Name: application of high-speed super-resolution speed microscopy in live primary cilium
Uploaded: 2018-01-16T12:30:00
Description: Watch this Scientific Journal Video about Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium at JoVE.com

Vi takker Dr. Kristen Verhey (University of Michigan, Ann Arbor) og Dr. Gregory Pazour (University of Massachusetts Medical School) for å gi noen plasmider. Prosjektet ble støttet av tilskudd fra National Institutes of Health (NIH GM097037, GM116204 og GM122552 til W.Y.).

1. NIH-3T3 celle forberedelse til hastighet mikroskopi fra lager
<ol><li>1,5 uker før eksperimentet, gjenopprette en fersk kultur NIH-3T3 celler fra en frossen lager ved tining på 37 ° C og overføre cellene til en 25 cm2 celle kultur kolbe med 3 mL av Dulbeccos endret Eagle's Medium (DMEM) med 110 mg/mL natrium pyruvate, 2 mM glutamin, 10% fosterets bovin serum og 1% penicillin/streptomycin.</li>
	<li>Inkuber cellene på 37 ° C i en 5% CO2 inkubator.</li>
	<li>Dele cellene på 80% confluency,...

<ol>
	<li>Huang, B., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+fluorescence+microscopy.">Super-resolution fluorescence microscopy.</a> Annu Rev Biochem. 78, 993-1016 (2009).</li><li>Leung, B. O., Chou, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+super-resolution+fluorescence+microscopy+for+biology.">Review of super-resolution fluorescence microscopy for biology.</a> Appl Spectrosc. 65, 967-980 (2011).</li><li>Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STED+microscopy+reveals+that+synaptotagmin+remains+clustered+after+synaptic+vesicle+exocytosis.">STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis.</a> Nature. 440, 935-939 (2006).</li><li>Betzig, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313, 1642-1645 (2006).</li><li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy+(STORM).">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).</a> Nat Meth. 3, 793-796 (2006).</li><li>Ma, J., Yang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+distribution+of+transient+interactions+in+the+nuclear+pore+complex+obtained+from+single-molecule+snapshots.">Three-dimensional distribution of transient interactions in the nuclear pore complex obtained from single-molecule snapshots.</a> Proc Natl Acad Sci USA. 107, 7305-7310 (2010).</li><li>Ma, J., Goryaynov, A., Sarma, A., Yang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-regulated+viscous+channel+in+the+nuclear+pore+complex.">Self-regulated viscous channel in the nuclear pore complex.</a> Proc Natl Acad Sci USA. 109, 7326-7331 (2012).</li><li>Ma, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+three-dimensional+mapping+of+mRNA+export+through+the+nuclear+pore.">High-resolution three-dimensional mapping of mRNA export through the nuclear pore.</a> Nat Comm. 4, (2013).</li><li>Akey, C. W., Radermacher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+the+Xenopus+nuclear+pore+complex+revealed+by+three-dimensional+cryo-electron+microscopy.">Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy.</a> J Cell Biol. 122, 1-19 (1993).</li><li>Akey, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+and+structure+of+the+nuclear+pore+complex+revealed+by+cryo-electron+microscopy.">Interactions and structure of the nuclear pore complex revealed by cryo-electron microscopy.</a> J Cell Biol. 109, 955-970 (1989).</li><li>Czarnecki, P. G., Shah, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ciliary+transition+zone:+from+morphology+and+molecules+to+medicine.">The ciliary transition zone: from morphology and molecules to medicine.</a> Trends Cell Biol. 22, 201-210 (2012).</li><li>Elf, J., Li, G. -. W., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+transcription+factor+dynamics+at+the+single-molecule+level+in+a+living+cell.">Probing transcription factor dynamics at the single-molecule level in a living cell.</a> Science. 316, 1191-1194 (2007).</li><li>Anzalone, A., Annibale, P., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+orbital+tracking+in+a+modified+two-photon+microscope:+an+application+to+the+tracking+of+intracellular+vesicles.">3D orbital tracking in a modified two-photon microscope: an application to the tracking of intracellular vesicles.</a> J Vis Exp. , (2014).