Name: light-sheet fluorescence microscopy for the study of the murine heart
Uploaded: 2018-09-15T14:00:00
Description: Watch this Scientific Journal Video about Light-sheet Fluorescence Microscopy for the Study of the Murine Heart at JoVE.com

Forfatterne ønsker å uttrykke takknemlighet til Thao Nguyen og Atsushi Nakano fra UCLA for å gi mus prøven bildet. Denne studien ble støttet av tilskudd NIH HL118650 (til Tzung K. Hsiai), HL083015 (til Tzung K. Hsiai), HD069305 (til N. C. Chi og Tzung K. Hsiai.), HL111437 (til Tzung K. Hsiai og N. C. Chi), HL129727 (til Tzung K. Hsiai) og University of Texas stjerner midler (til Juhyun Lee).

Alle prosedyrer som involverer bruk av dyr har blitt godkjent av institusjonelle gjennomgang komiteer (IACUC) ved University of California, Los Angeles, California.
1. imaging System Setup
Merk: Se figur 1 og figur 2.
<ol><li>Hente med 3 bølgelengder kontinuerlige bølgen (CW) laser: 405 nm, 473 nm og 532 nm. Sted 2 speil (M1 og M2) 150 mm fra hverandre og justere dem med flyene sine speil på 45° av st...

<ol>
	<li>Richardson, D. S., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clarifying+tissue+clearing.">Clarifying tissue clearing.</a> Cell. 162 (2), 246-257 (2015).</li><li>Lee, 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=4-Dimensional+light-sheet+microscopy+to+elucidate+shear+stress+modulation+of+cardiac+trabeculation.">4-Dimensional light-sheet microscopy to elucidate shear stress modulation of cardiac trabeculation.</a> Journal of Clinical Investigation. 126 (5), 1679-1690 (2016).</li><li>Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J., Stelzer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+sectioning+deep+inside+live+embryos+by+selective+plane+illumination+microscopy.">Optical sectioning deep inside live embryos by selective plane illumination microscopy.</a> Science. 305 (5686), 1007-1009 (2004).</li><li>Huisken, J., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+plane+illumination+microscopy+techniques+in+developmental+biology.">Selective plane illumination microscopy techniques in developmental biology.</a> Development. 136 (12), 1963-1975 (2009).</li><li>Bakkers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+to+study+cardiac+development+and+human+cardiac+disease.">Zebrafish as a model to study cardiac development and human cardiac disease.</a> Cardiovascular Research. 91 (2), 279-288 (2011).</li><li>High, F. A., Epstein, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multifaceted+role+of+Notch+in+cardiac+development+and+disease.">The multifaceted role of Notch in cardiac development and disease.</a> Nature Reviews Genetics. 9 (1), 49-61 (2008).</li><li>Sachinidis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+specific+differentiation+of+mouse+embryonic+stem+cells.">Cardiac specific differentiation of mouse embryonic stem cells.</a> Cardiovascular Research. 58 (2), 278-291 (2003).</li><li>Ding, Y., 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=Light-sheet+fluorescence+imaging+to+localize+cardiac+lineage+and+protein+distribution.">Light-sheet fluorescence imaging to localize cardiac lineage and protein distribution.</a> Scientific Reports. 7, 42209 (2017).</li><li>Tomer, R., Ye, L., Hsueh, B., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+CLARITY+for+rapid+and+high-resolution+imaging+of+intact+tissues.">Advanced CLARITY for rapid and high-resolution imaging of intact tissues.</a> Nature Protocols. 9 (7), 1682-1697 (2014).</li><li>Robbins, N., Thompson, A., Mann, A., Blomkalns, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+excision+of+murine+aorta;+a+versatile+technique+in+the+study+of+cardiovascular+disease.">Isolation and excision of murine aorta; a versatile technique in the study of cardiovascular disease.</a> Journal of Visualized Experiments. (93), e52172 (2014).</li><li>National Heart, L., Blood Institute, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Standard Operating Procedures (SOP's) for Duchenne Animal Models. , (2015).</li><li>Sung, K., 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=Simplified+three-dimensional+tissue+clearing+and+incorporation+of+colorimetric+phenotyping.">Simplified three-dimensional tissue clearing and incorporation of colorimetric phenotyping.</a> Scientific Reports. 6, 30736 (2016).</li><li>Yuan, Z., Qiao, C., Hu, P., Li, J., Xiao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+adeno-associated+virus+vector+producer+cell+line+method+for+scalable+vector+production+of+different+serotypes.">A versatile adeno-associated virus vector producer cell line method for scalable vector production of different serotypes.</a> Human Gene Therapy. 22 (5), 613-624 (2011).</li><li>FEI, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Amira User's Guide. , 59-138 (2017).</li><li>Gao, 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=Noninvasive+imaging+of+3D+dynamics+in+thickly+fluorescent+specimens+beyond+the+diffraction+limit.">Noninvasive imaging of 3D dynamics in thickly fluorescent specimens beyond the diffraction limit.</a> Cell. 151 (6), 1370-1385 (2012).</li><li>Truong, T. V., Supatto, W., Koos, D. S., Choi, J. M., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+and+fast+live+imaging+with+two-photon+scanned+light-sheet+microscopy.">Deep and fast live imaging with two-photon scanned light-sheet microscopy.</a> Nature Methods. 8 (9), 757-760 (2011).</li><li>Ding, Y., 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=Integrating+light-sheet+imaging+with+virtual+reality+to+recapitulate+developmental+cardiac+mechanics.">Integrating light-sheet imaging with virtual reality to recapitulate developmental cardiac mechanics.</a> JCI Insight. 2 (22), (2017).</li></ol>

LSFM systemet og teknikken beskrevet her benytter en tosidig belysning bjelke kombinert med en optisk avregning bildet musen hjertet på postnatal og voksen stadier av sin utvikling. En tradisjonell enkelt belysning bjelke lider Foton spredning og absorpsjon gjennom tykkere og større vev prøver1,3. Tosidig bjelker gir en jevnere belysning av prøven, og dermed minimere effekten av striping og andre gjenstander som er ofte sett i bilder produsert av en ensidig b...

Lys arks fluorescens mikroskopi var en teknikk utviklet i 1903, og brukes i dag som å studere genuttrykk og også å produsere 3D eller 4 D modeller vev prøver1,2,3. Denne tenkelig metoden bruker et tynt ark av lys til å lyse opp et enkelt fly av en prøve slik at bare det flyet er fanget av detektoren. Prøven kan deretter flyttes i aksial-retning å fange hvert lag, ett avsnitt om gangen, og gjengi en 3D modell etter post-prosessering av ervervet bilder4. Men absorpsjon og spredning av fotoner, har LSFM vært begrenset til prøvene som enten få mikrometer tykke eller optisk gjennomsiktig1.
Begrensningene for LSFM har ført til omfattende studier av organismer som har vev som er optisk gjennomsiktige, som sebrafisk. Studier med hjerte utvikling og differensiering er ofte utført på sebrafisk siden det er bevart gener mellom mennesker og sebrafisk5,6. Selv om disse studiene har ført til fremskritt i cardiac forskning knyttet til cardiomyopathies6,7, er det fortsatt behov for å utføre lignende forskning på høyere nivå organismer som pattedyr.
Pattedyr hjerte vev presenterer en utfordring på grunn av tykkelse og tetthet av vev, opptaket på grunn av hemoglobin i røde blod celler og striping som oppstår grunnet ensidig belysning av prøven under tradisjonelle LSFM metoder1 , 8. for å kompensere for disse begrensningene, vi foreslått å bruke tosidig belysning og en forenklet versjon av KLARHET teknikk9 kombinert med en brytningsindeks matchende løsning (FELGER). Derfor lar dette systemet for avbilding av et utvalg som er større enn 10 x 10 x 10 mm3 mens opprettholde en god oppløsning i aksial og lateral fly8.
Dette systemet ble først kalibrert med fluorescerende perler ordnet i forskjellige konfigurasjoner i glass slangen. Da ble systemet brukt å image postnatal og voksen murine hjerter. Først ble postnatal musen hjertet avbildet på 7 dager (P7) å avsløre ventrikkel hulrom, tykkelsen på ventrikkel veggen, ventil strukturer og tilstedeværelsen av trabeculation. For det andre, en studie ble utført for å identifisere celler som vil differensiere i cardiomyocytes ved å bruke et postnatal musen hjerte i 1 dag (P1) med grobunn-merket cardiomyocytes og gule fluorescerende protein (YFP). Til slutt, voksen mus på 7,5 måneder ble fotografert for å observere tilstedeværelsen av nyre ytre medullær kalium (ROMK)-kanaler etter gene terapi8.

