tr><td> EGTA </td><td> 0,75 mm </td><td> - </td><td> - </td><td> 0,5 mM </td></tr><tr><td> Glucosio </td><td> 10 mM </td><td> 10 mM </td><td> - </td><td> - </td></tr><tr><td> Glutatione </td><td> 2 mM </td><td> 2 mM </td><td> - </td><td> 1 mM </td></tr><tr><td> * CH 3 CSO 3 S </td><td> - </td><td> - </td><td> - </td><td> 100 mM </td></tr><tr><td> TEACl </td><td> - </td><td> - </td><td> - </td><td> 10 mM </td></tr><tr><td> ATP-Mg </td><td> - </td><td> - </td><td> - </td><td> 10 mM </td></tr><tr><td> GTP-Li </td><td> - </td><td> - </td><td> - </td><td> 1 mM </td></tr><tr><td> Rpep-Rhod ** </td><td> - </td><td> - </td><td> - </td><td> 5 mM </td></tr><tr><td> Volume </td><td> 200 mL </td><td> 100 ml </td><td> 5 microlitri </td><td> 100 ul </td></tr></tbody></table> * Metanosolfonato cesio. ** Ribeye vincolante peptide: rodamina + EQTVPVDLSVARDR-COOH (mw 1.997,75).

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I vantaggi di tipo oggettivo-microscopia TIRF sono che: 1) fornisce un&#39;eccellente sezionamento ottico limitando luce di eccitazione ad una regione ristretta all&#39;interno del piano focale dell&#39;obiettivo, minimizzando così out-of-focus luce, 2) in quanto la luce diminuisce esponenzialmente con la distanza , il movimento in direzione verticale può essere monitorato come un cambiamento di intensità di fluorescenza, 3) la raccolta efficiente luce attraverso l&#39;obiettivo ad alta apertura numerica 1,5. Il princi...

imaging exocytosis in retinal bipolar cells with tirf microscopy

Fluorescenza di riflessione interna totale (TIRF) microscopia è una tecnica che permette lo studio di eventi che accadono a livello della membrana cellulare, con immagini selettivo di molecole fluorescenti che sono più vicini ad una sostanza alto indice di rifrazione come il vetro<sup&gt; 1</sup&gt;. In questo articolo, si applica questa tecnica per esocitosi immagine delle vescicole sinaptiche nelle cellule bipolari della retina isolata dalla retina pesci rossi. Questi neuroni sono molto adatti per questo tipo di studio a causa della loro terminali degli assoni di grandi dimensioni. Contemporaneamente cerotto di fissaggio le cellule bipolari, è possibile indagare il rapporto tra pre-sinaptica di tensione e rilascio sinaptico<sup&gt; 2,3</sup&gt;. Vescicole sinaptiche terminali all&#39;interno delle cellule del bipolare sono caricati con un colorante fluorescente (FM 1-43 ®) per co-sbuffando il colorante e una soluzione contenente una suoneria alta K<sup&gt; +</supConcentrazione&gt; sui terminali sinaptici. Questo depolarizza le cellule e stimola endocitosi e conseguente assorbimento colorante in vescicole glutamatergica. Dopo aver lavato il colorante in eccesso per circa 30 minuti, le cellule sono pronte per essere cerotto bloccato e ripreso in contemporanea con un laser a 488 nm. La soluzione contiene una pipetta di patch rodamina basata peptide che si lega selettivamente alla proteina sinaptica Ribeye nastro<sup&gt; 4</sup&gt;, In tal modo l&#39;etichettatura nastri specificamente quando i terminali sono ripreso con un laser a 561 nm. Questo permette la localizzazione precisa delle zone attive e la separazione della sinaptica da eventi extra-sinaptico.

Watch this Scientific Journal Video about Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy at JoVE.com

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

In questo video ci dimostra come etichettare e visualizzare singole esocitosi delle vescicole sinaptiche e la tratta di pesci rossi cellule bipolari della retina mediante fluorescenza di riflessione interna totale (TIRF) microscopia.

Esocitosi imaging in cellule bipolari della retina con microscopia TIRF

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

imaging-exocytosis-in-retinal-bipolar-cells-with-tirf-microscopy

Research

JoVE Journal

Biology

11.6K Views.  Yale University School of Medicine. In questo video ci dimostra come etichettare e visualizzare singole esocitosi delle vescicole sinaptiche e la tratta di pesci rossi cellule bipolari della retina mediante fluorescenza di riflessione interna totale (TIRF) microscopia.

Video: Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

Misurare esocitosi nei neuroni Utilizzando Etichettatura FM

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

Ricerca

Biologia

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

A lungo termine Imaging cellule di mammifero utilizzando Wide-Field Microscopia

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Time-lapse imaging di mitosi dopo la trasfezione di siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Basato sulla fluorescenza Misura Store-Entry operato di calcio in cellule vive: dal Cancer Cell coltura per fibra muscolare scheletrica

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

Imaging di fluorescenza con One nanometri Precisione (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

Visualizzazione Clathrin endocitosi mediata da G recettori accoppiati a proteine ​​a risoluzione singolo evento tramite microscopia TIRF

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Un&#39;interfaccia utente grafica per il monitoraggio assistito della concentrazione di proteine ​​in protrusioni cellulari dinamiche

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

FITC-Destrano di imaging come Reporter per esocitosi regolata

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

Preparazione del campione biologico di congelamento ad alta pressione, aumento di contrasto assistita da microonde e Minimal resina incorporamento per Imaging con volumi

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...