</li><li>Ritter, J. G., Veith, R., Veenendaal, A., Siebrasse, J. P., Kubitscheck, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+sheet+microscopy+for+single+molecule+tracking+in+living+tissue.">Light sheet microscopy for single molecule tracking in living tissue.</a> PloS one. 5, 11639 (2010).</li><li>Marshall, W. F., Nonaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cilia:+tuning+in+to+the+cell's+antenna.">Cilia: tuning in to the cell's antenna.</a> Curr Biol. 16, 604-614 (2006).</li><li>Scholey, J. M., Anderson, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraflagellar+transport+and+cilium-based+signaling.">Intraflagellar transport and cilium-based signaling.</a> Cell. 125, 439-442 (2006).</li><li>Yang, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superresolution+pattern+recognition+reveals+the+architectural+map+of+the+ciliary+transition+zone.">Superresolution pattern recognition reveals the architectural map of the ciliary transition zone.</a> Sci Rep. 5, 14096 (2015).</li><li>Craige, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CEP290+tethers+flagellar+transition+zone+microtubules+to+the+membrane+and+regulates+flagellar+protein+content.">CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content.</a> J Cell Biol. 190, 927-940 (2010).</li><li>Kee, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+size-exclusion+permeability+barrier+and+nucleoporins+characterize+a+ciliary+pore+complex+that+regulates+transport+into+cilia.">A size-exclusion permeability barrier and nucleoporins characterize a ciliary pore complex that regulates transport into cilia.</a> Nat Cell Biol. 14, 431-437 (2012).</li><li>Najafi, M., Maza, N. A., Calvert, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steric+volume+exclusion+sets+soluble+protein+concentrations+in+photoreceptor+sensory+cilia.">Steric volume exclusion sets soluble protein concentrations in photoreceptor sensory cilia.</a> Proc Natl Acad Sci USA. 109, 203-208 (2012).</li><li>Nachury, M. V., Seeley, E. S., Jin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trafficking+to+the+ciliary+membrane:+how+to+get+across+the+periciliary+diffusion+barrier.">Trafficking to the ciliary membrane: how to get across the periciliary diffusion barrier.</a> Annu Rev Cell Dev Biol. 26, 59-87 (2010).</li><li>Ye, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+molecule+imaging+reveals+a+major+role+for+diffusion+in+the+exploration+of+ciliary+space+by+signaling+receptors.">Single molecule imaging reveals a major role for diffusion in the exploration of ciliary space by signaling receptors.</a> Elife. 2, 00654 (2013).</li><li>Ross, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Bardet-Biedl+syndrome+ciliary+proteins+perturbs+planar+cell+polarity+in+vertebrates.">Disruption of Bardet-Biedl syndrome ciliary proteins perturbs planar cell polarity in vertebrates.</a> Nat Genetics. 37, 1135-1140 (2005).</li><li>Handel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+targeting+of+somatostatin+receptor+3+to+neuronal+cilia.">Selective targeting of somatostatin receptor 3 to neuronal cilia.</a> Neuroscience. 89, 909-926 (1999).</li><li>Follit, J. A., Tuft, R. A., Fogarty, K. E., Pazour, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intraflagellar+transport+protein+IFT20+is+associated+with+the+Golgi+complex+and+is+required+for+cilia+assembly.">The intraflagellar transport protein IFT20 is associated with the Golgi complex and is required for cilia assembly.</a> Mol Biol Cell. 17, 3781-3792 (2006).</li><li>Awata, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NPHP4+controls+ciliary+trafficking+of+membrane+proteins+and+large+soluble+proteins+at+the+transition+zone.">NPHP4 controls ciliary trafficking of membrane proteins and large soluble proteins at the transition zone.</a> J Cell Sci. 127, 4714-4727 (2014).</li><li>Howarth, M., Ting, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+proteins+in+live+mammalian+cells+with+biotin+ligase+and+monovalent+streptavidin.">Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin.</a> Nat Protoc. 3, 534-545 (2008).</li><li>Huang, B., Wang, W., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+super-resolution+imaging+by+stochastic+optical+reconstruction+microscopy.">Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy.</a> Science. 319, 810-813 (2008).</li></ol>