<table border="0" cellpadding="0" cellspacing="0" style="width:967px;" width="967">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">CW Laser</td>
			<td style="width:136px;">Laserglow Technologies</td>
			<td style="width:393px;">LMM-GBV1-PF3-00300-05</td>
			<td style="width:191px;">Excitation of fluorophores</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Neutral density filter</td>
			<td style="width:136px;">Thorlabs</td>
			<td style="width:393px;">NDC-50C-4M</td>
			<td style="width:191px;">Controls amount of light entering system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Achromatic beam expander</td>
			<td style="width:136px;">Thorlabs</td>
			<td style="width:393px;">GBE05-A</td>
			<td style="width:191px;">Expands the beam of light</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Mechanical slit</td>
			<td style="width:136px;">Thorlabs</td>
			<td style="width:393px;">VA100C</td>
			<td style="width:191px;">Controls width of beam</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Beam splitter</td>
			<td style="width:136px;">Thorlabs</td>
			<td style="width:393px;">BS013</td>
			<td style="width:191px;">Forms dual-illumination beam</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Stereo microscope with 1X objective lense</td>
			<td style="width:136px;">Olympus</td>
			<td style="width:393px;">MVX10</td>
			<td style="width:191px;">Used for observation of sample</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">ORCA-Flash4.0 LT sCMOS camera</td>
			<td style="width:136px;">Hamamatsu Photonics</td>
			<td style="width:393px;">C11440-42U</td>
			<td style="width:191px;">Used to capture Images</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Acrylonitrile butadiene styrene (ABS)</td>
			<td style="width:136px;">Stratasys</td>
			<td style="width:393px;">uPrint</td>
			<td style="width:191px;">Material used to 3-D print a sample holder</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Fluorescent polystyrene beads</td>
			<td style="width:136px;">Spherotech Inc</td>
			<td style="width:393px;">PP-05-10</td>
			<td style="width:191px;">Used for imaging system calibration</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Borosilicate glass tubing</td>
			<td style="width:136px;">Corning</td>
			<td style="width:393px;">Pyrex 7740</td>
			<td style="width:191px;">Tubing for sample embedding</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Glycerol</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:393px;">BP229-4</td>
			<td style="width:191px;">Fill for sample chamber</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Phosphate-buffered saline</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:393px;">BP39920</td>
			<td style="width:191px;">Rinse solution for mouse hearts</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Paraformaldehyde</td>
			<td style="width:136px;">Electron microscopy sciences</td>
			<td style="width:393px;">RT-15700</td>
			<td style="width:191px;">First incubation solution</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:247px;">Acrylamide</td>
			<td style="width:136px;">Wako Chemicals</td>
			<td style="width:393px;">AAL-107</td>
			<td style="width:191px;">Mixed with 2,2&#39;-Azobis dihydrochloride for second incubation solution for mouse hearts</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">2,2&#39;-Azobis dihydrochloride</td>
			<td style="width:136px;">Wako Chemicals</td>
			<td style="width:393px;">VA-044</td>
			<td style="width:191px;">Mixed with Acrylamide for second incubation solution for mouse hearts</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Sodium dodecyl sulfate&#160;</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:393px;">71725</td>
			<td style="width:191px;">Mixed with Boric acid for third incubtion solution for mouse hearts</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Boric acid</td>
			<td style="width:136px;">Fischer Scientific</td>
			<td style="width:393px;">A74-1</td>
			<td style="width:191px;">Mixed with Sodium dodecyl sulfate for third incubtion solution for mouse hearts</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:247px;">Sigma D2158</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:393px;">D2158</td>
			<td style="width:191px;">Mixed with PB, Tween-20, and Sodium azide as a refractive index matching solution</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:247px;">Tween-20</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td>11332465001</td>
			<td style="width:191px;">Mixed with Sigma D2158, PB, and Sodium azide as a refractive index matching solution</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Sodium azide</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td>S2002</td>
			<td style="width:191px;">Mixed with Sigma D2158, PB, and Tween-20 as a refractive index matching solution</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Adeno-associated virus vector 9 with a cardiac-specific Troponin T promoter tagged with GFP</td>
			<td style="width:136px;">Vector Biolabs</td>
			<td style="width:393px;">VB2045</td>
			<td style="width:191px;">Expresses GFP when bound to ROMK</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">DC Servo Motor Actuator</td>
			<td style="width:136px;">Thorlabs</td>
			<td style="width:393px;">Z825B</td>
			<td style="width:191px;">Used for movement of sample in axial direction within light sheet</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">K-Cube Brushed DC Servo Motor Controller</td>
			<td style="width:136px;">Thorlabs</td>
			<td style="width:393px;">KDC101</td>
			<td style="width:191px;">Connects to motor actuator and controls movement of the actuator</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Amira</td>
			<td style="width:136px;">FEI Software</td>
			<td style="width:393px;">N/A</td>
			<td style="width:191px;">Visualization software for producing 2-D and 3-D images</td>
		</tr>
	</tbody>
</table>

