Name: fret microscopy for real-time monitoring of signaling events in live cells using unimolecular biosensors
Uploaded: 2012-08-20T13:00:00
Description: Watch this Scientific Journal Video about FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors at JoVE.com

Forfatterne vil gerne takke Anke Rüttgeroth og Karina Zimmermann til teknisk bistand. Dette arbejde blev støttet af Deutsche Forschungsgemeinschaft (tilskud NI 1301/1-1 til VON) og universitetet i Göttingen Medical Center (&quot;pro Futura&quot; tilskud til VON).

1. Opsætning af en FRET Imaging mikroskop I princippet kan en inverteret fluorescensmikroskop, som er tilgængelig i laboratoriet og har et kamera port tilpasses til FRET billeddannelse. Den endelige setup skal indeholde følgende vigtige komponenter: et mikroskop, en lyskilde med eller uden yderligere lukker, en bjælke-splitter for emission lys og en CCD-kamera (se figur 1). De hardwareenheder, især den lyskilde, lukkeren og kameraet er integreret i og styret af imaging so...

<ol>
	<li>Zaccolo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+chimeric+fluorescent+proteins+and+fluorescence+resonance+energy+transfer+to+monitor+cellular+responses.">Use of chimeric fluorescent proteins and fluorescence resonance energy transfer to monitor cellular responses.</a> Circ. Res. 94, 866-873 (2004).</li><li>Mehta, S., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reporting+from+the+field:+genetically+encoded+fluorescent+reporters+uncover+signaling+dynamics+in+living+biological+systems.">Reporting from the field: genetically encoded fluorescent reporters uncover signaling dynamics in living biological systems.</a> Annu. Rev. Biochem. 80, 375-401 (2011).</li><li>Zhang, J., Campbell, R. E., Ting, A. Y., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+new+fluorescent+probes+for+cell+biology.">Creating new fluorescent probes for cell biology.</a> Nat. Rev. Mol. Cell Biol. 3, 906-918 (2002).</li><li>Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+spatial+and+temporal+dynamics+of+intracellular+signaling.">Visualization of the spatial and temporal dynamics of intracellular signaling.</a> Dev. Cell. 4, 295-305 (2003).</li><li>Bunemann, M., Frank, M., Lohse, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gi+protein+activation+in+intact+cells+involves+subunit+rearrangement+rather+than+dissociation.">Gi protein activation in intact cells involves subunit rearrangement rather than dissociation.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 16077-16082 (2003).</li><li>Kotlikoff, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+Ca2++indicators:+using+genetics+and+molecular+design+to+understand+complex+physiology.">Genetically encoded Ca2+ indicators: using genetics and molecular design to understand complex physiology.</a> J. Physiol. 578, 55-67 (2007).</li><li>Willoughby, D., Cooper, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+of+cAMP+dynamics.">Live-cell imaging of cAMP dynamics.</a> Nat. Methods. 5, 29-36 (2008).</li><li>Nikolaev, V. O., Lohse, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+cAMP+synthesis+and+degradation+in+living+cells.">Monitoring of cAMP synthesis and degradation in living cells.</a> Physiology (Bethesda). 21, 86-92 (2006).</li><li>Tanimura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Fluorescence+Resonance+Energy+Transfer-based+Biosensors+for+the+Quantitative+Analysis+of+Inositol+1,4,5-Trisphosphate+Dynamics+in+Calcium+Oscillations.">Use of Fluorescence Resonance Energy Transfer-based Biosensors for the Quantitative Analysis of Inositol 1,4,5-Trisphosphate Dynamics in Calcium Oscillations.</a> J. Biol. Chem. 284, 8910-8917 (2009).</li><li>Nikolaev, V. O., Lohse, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+techniques+for+real-time+monitoring+of+cGMP+in+living+cells.">Novel techniques for real-time monitoring of cGMP in living cells.</a> Handb. Exp. Pharmacol. , 229-243 (2009).</li><li>Nausch, L. W., Ledoux, J., Bonev, A. D., Nelson, M. T., Dostmann, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+patterning+of+cGMP+in+vascular+smooth+muscle+cells+revealed+by+single+GFP-linked+biosensors.">Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 365-370 (2008).</li><li>Borner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET+measurements+of+intracellular+cAMP+concentrations+and+cAMP+analog+permeability+in+intact+cells.">FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells.</a> Nat. Protoc. 6, 427-438 (2011).</li><li>Nikolaev, V. O., Bunemann, M., Hein, L., Hannawacker, A., Lohse, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+single+chain+cAMP+sensors+for+receptor-induced+signal+propagation.">Novel single chain cAMP sensors for receptor-induced signal propagation.</a> J. Biol. Chem. 279, 37215-37218 (2004).</li><li>Hong, K. P., Spitzer, N. C., Nicol, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+molecular+toolkit+for+cAMP+studies+in+live+cells.">Improved molecular toolkit for cAMP studies in live cells.</a> BMC Res. Notes. 4, 241-24 (2011).</li><li>Niino, Y., Hotta, K., Oka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+live+cell+imaging+using+dual+FRET+sensors+with+a+single+excitation+light.">Simultaneous live cell imaging using dual FRET sensors with a single excitation light.</a> PLoS One. 4, e6036 (2009).</li><li>Palmer, A. E., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+calcium+signaling+using+genetically+targetable+fluorescent+indicators.">Measuring calcium signaling using genetically targetable fluorescent indicators.</a> Nat. Protoc. 1, 1057-1065 (2006).</li><li>Brumbaugh, J., Schleifenbaum, A., Stier, G., Sattler, M., Schultz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-+and+dual-parameter+FRET+kinase+probes+based+on+pleckstrin.">Single- and dual-parameter FRET kinase probes based on pleckstrin.</a> Nat. Protoc. 1, 1044-1055 (2006).</li><li>Aoki, K., Matsuda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+small+GTPase+activity+with+fluorescence+resonance+energy+transfer-based+biosensors.">Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors.</a> Nat. Protoc. 4, 1623-1631 (2009).</li></ol>

