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 gerne takke Jacob Solis for hans assistance i videoredigering. Dette arbejde blev finansieret delvist af en NSF KARRIERE Award DBI-0953902 til MJ Levene.

1. Animal Perfusion 5 og Whole Mouse Brain Clearing <ol><li> Hele længden af ​​proceduren kan variere afhængigt af, hvor lang tid anvendt pr dehydratiseringstrin, men i alt hele processen kan udføres i to dage. </li><li> Afvej YFP mus og derefter dybt bedøve med en intraperitoneal injektion af ketamin / xylazin (100 mg / kg: 10 mg / kg). </li><li> Bekræft et kirurgisk plan af dyb anæstesi før man går videre til kirurgi. Kontroller dyret hver 5 min for at se om den reagerer på en fast tå eller ...

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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 farvestoffer er kompatibel med en række af organiske opløsningsmidler, og derfor ikke udgør en særlig udfordring for clearing protokoller, fluorescerende proteiner er ofte mindre tolerante over for ændringer i opløsningsmiddel. 4 Målet med det foreliggende arbejde var at overvinde en alvorlig begrænsning af tidligere optiske clearing protokoller, hvor fluorescens af XFPs blev tabt eller alvorligt forringet. De billeder, der er præsenteret her, viser, at fluorescens fra YFP ble...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Phosphatbufret saltvand </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> 15.710 </td><td> 10 x 10 ml, 16% paraformaldehyd </td></tr><tr><td> Ethylalkohol </td><td> Amerikansk Bioanalytical </td><td> AB00515-00.500 </td><td> 500 ml, 200 proof </td></tr><tr><td> Ethylalkohol </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> 402.834 </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 Mel 5X </td><td> 15 WD </td></tr></tbody></table>

multiphoton microscopy of cleared mouse brain expressing yfp

Multiphoton microscopy af iboende fluorescens og anden harmoniske generation (SHG) af hele muse organer er gjort mulig ved optisk fjerne organ før billeddannelse. 1,2 For organer, der indeholder fluorescerende proteiner, såsom GFP og YFP, optiske clearing protokoller, der anvender methanol dehydrering og tydeligt ved hjælp af benzylalkohol: benzylbenzoat (Babb), mens ubeskyttet mod lys 3 ikke bevarer den fluorescerende signal. Protokollen præsenteres her er en hidtil ukendt måde at udføre helt organ optiske clearing på musehjerne samtidig fluorescenssignalet for YFP udtrykkes i neuroner. Ændring af den optiske clearing protokol, således at organet dehydratiseres under anvendelse af en ethanol gradueret række har vist sig at reducere beskadigelse af fluorescerende proteiner og bevare deres fluorescerende signal for multiphoton billeddannelse. 4. Brug en optimeret fremgangsmåde til optisk clearing med ethanol-baseret dehydrering og clearing af Babbmens afskærmet fra lys, viser vi høj opløsning multiphoton billeder af gult fluorescerende protein (YFP) ekspression i neuronerne i en musehjerne mere end 2 mm neden under vævsoverfladen.

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 microscopy af hele muse organer er mulig ved optisk fjerne organ før billeddannelse, men ikke alle protokoller bevare det fluorescerende signal af fluorescerende proteiner. Brug af et optisk clearing metode med ethanol-baseret dehydrering og benzylalkohol: benzylbenzoat clearing, viser vi i høj opløsning multiphoton billeder af hele musehjerne udtrykker YFP.

Multiphoton Mikroskopi af Cleared Mouse Brain udtrykker YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the ...

Research

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