Denne protokollen beskriver bruken av hastighet mikroskopi til den primære Flimmerhår, en cellular signalnettverk organelle som er svært avhengig av effektiv protein transport. HASTIGHET mikroskopi kan gi høy oppløsning (< 10 nm) steder for fluorescently-merket molekyler idet de passerer gjennom enkeltpunkt belysning sentrert på TZ. Tidligere har det vært brukt for å studere protein smuglingen via NPC6,7,8. Men kan det u...

Siden oppgitt av Ernst Abbe i 1873, har oppløsningen av konvensjonelle * lys vært antatt å være begrenset til ca 200 nm på grunn av lys Diffraksjon fra objektive1,2. Foreløpig super-oppløsning * lys teknikker bryte denne begrensningen og tillate fangst av dynamiske bilder med sub Diffraksjon (< 200 nm) oppløsning. Teknikkene generelt faller inn i to hovedkategorier: stimulert utslipp nedbryting (STED) mikroskopi basert tilnærminger, som genererer sub Diffraksjon belysning volum på grunn av lineære optisk reaksjon av fluorophores i prøver3; og photoactivated * lys (PALM) og Stokastisk optisk rekonstruksjon mikroskopi (STORM)-basert super-oppløsning teknikker, som bruker matematiske funksjoner for å lokalisere centroids av fluorophores og deretter gjeninnføre disse centroids til super-oppløsning bilder4,5. For tiden, på grunn av relativt ukomplisert optisk stilling, er PALM og STORM mye ansatt av bare en undergruppe av fluorophores i hvert en lang video av en biologisk forberedelse. Dette gir mer nøyaktig lokalisering av 2D Gaussian montering av lysrør stedet, kalt funksjonen Poenget spredning (PSF), fluorescently-merket proteiner i rammene av videoen. 2D plasseringen av hver fluorescently-merket molekyl kan deretter bli oppå et enkelt tenkelig fly å produsere en super-oppløsning bildet av biologiske forberedelse1,2. Mens disse single-molekylet lokalisering, super-oppløsning tilnærminger til mikroskopi sikkert revolusjonert hvordan avbilding av biologiske prøver ble utført, det er fortsatt utfordringer å overvinne. For eksempel kan STORM og PALM oppnå sine beste romlig oppløsning etter fiksering av biologiske prøver og dermed presentere en statisk fremstilling av fluorescently-merket proteiner, som er en lignende begrensning av elektronmikroskop. I tillegg for å oppnå høy romlig oppløsning for hver fluorescently-merket protein i lever celler, må prøver avbildes på svært lang framerates som ikke kan fange protein dynamics. Derfor er det nødvendig å overvinne disse viktigste tekniske hindrene.
For å oppnå en høy spatiotemporal oppløsning som er velegnet for å oppdage raske proteiner eller RNAs i levende celler, har vi utviklet super-oppløsning mikroskopi hastighet i vårt laboratorium (figur 1)6,7, 8. flere store tekniske fremskritt i hastighet mikroskopi tidligere har aktivert oss å spore nucleocytoplasmic transport av små molekyler, proteiner, mRNA og virus igjennom innfødt kjernefysiske pore komplekser (NPC)6, 7 , 8. kort, følgende funksjoner i hastighet mikroskopi brukes til å spore raske makromolekyler gjennom sub mikrometer rotasjons symmetrisk strukturer i levende celler, for eksempel NPCer og primære flimmerhårene: (1) en tilbøyelig eller vertikal belysning PSF gjør magnetisering av enkelt molekyler innenfor et lite Diffraksjon limit volum i fokalplanet (figur 1). (2) tilbøyelig PSF kan unngå sterkt ut-av-fokus fluorescens og dermed forbedre signal-til-støy-forhold. (3) optisk tetthet på 100-500 kW / cm2 i belysning PSF lar tusenvis av fotoner inn fra enkelt fluorophores med rask oppdagelsen hastigheter (> 500 Hz). (4) rask oppdagelsen hastighet også reduserer single-molekylet romlige lokalisering feilen (< 10 nm) å bestemme