i denne protokol, viser vi, hvordan man opbygger en simpel billig, men kraftig FRET imaging system til rutinemæssige applikationer med en bred vifte af tilgængelige biosensorer. Systemet præsenteres her er designet til CFP og YFP eller lignende typer fluorescerende proteiner, som donor-acceptor-par. Samtidig har andre individuelle biosensorer bliver tilgængelige som bruger for eksempel grønne og røde fluorescerende proteiner 14. At tilpasse det beskrevne system for andre farver, skal passende lyskilder ...

<table border="1"><tbody><tr><td> Navn på reagens / udstyr </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> BES Buffer Grade </td><td> APPLICHEM </td><td> A1062 </td><td></td></tr><tr><td> CaCl2-dihydrat </td><td> Sigma-Aldrich </td><td> C5010 </td><td></td></tr><tr><td> Glas coverslides </td><td> Thermo Scientific </td><td> 004710781 </td><td> Diameter 24 mm </td></tr><tr><td> Glas-bund celle-dyrkningsskåle </td><td> Verden Precision Instruments </td><td> FD3510-100 </td><td></td></tr><tr><td> D-MEM-medium </td><td> Biochrom AG </td><td> F0445 </td><td></td></tr><tr><td> Føtalt kalveserum (FCS) </td><td> Thermo Scientific </td><td> SH30073.02 </td><td></td></tr><tr><td> L-Glutamin </td><td> Biochrom AG </td><td> K0283 </td><td></td></tr><tr><td> HEPES </td><td> Sigma </td><td> H4034 </td><td></td></tr><tr><td> KCl </td><td> Sigma </td><td> P5405 </td><td></td></tr><tr><td> MgCl2 hexahydrat </td><td> APPLICHEM </td><td> A4425 </td><td></td></tr><tr><td> NaCl </td><td> APPLICHEM </td><td> A1149 </td><td></td></tr><tr><td> Na 2 HPO 4 </td><td> Sigma-Aldrich </td><td> S9707 </td><td></td></tr><tr><td> Penicillin / streptomycin </td><td> Biochrom AG </td><td> A2213 </td><td></td></tr><tr><td> Omvendt fluorescerende mikroskop </td><td> fx Nikon </td><td> Anmodning hos Nikon </td><td></td></tr><tr><td> CoolLED </td><td> CoolLED </td><td> p E-100 </td><td> 440 nm </td></tr><tr><td> DualView </td><td> Photometrics </td&gt; <td> DV2-SYS </td><td></td></tr><tr><td> DualView filter skyderen </td><td> Photometrics </td><td> 05-EM </td><td></td></tr><tr><td> CFP / YFP filtersæt </td><td> Chroma Technology </td><td> 49.052 </td><td> uden emissionsfilter </td></tr><tr><td> ORCA-03G kamera </td><td> Hamamatsu Photonics </td><td> C8484-03G02 </td><td></td></tr><tr><td> Arduino I / O-kort </td><td> Sparkfun Electronics </td><td> DEV-00.666 </td><td></td></tr><tr><td> Attofluor cellekammer </td><td> Invitrogen </td><td> A-7816 </td><td></td></tr><tr><td> Personlig computer med Windows XP eller Windows7-system </td><td> Enhver leverandør </td><td></td><td> Medtag harddisk med høj kapacitet </td></tr></tbody></table>

fret microscopy for real-time monitoring of signaling events in live cells using unimolecular biosensors

