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 medlemmer af vores team, især MC Blache for teknisk bistand samt M Irla og B Imhof, for deres støtte og frugtbare diskussioner. Tal for deflation og indespærring gengives udlånt af Nature Methods. Dette projekt er støttet af institutionelle tilskud fra CNRS, INSERM og Marseille Universitet, og ved hjælp af specifikke tilskud fra regionen Provence-Alpes-Côte-d&#39;Azur, Institut National du Cancer, Agence Nationale de la Recherche (ANR-08-PCVI- 0034-02, ANR 2010 BLAN 1214 01) &amp; Fondation pour la Recherche Medicale (Equipe labélisée FRM-2009). VR understøttes af et fællesskab fra Ligue Nationale Contre le Cancer.

I denne video, præsenterer vi en helt enkelt partikel sporing eksperiment, ved hjælp af kvante-prikker målrettet til en specifik membran receptor. Hovedmålet med dette eksperiment består i kræsne forskellige typer molekylær diffusion adfærd målt i plasmamembranen af ​​levende celler. Faktisk kan molekylære bevægelser, der opstår på membranen typisk afvige fra Brownsk diffusion ved at blive lineært rettet eller begrænset i nanodomains 26-29, f.eks. Vi stræber efter at samtidig efter så man...

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I enkelt-partikel tracking,, ved siden af ​​de celle og mikroskopi aspekter analysen udgør en væsentlig del af arbejdet. Dette omhandler algoritme, der anvendes til at udføre de tre hovedopgaver: afsløre, estimering og tilslutte toppe over hver frame. Men den deraf følgende aspekter af dette arbejde ligger i udarbejdelsen af ​​algoritmen selv, som måske skal tilpasses til en ny dedikeret undersøgelse, hovedsagelig i de sidste og ekstra skridt (såsom at dechifrere former for bevægelse, interaktion eller ...

<table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Katalog nummer </td><td> Mængde </td></tr><tr><td> COS-7-cellelinje </td><td> ATCC </td><td> CRL-1651 </td><td> 5.000 celler / brønd </td></tr><tr><td> HBSS uden Ca2 + </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-brønds 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"> Biotinyleret Fab (for Fab-syntese, henvises 21 se) </td></tr><tr><td> Fab fra mAb 108 </td><td> ATCC </td><td> HB-9764 </td><td> 200 ug </td></tr><tr><td> NHS-Biotin<html></td><td> Thermo Scientific </td><td> 21435 </td><td> 18,5 mg </td></tr><tr><td colspan="4"> Komplet medium </td></tr><tr><td> DMEM </td><td> GIBCO </td><td> 41965 </td><td> 500 ml </td></tr><tr><td> Føtalt bovint 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> Natriumpyruvat </td><td> GIBCO </td><td> 11360 </td><td> 5 ml </td></tr><tr><td colspan="4"> Billeddannende medie </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 ul </td></tr></tbody></table>En &quot;&gt; <tbody><tr><td> Udstyr </td><td> Firma </td><td> Reference </td></tr><tr><td> Inverteret mikroskop </td><td> Nikon </td><td> Eclipse TE2000U </td></tr><tr><td> Lysstofrør </td><td> Nikon </td><td> Intensilight C-HGFIE </td></tr><tr><td> 1,3 NA 100x mål </td><td> Nikon </td><td> Planlæg Fluor 1,30 </td></tr><tr><td> 1,49 NA 100x mål </td><td> Nikon </td><td> APO TIRF 1,49 </td></tr><tr><td> Kamera </td><td> Roper Videnskabelig </td><td> Cascade 512 B </td></tr><tr><td> Termostateres box </td><td> Livet Imaging Services </td><td> The Box </td></tr></tbody></table> Bilag: eksempel Script af MTT supplerende analyse Funktionen MTT_example (filnavn) %%% Basale eksempler viser, hvordan du gendanner MTT output resultater %%% Til plotte hver trace og opbygge histogrammet %%% Af fluorescensintensiteter hvis nargin &lt;1% nej file_name forudsat? files = dir (&#39;* Stk.&#39;); hvis isEmpty (filer), DISP (&quot;ingen data i den nuværende dir &#39;), tilbage, slut . file_name =-filer (1) navn% standard: For det første Stk fil DISP ([&#39;bruger&#39; filnavn &#39;som standard&#39;]) ende file_param = [filnavn &#39;_tab_param.dat&#39;];% outputfil %% Belastning data CD (&quot;output23)% eller (&#39;output22) ifølge version, der anvendes % Disclaimer: Version 2,2 kun genererer 7 parametre, % Ekstra parameter, støj, blev tilsat i version 2.3 % Hvis du vil læse alle parametre på én gang, i en enkelt tabel % Tab_param = fread_all_param (file_param); % Tab_i = tab_param (2:8: ende :); tab_j = ... % Hvis du vil læse alle parametre (undtagen 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),%-indekset er 3, fordi spor nummer og stelnummer, ikke informativ, ignoreres! 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 løbet af spor N_traces = størrelse (tab_i, 1); % Tabeller er N_traces linjer ved N_frames søjler for itrc = 1: N_traces No_blk_index = tab_blk (itrc, :)&gt; 0;% ikke blinkende trin kun plot (tab_i (itrc, No_blk_index), tab_j (itrc, No_blk_index)) &amp; Nbsp; xlabel (&#39;i (pixel) &quot;), ylabel (&#39; j (pixel) &#39;) title ([&#39;spor #&#39; num2str (itrc)]) DISP (&#39;Vær ramme enhver nøgle til næste spor&#39;), pause ende %% Fluo histogram N_datapoints = SUM (tab_blk (:)&gt; 0);% ikke blinkende trin kun hist (tab_alpha (tab_blk&gt; 0), 2 * sqrt (N_datapoints))% bruger 2sqrt (N) skraldespande xlabel (&#39;intensitet (au)&#39;), ylabel (&#39;hændelse&#39;) titel (&quot;histogram af partikler fluorescensintensitet)

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

Vores mål er at opnå en omfattende beskrivelse af molekylære processer finder sted på cellemembraner i forskellige biologiske funktioner. Vi stræber efter at karakterisere den komplekse organisation og dynamikken i plasmamembranen på enkelt-molekyle niveau, ved at udvikle analytiske værktøjer dedikeret til enkelt-partikel Tracking (SPT) ved høj tæthed: Multiple-Target Tracing (MTT) 1. Enkelt molekyle videomicroscopy, giver millisekund og nanometrisk opløsning 1-11, tillader en detaljeret afbildning af membranen organisation 12-14 ved nøjagtig kortlægning deskriptorer såsom celle-receptorer lokalisering, mobilitet, indespærring eller interaktioner. Vi fornyet SPT, både eksperimentelt og algoritmer. Eksperimentelle aspekter omfattede optimering opsætning og celle mærkning, med særlig vægt på at nå den højest mulige mærkning tæthed, for at skabe en dynamisk øjebliksbillede af molekylær dynamik ens det forekommer inde i membranen. Algoritmiske problemer pågældende hvert trin anvendes til at genopbygge baner: toppe detektion, estimering og gentilslutning, behandles af specifikke værktøjer fra billedanalyse 15,16. Implementering deflation efter afsløring giver redde toppe i første omgang skjult af tilstødende, stærkere toppe. Det skal bemærkes, direkte forbedre afsløring påvirker gentilslutning, ved at reducere huller i baner. Forestillinger er blevet evalueret ved brug Monte-Carlo simuleringer for forskellige mærkning tæthed og støj værdier, som typisk udgør de to store begrænsninger for parallelle målinger med høj spatiotemporal opløsning. Den nanometrisk nøjagtighed 17 opnået for enkelte molekyler, enten ved hjælp af successive on / off photoswitching eller ikke-lineær optik, kan levere udtømmende observationer. Dette er grundlaget for Nanoscopy metoder 17, såsom STORM 18, PALM 19,20, RESOLFT 21 eller place 22,23, whiCH kan kræver ofte billeddannende faste prøver. Den centrale opgave er at afsløre og vurdering af diffraktion-begrænsede toppe stammer fra enkelt-molekyler. Derfor giver tilstrækkelige forudsætninger, såsom håndtering af et konstant positionsnøjagtighed i stedet for Brownsk bevægelse, er MTT ligefrem velegnet til nanoskopiske analyser. Endvidere kan MTT fundamentalt anvendes i enhver skala: ikke blot molekyler, men også for celler eller dyr, f.eks. Derfor MTT er en effektiv sporing algoritme, der finder anvendelse 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

Multipel-Target Sporingen er en hjemmelavet algoritme udviklet til sporing individuelt mærkede molekyler i plasmamembranen af ​​levende celler. Effektiv opdage, vurdere og sporing molekyler over tid ved høj densitet giver et brugervenligt, omfattende værktøj til at undersøge nanoskala membran dynamik.

Kortlægning af 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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