romlige baner av flytte fluorescerende molekyler i levende celler, fordi Diffusjon er en av viktigste faktorene forårsaker feil av enkelt-molekylet lokalisering for å flytte molekyler. (5) well-established 2D til 3D transformasjon algoritmer gir oss å gi 3D romlige sannsynlighet tetthet kart av transportveier for molekyler i NPC eller den primære Flimmerhår. Det er bemerkelsesverdig at våre konverteringen mellom det kartesiske og sylindriske koordinering systemet brukes til å generere en 3D romlige sannsynlighet tetthet kart i stedet for 3D single-molekylet sporing (figur 2). Tidligere har elektronmikroskop data avdekket at NPC9,10 og primære Flimmerhår11 begge har en rotasjons symmetrisk struktur. I prinsippet bør tilfeldig spre molekyler flytte gjennom NPC eller primære Flimmerhår også ha rotasjons symmetrisk distribusjoner. Som vist i figur 2, et høyt antall tilfeldig spre molekyler inne i sylinderen vil generere rotasjons symmetrisk distribusjoner på tverrsnittvisningen som i NPC, ytterligere resulterer i en ca uniform romlige distribusjon i hver liten sub region mellom to nabokommunene ringene (figur 2E). Denne jevn fordeling fører at den romlige fordelingen langs θ dimensjon i sylindriske systemet er konstant. Deretter kan 3D-koordinatene (R, X, θ) forenkles skal 2D-koordinater (R, X, konstant). Egentlig er vår konverteringen mellom det kartesiske og sylindriske systemer fra 2D (X, Y) til 2D (R, X, konstant). Den konstant θ, refererer til de romlige tetthet p i figur 2E, beregnes ved hjelp av ligning A<img alt="Equation 1" src="/files/ftp_upload/56475/56475eq1.jpg">.
Til slutt, enkelt-molekylet sporing har bred anvendelse i biologiske, dermed er det naturlig at en mengde teknikker vil bli utviklet for å fylle spesifikke biologiske nisjer12,13,14. Slik er tilfellet med hastighet mikroskopi. Tidligere, når kombinert med en 3D transformasjon algoritme, denne teknikken ble utviklet for å løse 3D transport ruter av transitt molekyler gjennom NPCer, en sub-diffraction-størrelse og rotasjons symmetrisk biologiske struktur6. I dette papiret vist primære flimmerhårene å være utmerket modell organeller også. Primære flimmerhårene er sylindrisk, antenne-lignende organelles (~ 125 nm radius) at prosjektet fra overflaten av mest pattedyr celler15,16,17. De er ansvarlig for å motta eksterne signaler og oversender en intracellulær respons vanligvis assosiert med vekst og metabolisme15,16. Derfor flux strukturelle proteiner, resirkulering av transmembrane reseptorer, og overføring av intracellulær budbringere er viktig ansvar primære flimmerhårene. Tidspunktet mellom primære flimmerhårene og celle kroppen er en kritisk selektivitet barriere, kalt overgang sone eller TZ, som alle denne protein transporten må være11,18,19, 20. I tillegg til TZ gating funksjon antas minst to transport prosesser, intraflagellar transport og passiv diffusjon, å være ansvarlig for flytting av protein gjennom denne regionen16,21, 22. fra en helse-synspunkt, tap av primære flimmerhårene og påfølgende dereguleringen nedstrøms signalnettverk er karakteristisk for mange kreft. I tillegg er mange genetiske sykdommer, som Bardet-Biedl syndrom og polycystic nyresykdom, forbundet med defekt protein transport23. Både sub Diffraksjon grensen størrelse og den komplekse prosessen med selektiv protein transport gjennom til TZ gjør primære flimmerhårene en ettertraktet mål for denne teknikken. I denne metoder papir, vil vi vise sporing av en ciliary transmembrane protein, somatostatin reseptor 3 (SSTR3)24, merket eksternt med Alexa Fluor 647 og en del av IFT, IFT2025, merket med et smeltet GFP molekyl.