Förster resonans (FRET) mikroskopi fortsætter med at vinde stigende interesse som en teknik til real-time overvågning af biokemiske og signalere begivenheder i levende celler og væv. Sammenlignet med klassiske biokemiske metoder, er denne nye teknologi kendetegnet ved høj tidslig og rumlig opløsning. FRET forsøg bruge forskellige genetisk kodede biosensorer, som kan udtrykkes og afbildet over tid in situ eller in vivo 1-2. Typiske biosensorer kan enten rapportere protein-protein-interaktioner ved at måle FRET mellem en fluorofor-mærket par proteiner eller konformationelle ændringer i et enkelt protein, der huser donor-og acceptor-fluoroforer indbyrdes forbundet med en bindingsdel til et molekyle af interesse 3-4. Bimolekylære biosensorer for protein-protein interaktioner indbefatter for eksempel, konstruktioner designet til at overvåge G-protein-aktivering i celler 5, mens de unimolekylær sensorer meaSuring konformationelle ændringer er almindeligt anvendt til billede second messengers, såsom calcium 6, cAMP 7-8, inositolphosphater 9 og cGMP 10-11. Her beskriver vi, hvordan man opbygger et tilpasset epifluorescens FRET imaging system fra enkelte kommercielt tilgængelige komponenter og hvordan du kan styre hele opsætningen ved hjælp af Micro-Manager freeware. Denne enkle, men kraftfulde instrument er designet til rutinemæssige eller mere sofistikerede FRET målinger i levende celler. Erhvervede billeder behandles ved hjælp af selvstændig skriftlig plug-ins til at visualisere ændringer i FRET forhold i real-tid under alle eksperimenter, før de lagres i et grafikprogram format kompatibelt med den indbyggede ImageJ freeware anvendes til efterfølgende dataanalyse. Dette billige system er kendetegnet ved høj fleksibilitet og kan med succes anvendes til at overvåge forskellige biokemiske begivenheder og signaleringsmolekyler af et væld af tilgængelige FRET biosensorer i levende celler og væv. Som et eksempel udviser vi how at bruge denne afbildningssystem til at udføre tidstro overvågning af cAMP i levende 293A-celler ved stimulering med en β-adrenerg receptoragonist og blokker.

Watch this Scientific Journal Video about FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors at JoVE.com

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

Förster resonans (FRET) mikroskopi er en kraftfuld teknik til real-time overvågning af signalering begivenheder i levende celler ved hjælp af forskellige biosensorer som reportere. Her beskriver vi, hvordan man opbygger et tilpasset epifluorescens FRET imaging system fra kommercielt tilgængelige komponenter, og hvordan man bruger det til FRET eksperimenter.

FRET mikroskopi af Real-time overvågning af signalering begivenheder i levende celler Brug unimolekylær Biosensorer

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Research

JoVE Journal

Biology

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

Real-time overvågning af ligand-receptor interaktioner med Fluorescence Resonance Energy Transfer

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Overvågning plasmidreplikation Live pattedvrceller over flere generationer ved fluorescensmikroskopi

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Autonomt Bioluminescent pattedvrceller for vedvarende og Real-time overvågning af cytotoksicitet

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

Real-time Imaging af Single Engineered RNA-transkripter i levende celler Brug Ratiometrisk bimolekylær Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

Påvisning af Intracellulær genekspression i levende celler af murine, Human og porcin oprindelse anvendelse af fluorescens-mærkede Nanopartikler

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Imaging G-protein-koblet receptor-medieret Kemotaksi og dens signaleringshændelser i Neutrofil-lignende HL60-celler

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Identifikation af intracellulær signalering begivenheder induceret i levedygtige celler ved interaktion med naboceller Gennemgår apoptotisk celledød

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Karakterisering af Forkalkning Events med Live Optiske og elektronmikroskopi teknikker i en Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells

Foto stimulation af Femtosecond laser aktiverer ekstracellulær-signal-reguleret kinase (ERK) signalering eller mitokondrie hændelser i målcellerne

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation ...