Name: multiphoton microscopy of cleared mouse brain expressing yfp
Uploaded: 2012-09-23T14:00:00
Description: Watch this Scientific Journal Video about Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP at JoVE.com

Vi vil gjerne takke Jacob Solis for hans hjelp i videoredigering. Dette arbeidet ble finansiert delvis av en NSF KARRIERE Award DBI-0953902 til MJ Levene.

1. Animal Perfusion 5 og Whole Mouse Brain Clearing <ol><li> Hele lengden av prosedyren kan variere avhengig av lengden av tid som brukes per dehydrering trinn, men totalt hele prosessen kan gjennomføres i to dager. </li><li> Vei YFP mus og deretter dypt anesthetize med en intraperitoneal injeksjon av ketamin / xylazin (100 mg / kg: 10 mg / kg). </li><li> Bekreft et kirurgisk plan dyp anestesi før du fortsetter til kirurgi. Sjekk dyret hver 5 min for å se om den reagerer på en fast tå eller hale klemme...

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	<li>Parra, S. G., Chia, T. H., Zinter, J. P., Levene, 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=Multiphoton+microscopy+of+cleared+mouse+organs.">Multiphoton microscopy of cleared mouse organs.</a> J. Biomed. Opt. 15, 036017 (2010).</li><li>Vesuna, S., Torres, R., Levene, 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=Multiphoton+fluorescence,+second+harmonic+generation,+and+fluorescence+lifetime+imaging+of+whole+cleared+mouse+organs.">Multiphoton fluorescence, second harmonic generation, and fluorescence lifetime imaging of whole cleared mouse organs.</a> J. Biomed. Opt. 16, 106009 (2011).</li><li>Zucker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+insect+and+mammalian+embryo+imaging+with+confocal+microscopy:+Morphology+and+apoptosis.">Whole insect and mammalian embryo imaging with confocal microscopy: Morphology and apoptosis.</a> Cytometry. A69, 1143-1152 (2006).</li><li>Sakhalkar, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+in+bulk+tissue+specimens+using+optical+emission+tomography:+Fluorescence+preservation+during+optical+clearing.">Functional imaging in bulk tissue specimens using optical emission tomography: Fluorescence preservation during optical clearing.</a> Phys. Med. Biol. 52, 2035-2054 (2007).</li><li>Dazai, J., Spring, S., Cahill, L. S., Henkelman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple-mouse+Neuroanatomical+Magnetic+Resonance+Imaging.">Multiple-mouse Neuroanatomical Magnetic Resonance Imaging.</a> J. Vis. Exp. (48), e2497 (2011).</li><li>Pologruto, T. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.">ScanImage: flexible software for operating laser scanning microscopes.</a> Biomed. Eng. Online. 2, (2003).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (2001).</li><li>Abramoff, M. D., Magelhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11, 36-42 (2004).</li><li>Feng, G., Mellor, R. H., Bernstein, M., Keller-Peck, C., Nguyen, Q. T., Wallace, M., Nerbonne, J. M., Lichtman, J. W., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Neuronal+Subsets+in+Transgenic+Mice+Expressing+Multiple+Spectral+Variants+of+GFP.">Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Hama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale:+a+chemical+approach+for+fluorescence+imaging+and+reconstruction+of+transparent+mouse+brain.">Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.</a> Nat. Neuro. 14, 1481-1488 (2011).</li></ol>

Mens standard organiske fargestoffer er kompatibel med en rekke av organiske løsemidler, og derfor ikke utgjør en særlig utfordring for clearing protokoller, fluorescerende proteiner er ofte mindre tolerante overfor endringer i løsemiddel. 4 Målet med dette arbeidet var å overvinne en alvorlig begrensning av tidligere optiske clearing protokoller hvor fluorescens av xfps gikk tapt eller sterkt forringet. Bildene som presenteres her viser at fluorescens fra YFP ble bevart. Den optiske Skrapeteknikken bes...

<table border="1"><tbody><tr><td> Navnet på reagensen </td><td> Selskapet </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Fosfatbufret saltoppløsning </td><td> Sigma-Aldrich, Inc. </td><td> D8537 </td><td> 500 ml, pH 7,2 </td></tr><tr><td> Paraformaldehyd </td><td> Elektronmikroskopi Sciences </td><td> 15710 </td><td> 10 x 10 ml, 16% paraformaldehyd </td></tr><tr><td> Etylalkohol </td><td> American Bioanalytical </td><td> AB00515-00500 </td><td> 500 ml, 200 proof </td></tr><tr><td> Etylalkohol </td><td> Pharmco Products, Inc. </td><td> 111000190 </td><td> 1 gal., 190 proof </td></tr><tr><td> Benzylalkohol </td><td> Sigma-Aldrich, Inc. </td><td> 402834 </td><td> 500 ml, 99 +% </td></tr><tr><td> Benzylbenzoat </td><td> Sigma-Aldrich, Inc. </td><td> B6630-IL </td><td> 500 ml, ≥ 99% </td></tr><tr><td> 5X/0.5 NA mål </td><td> Nikon </td><td> AZ Plan Flour 5X </td><td> 15 WD </td></tr></tbody></table>

multiphoton microscopy of cleared mouse brain expressing yfp

Multiphoton mikroskopi av iboende fluorescens og andre harmonisk generering (SHG) av hele mus organer er gjort mulig av optisk tømme orgelet før avbildning. 1,2 Men for organer som inneholder fluorescerende proteiner som GFP og YFP, optiske clearing protokoller som bruker metanol dehydrering og tydelig med benzylalkohol: benzylbenzoat (Babb) mens ubeskyttet mot lys 3 ikke bevare den fluorescerende signal. Protokollen som presenteres her er en ny måte i å utføre hele organet optisk clearing på musen hjernen samtidig bevare fluorescens signal YFP uttrykt i nevroner. Endring av optiske clearing protokollen slik at organet er dehydrert ved hjelp av en etanol graderte serier har blitt funnet å redusere skadene på det fluorescerende proteiner og bevare sin fluorescenssignalet for multiphoton imaging. 4 Bruke en optimalisert fremgangsmåte for optisk clearing med etanol-basert dehydrering og clearing av Babbmens skjermet mot lys, viser vi høyoppløselige multiphoton bilder av gule fluorescerende protein (YFP) uttrykk i nervecellene av en mus hjernen mer enn 2 mm under vevsoverflaten.

Watch this Scientific Journal Video about Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP at JoVE.com

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Multiphoton mikroskopi av hele mus organer er mulig ved optisk fjerne organ før bildebehandling, men ikke alle protokoller bevare den fluorescerende signal av fluorescerende proteiner. Bruke en optisk clearing metoden med etanol-basert dehydrering og benzylalkohol: benzylbenzoat clearing, viser vi høyoppløselige multiphoton bilder av hele mus hjernen uttrykker YFP.

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