<table border="0" cellpadding="0" cellspacing="0" style="width:619px;" width="618">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="47">
			<td height="47" style="height:47px;width:123px;">25 cm2 tissue culture dish</td>
			<td style="width:204px;">Corning</td>
			<td style="width:101px;">VV-01936-00</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">Penicillin/streptomycin</td>
			<td style="width:204px;">ThermoFisher</td>
			<td style="width:101px;">15140122</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">Fetal bovine serum</td>
			<td style="width:204px;">ThermoFisher</td>
			<td style="width:101px;">10438018</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:123px;">DMEM</td>
			<td style="width:204px;">ThermoFisher</td>
			<td style="width:101px;">10566-016</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:123px;">OPTIMEM</td>
			<td style="width:204px;">ThermoFisher</td>
			<td style="width:101px;">31985062</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:123px;">Trypsin</td>
			<td style="width:204px;">ThermoFisher</td>
			<td style="width:101px;">25300054</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">Phosphate buffered saline</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td style="width:101px;">P3813-1PAK</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:123px;">Transit LT1</td>
			<td style="width:204px;">Mirus</td>
			<td style="width:101px;">MIR 2300</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">35 mm glass bottom dish</td>
			<td style="width:204px;">MatTek</td>
			<td style="width:101px;">P35GCOL-0-14-C</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:123px;">AlexaFluor 647-conjugated streptavidin</td>
			<td style="width:204px;">ThermoFisher</td>
			<td style="width:101px;">S21374</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:123px;">Biotin</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td style="width:101px;">B4501-100MG</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">633 nm He-Ne laser</td>
			<td style="width:204px;">Melles Griot</td>
			<td style="width:101px;">25-LHP-928-249</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">561 nm solid state laser</td>
			<td style="width:204px;">Coherent</td>
			<td style="width:101px;">OBIS 561-50 LS</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">488 nm solid state laser</td>
			<td style="width:204px;">Coherent</td>
			<td style="width:101px;">1185053</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:123px;">Inverted fluorescence microscope</td>
			<td style="width:204px;">Olympus</td>
			<td style="width:101px;">IX81</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:123px;">1.4-NA 100&#215; oil-immersion apochromatic objective</td>
			<td style="width:204px;">Olympus</td>
			<td style="width:101px;">UPLSAPO 100&#215;</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:123px;">On-chip multiplication gain charge-coupled-device camera</td>
			<td style="width:204px;">Roper Scientific</td>
			<td style="width:101px;">Cascade 128+</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:123px;">Dichroic filter</td>
			<td style="width:204px;">Semrock</td>
			<td style="width:101px;">Di01- R405/488/561/635-25x36</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:123px;">Emission filter</td>
			<td style="width:204px;">Semrock</td>
			<td style="width:101px;">NF01-405/488/561/635-25X5.0</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:123px;">Slidebook 6.0</td>
			<td>Intelligent Imaging Innovations</td>
			<td></td>
			<td style="width:191px;">digital microscopy software</td>
		</tr>
	</tbody>
</table>

application of high-speed super-resolution speed microscopy in live primary cilium

Den primære Flimmerhår er en microtubule-baserte protrusion på overflaten av mange eukaryote celler og inneholder et unikt tilbud av proteiner som fungerer kritisk i cellen motilitet og signalering. Siden flimmerhårene er i stand til å syntetisere sin egen protein, må nesten 200 unike ciliary proteiner være trafikkerte mellom stoffer og primære flimmerhårene. Men det er fortsatt en teknisk utfordring å kartlegge tredimensjonale (3D) steder av transport for disse proteinene i live primære flimmerhårene på grunn av begrensningene av aktuelle eksisterende teknikker. For å erobre utfordringen, har nylig vi utviklet og ansatt en høyhastighets virtuelle 3D super-oppløsning mikroskopi, kalt ett-punkts kanten-eksitasjon sub Diffraksjon (hastighet) mikroskopi, for å finne transport trasé for 3D romlige hvor begge cytosolic og membran proteiner i primære flimmerhårene levende celler. I denne artikkelen vil vi vise detaljert oppsett av hastighet mikroskopi, utarbeidelse av celler som uttrykker fluorescens-protein-merket ciliary proteiner, enkelt-molekylet sanntidssporing av personlige proteiner i live Flimmerhår og prestasjoner 3D romlige sannsynlighet tetthet kart av transportveier for ciliary proteiner.

Denne delen viser dataene innhentet utføre hastighet mikroskopi på TZ av primære flimmerhårene å studere transportruten av SSTR3 forbundet med en ~ 15 nm eksterne linker til Alexa647 (Figur 3A). Det tjener dobbelt formål på bekrefter 3D transformasjon algoritmen. Alexa647 bør bare etiketten i ytre overflaten av den primære Flimmerhår og derfor 3D transportruten skulle avsløre en høy tetthet transportrute på denne plasseringen. NI...

Watch this Scientific Journal Video about Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium at JoVE.com

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

Nylig kartlagt vi tredimensjonale (3D) romlige plasseringen av transportveier for ulike proteiner translocating inne primære flimmerhårene i lever celler. Her live denne papir detaljer eksperimentelle oppsett, prosessen med biologiske prøver og data-analyser for 3D super-oppløsning fluorescens imaging tilnærming nylig brukt i primære flimmerhårene.

Anvendelse av høyhastighets super-oppløsning hastighet mikroskopi i Live primære Flimmerhår

