Name: mapping molecular diffusion in the plasma membrane by multiple-target tracing (mtt)
Uploaded: 2012-05-27T14:00:00
Description: Watch this Scientific Journal Video about Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT at JoVE.com

Vi takker medlemmene av teamet vårt, særlig MC Blaché for teknisk assistanse, samt M Irla og B Imhof, for deres støtte og fruktbare diskusjoner. Tall for deflasjon og innesperring reprodusert courtesy of Nature Methods. Dette prosjektet er støttet av institusjonelle tilskudd fra CNRS og INSERM og Marseille universitet, og ved spesielle tilskudd fra regionen Provence-Alpes-Côte-d&#39;Azur, Institut National du Cancer, Agence Nationale de la Recherche (ANR-08-PCVI- 0034-02, 2010 ANR BLAN 1214 01) og Fondation pour la Recherche Médicale (Equipe labélisée FRM-2009). VR er støttet av et fellesskap fra Ligue Nationale Contre le Cancer.

I denne videoen presenterer vi en full enkelt partikkel sporing eksperiment ved hjelp av kvante-prikker rettet mot en spesifikk membran reseptor. Hovedmålet dette eksperimentet består i diskriminerende ulike typer molekylære diffusjon atferd målt i plasma membran av levende celler. Faktisk kan molekylære bevegelser som oppstår på membranen typisk avvike fra Brownske diffusjon ved å være lineært mot eller innelukket i nanodomains 26-29, for eksempel. Vi tar sikte på samtidig å følge så mange rese...

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I single-partikkel sporing, ved siden av cellen og mikroskopi aspekter, representerer analysen en vesentlig del av arbeidet. Dette løser algoritmen som brukes til å utføre de tre hovedoppgaver: å oppdage, estimering og koble topper over hver ramme. Men påfølgende del av dette arbeidet ligger i å utarbeide algoritmen selv, som kanskje må være tilrettelagt for eventuell ny dedikert etterforskning, i hovedsak for de siste, ekstra trinn (for eksempel tyde moduser bevegelse, interaksjoner eller støkiometri). <p...

<table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Katalognummer </td><td> Antall </td></tr><tr><td> Cos-7 cellelinje </td><td> ATCC </td><td> CRL-1651 </td><td> 5000 celler / brønn </td></tr><tr><td> HBSS uten Ca 2 + </td><td> GIBCO </td><td> 14175 </td><td> 1 ml </td></tr><tr><td> 0,05% Trypsin EDTA </td><td> GIBCO </td><td> 25300 </td><td> 1 ml </td></tr><tr><td> 8-brønn Lab-tek </td><td> Nunc </td><td> 155441 </td><td> 1 </td></tr><tr><td> QDot-605 streptavidin </td><td> Invitrogen </td><td> Q10101MP </td><td> 20 mm </td></tr><tr><td colspan="4"> Biotinylated Fab (for Fab syntese, se referanse 21) </td></tr><tr><td> Fab fra MAB 108 </td><td> ATCC </td><td> HB-9764 </td><td> 200 mikrogram </td></tr><tr><td> NHS-Biotin<html></td><td> Thermo Scientific </td><td> 21435 </td><td> 18,5 mikrogram </td></tr><tr><td colspan="4"> Komplett medium </td></tr><tr><td> DMEM </td><td> GIBCO </td><td> 41965 </td><td> 500 ml </td></tr><tr><td> Fetal bovin serum </td><td> SIGMA </td><td> F7524 </td><td> 50 ml </td></tr><tr><td> L-Glutamin </td><td> GIBCO </td><td> 25030 </td><td> 5 ml </td></tr><tr><td> HEPES </td><td> GIBCO </td><td> 15630 </td><td> 5 ml </td></tr><tr><td> Sodium Pyruvat </td><td> GIBCO </td><td> 11360 </td><td> 5 ml </td></tr><tr><td colspan="4"> Imaging medium </td></tr><tr><td> HBSS med Ca 2 + </td><td> GIBCO </td><td> 14025 </td><td> 25 ml </td></tr><tr><td> HEPES </td><td> GIBCO </td><td> 15630 </td><td> 250 mL </td></tr></tbody></table>1 &quot;&gt; <tbody><tr><td> Utstyr </td><td> Firma </td><td> Referanse </td></tr><tr><td> Inverted mikroskop </td><td> Nikon </td><td> Eclipse TE2000U </td></tr><tr><td> Lysrörslampa </td><td> Nikon </td><td> Intensilight C-HGFIE </td></tr><tr><td> 1,3 NA 100x objektiv </td><td> Nikon </td><td> Planlegg Fluor 1,30 </td></tr><tr><td> 1,49 NA 100x objektiv </td><td> Nikon </td><td> APO TIRF 1,49 </td></tr><tr><td> Kamera </td><td> Roper Scientific </td><td> Cascade 512 B </td></tr><tr><td> Thermostated boks </td><td> Livet Imaging Services </td><td> The Box </td></tr></tbody></table> Vedlegg: eksempel Script av MTT supplerende analyse funksjon MTT_example (filnavn) %%% Grunnleggende eksempler som viser hvordan du kan gjenopprette MTT utgang resultater %%% Til plotte hver trace og å bygge histogrammet %%% Av fluorescens intensiteter hvis nargin &lt;1% nei file_name gitt? filene = dir (&#39;* stk.&#39;); hvis isempty (filer), disp (&#39;ingen data i dagens dir&#39;), retur, slutt . file_name = filer (1) navn;% standard: første stk fil disp ([&#39;hjelp&#39; filnavn &#39;standard&#39;]) slutt file_param = [filnavn &#39;_tab_param.dat&#39;];% utdatafil %% Belastning data CD (&#39;output23&#39;)% eller (&#39;output22&#39;), ifølge versjon brukt % Ansvarsfraskrivelse: versjon 2.2 genererer bare 7 parametre, % En ekstra parameter, støy, ble lagt i versjon 2.3 % For å lese alle parametere samtidig, i én tabell % Tab_param = fread_all_param (file_param); % TAb_i = tab_param (02:08: slutt :); tab_j = ... % For å lese alle parametere (unntatt frame_number) i separate tabeller % [Tab_i, tab_j, tab_alpha, tab_radius, tab_offset, tab_blk, tab_noise] = fread_all_data_spt (file_param); tab_i = fread_data_spt (file_param, 3);% indeks er 3 fordi spor nummer &amp; ramme nummer, ikke informativ, forkastes! tab_j = fread_data_spt (file_param, 4); tab_alpha = fread_data_spt (file_param, 5); tab_blk = fread_data_spt (file_param, 8); %% Loop løpet spor N_traces = størrelse (tab_i, 1); % Tabeller er N_traces linjer ved N_frames søyler for itrc = 1: N_traces No_blk_index = tab_blk (itrc, :)&gt; 0;% ikke blinkende trinnene bare plot (tab_i (itrc, No_blk_index), tab_j (itrc, No_blk_index)) &amp; Nbsp; xlabel (&#39;i (piksel)&#39;), ylabel (&#39;j (piksel)&#39;) title ([&#39;spor #&#39; num2str (itrc)]) disp (&#39;Vennligst slå hvilken som helst tast for neste spor&#39;), pause slutt %% Fluo histogram N_datapoints = SUM (tab_blk (:)&gt; 0);% ikke blinkende trinnene bare hist (tab_alpha (tab_blk&gt; 0), 2 * sqrt (N_datapoints))% bruker 2sqrt (N) binger xlabel (&#39;intensitet (AU)&#39;), ylabel (&#39;forekomst&#39;) title (&#39;histogram av partikler fluorescensintensiteten&#39;)

mapping molecular diffusion in the plasma membrane by multiple-target tracing (mtt)

Vårt mål er å oppnå en omfattende beskrivelse av molekylære prosesser skjer på cellemembraner i forskjellige biologiske funksjoner. Vi tar sikte på å karakterisere kompleks organisasjon, og dynamikken i plasmamembranen ved enkelt-molekyl nivå, ved å utvikle analytiske verktøy som er spesielt Single-Particle Tracking (SPT) ved høy tetthet: Multiple-Target Tracing (MTT) 1. Single-molekyl videomicroscopy, tilbyr millisekund og nanometric oppløsning 1-11, gir en detaljert fremstilling av membran organisasjon 12-14 av nøyaktig kartlegging deskriptorer som celle reseptorer lokalisering, mobilitet, innesperring eller interaksjoner. Vi revisited SPT, både eksperimentelt og algoritmer. Eksperimentelle aspekter inkluderte optimalisere oppsett og celle merking, med særlig vekt på å nå høyest mulig merking tetthet, for å gi en dynamisk øyeblikksbilde av molekylære dynamikk ens det skjer innenfor membranen. Algoritmiske problemer bekymret hvert trinn brukes til ombygging baner: topper deteksjon, estimering og gjenkjenning, løses ved spesifikke verktøy fra bildeanalyse 15,16. Implementering deflasjon etter påvisning lar redde topper opprinnelig skjult av nabolandene, sterkere topper. Av notatet, bedre deteksjon påvirker direkte oppkobling, ved å redusere gap innen baner. Forestillinger har blitt evaluert ved hjelp av Monte-Carlo simuleringer for ulike merking tetthet og støy verdier, som typisk representerer de to store begrensninger for parallelle målinger ved høy spatiotemporal oppløsning. Den nanometric nøyaktighet 17 innhentet for enkle molekyler, enten ved hjelp påfølgende on / off photoswitching eller ikke-lineær optikk, kan levere grundige observasjoner. Dette er grunnlaget for nanoscopy metoder 17 som 18 STORM, PALM 19,20, RESOLFT 21 eller Sted 22,23, WHIch kan ofte kreve bildebehandlingsprogrammer faste prøver. Den sentrale oppgave er å oppdage og estimering av diffraksjon-begrensede topper kommer fra single-molekyler. Derfor gir tilstrekkelige forutsetninger for eksempel håndtering av en konstant posisjonelle nøyaktighet i stedet for Brownske bevegelser, er MTT oversiktlig egnet for nanoscopic analyser. Videre kan MTT fundamentalt brukes til enhver skala: ikke bare for molekyler, men også for celler eller dyr, for eksempel. Derfor er MTT en kraftig sporing algoritme som finner programmer på molekylære og cellulære skalaer.

Watch this Scientific Journal Video about Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT at JoVE.com

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing er en hjemmelaget algoritme utviklet for sporing individuelt merket molekyler i plasma membran av levende celler. Effektiv oppdage, estimering og sporing molekyler over tid ved høy tetthet gir et brukervennlig, omfattende verktøy for å undersøke nanoskala membran dynamikk.

Kartlegging Molekylær Diffusion i plasmamembranen ved Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

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Biology

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

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