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

The overall goal of this experiment is to identify protein transport routes in primary cilia. This method can help answer key question in the cilia field, such as protein transport route differences in normal and dysfunctional cilia. The main advantage of this technique is that it has high enough resolution to see subdiffraction protein transport routes in live cells.To begin, thaw frozen stock of specially engineered of NIH-3T3 cells at 37 degrees Celsius, one and a half weeks in advanced of the experiment. Transfer the thawed cells to a 25 centimeter squared cell cultured flask containing three milliliters of pre-warmed media. Maintain the cells at 37 degrees Celsius in a 5%carbon-dioxide incubator.Culture the cells until they reach 80%confluency and then split the cells. In order to split the cells, trypsinize them with 0.25%Trypsin for two minutes at 37 degrees Celsius. Following the brief incubation, aspirate the Trypsin and replace it with two milliliters of pre-warmed culture media.Pipette the media to break up any cell clusters, then remove 75%of the cells from the flask and bring total volume of media back up to three milliliters. Split the cells at least three times before the experiment to ensure homogeneity of their cell cycle. Two days before the experiment, split the cells and plate them 60-70%confluency on a 35 millimeter glass bottom plate with 1.5 milliliters of the same culture medium.Then, one day before the experiment, chemically transfect the cells with the desired plasmid. Mix 500-1000 nanograms of the plasmid in a one to 2.5 ratio with transfection reagent and 0.25 milliliters of reduced serum media without antibiotics for 30 minutes. Once the transfection complex has formed, aspirate the media from the 35 millimeter glass bottom dish and replace it with the 0.25 millimeters transfection mixture and an extra 1.25 millimeters of reduced serum media without antibiotics and return the plate to the incubator.For cells with externally labeled SSTR3 construct, remove the media from the glass bottom dish one hour before the experiment. Wash the cells five times with one milliliter of PBS and add one milliliter of reduced serum media supplemented with one micromolar of Alexa 647 conjugated streptavidin. No more than 15 minutes before the experiment, remove media from the glass bottom dish and wash the cells five times with one milliliter of PBS.Then add one milliliter of the imaging buffer into the glass bottom dish. Affix the glass bottom plate to the stage in the microscope. Find a GFP express cell using the GFP filter set and locate a cell that is properly expressing SSTR3-GFP in a primary cilia.Once a suitable cell has been found, align the NPHP4-mCherry spot at the base of the primary cilia with the location on the imaging plane that corresponds to the laser's single point illumination. Using the snap function, in the camera tab of the focus controls window, capture an epifluorescence image of NPHP4-mCherry and SSTR3 proteins tagged with green fluorescence protein. Once the reference images are obtained, locally reduce the concentration of labeled single molecules by photobleaching the transition zone with a one milliwatt laser illumination for 20 seconds.To prepare for single molecule tracking, reduce the laser illumination power to 0.5 milliwatts for molecules labeled with Alexa 647. Set the gain and intensification to maximum and the frame rate to two milliseconds. Then click the stream button in the camera tab of the focus controls window and engage the appropriate illumination laser.Record the non-photobleached labeled single molecules as they are transported through the photobleached region of the transition zone. And remember to record no more than two minutes of video to avoid the effects of ciliary drift. After capturing the single molecules, process the videos using a 2D Gaussian fitting algorithm such as Glimpse by the Gelles lab, which precisely localizes the centroid of each single molecule's excitation point, spread function, and an encompassing area of interest.Finally, select all single molecule locations with a precision better than 10 nanometers and correct the center of the cilia based on the distribution of single molecule locations fitted with a 2D Gaussian function. As described in this video, single point illumination can be used to track an AF647 tagged SSTR3 molecule through the transition zone marked by NPHP4-mCherry. Before data analysis, superimposing the single molecule video on the wide field illumination is a useful validation step to make sure the laser is properly aligned, and that there is a adequate signal-to-noise ratio.Imaged here is a cell with a primary cilium identified by the staining. IFT20 is component of the intraflagellar transport complex which carries cargo along microtubules inside primary cilia, marked by Arl13b-mCherry. Once identified, speed microscopy is used to track individual IFT20-GFP proteins.Next, and 2D to 3D transformation algorithm is used to produce the spatial probability density distribution of the protein inside primary cilia along a single dimension. This high density region likely colocalizes with the axonemal microtubules in agreement with the known location of the intraflagellar transport route. While attempting this procedure, it's important to precisely align the sample with the laser to minimize systematic error.After watching this video, you should have a good understanding of how to do single molecule tracking with speed microscopy and you will know how to obtain single molecule transport route in primary cilia.

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Biology

Unit 1

Cells and their Components

SCIENTISTS IN ACTION

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery ...

Super-resolution Imaging of the Bacterial Division Machinery

Super-oppløsning Imaging av Bakteriell divisjon Machinery

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

Test Prøver for Optimalisere STORM Super-oppløsning Mikros

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

Visualisering av G3BP Stress Granulat Dynamics i Levende primære celler

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Prøvepreparering for mikroskopi av enkeltvirionakroskopi og superoppløselig fluorescensavbildning

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Super-Resolution Imaging av Cytokinetic Z Ring i levende bakterier Bruke Fast 3D-Strukturert Illumination Mikros (F3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy iPALM

Tredimensjonal Super Resolution Mikroskopi av F-aktin filamenter av Interferometric PhotoActivated Lokalisering Mikroskopi (iPALM)

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Open-source Single-partikkel Analyse for Super-oppløsning Mikroskopi med VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Bakken staten uttømming super-oppløsning Imaging i pattedyrceller

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Undersøkelse av mitotisk og Meiotic fisjon gjær Nuclear Dynamics av fluorescens Live-Cell mikroskopi

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...