light-sheet fluorescence microscopy for the study of the murine heart

Lys arks fluorescens mikroskopi er mye brukt for rask bildeopptak med høy aksiell oppløsning fra mikrometer millimeter skala. Tradisjonelle lys arks teknikker innebærer bruk av en enkelt belysning bjelke ortogonalt rettet eksempel vev. Bilder av store prøver som er produsert ved hjelp av en enkelt belysning bjelke inneholder striper eller gjenstander og lider en redusert oppløsning spredning og absorpsjon av lys av vev. Denne studien bruker en tosidig belysning stråle og en forenklet KLARHET optisk fjerne teknikk for murine hjertet. Disse teknikkene tillater dypere bildebehandling ved å fjerne lipider fra hjertet og produsere et stort felt av bildebehandling, større enn 10 x 10 x 10 mm3. Som et resultat, denne strategien kan vi kvantifisere ventrikkel dimensjonene, spore cardiac slektslinje og lokalisere den romlige fordelingen av hjerte-spesifikke proteiner og ion-kanaler fra den postnatal til voksen mus hjerter med tilstrekkelig kontrast og oppløsning.

Teknikken er beskrevet brukt her en tosidig belysning bjelke kombinert med en optisk clearing av musen hjertet for å oppnå en dypere tenkelig dybde og en større tenkelig volum med tilstrekkelig tenkelig oppløsning (figur 1). Kalibrer systemet ble fluorescerende perler plassert i glass slangen og innen tenkelig system. Perler ble deretter fotografert og visualisert i x-y flyet, y-z-fly, og i x-z flyet, for å få poenget spre funksjon (PSF) for systemet<su...

Watch this Scientific Journal Video about Light-sheet Fluorescence Microscopy for the Study of the Murine Heart at JoVE.com

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Denne studien bruker en tosidig belysning lys arks fluorescens mikroskopi (LSFM) teknikk kombinert med optisk avregning for å studere murine hjertet.

Lys arks fluorescens mikroskopi for studier av Murine hjertet

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Den største fordelen med denne teknologien er at den gir rask avbildning av enten musehjertet. Og det kan brukes til å observere tilstedeværelsen av luftkanaler etter genterapi. Selv om denne metoden kan gi innsikt i utvikling kardiologi, kan den også brukes på andre systemer, for eksempel nevrologi og lungeologi.Vanligvis vil enkeltpersoner slite med denne teknikken fordi montering av et bilde av det voksne musehjertet kan være vanskelig, og det er forskjellig fra andre tradisjonelle teknikker, for eksempel konfokal og omvendt mikroskopi. Plasser en kontinuerlig bølgelaser med tre bølgelengder på 405 nanometer, 473 nanometer og 532 nanometer. Deretter feste speile en og justere den med speilplanet på 45 grader til strålen.Dette vil lede laseren mot strålesplitteren, som danner det dobbeltsidige belysningsoppsettet. Deretter passerer strålen gjennom et 50 millimeter diameter nøytralt tetthetsfilter, en stråleutvider og et 25 millimeter diameter pin hull, alle plassert 150 millimeter fra hverandre. Pass strålen gjennom en 50-50 strålesplitter, plassert 150 millimeter fra pinhole.Deretter plasserer du speil to 150 millimeter fra strålesplitteren, ved 90 grader til fremre stråle, og justerer den slik at speilplanet er i en 45 graders vinkel til strålen. Deretter plasserer du speil tre 100 millimeter fra speil to, og justerer det slik at speilplanet er i en 45 graders vinkel til strålen reflektert fra speil to. Bruk denne reflekterte strålen til å danne den ene siden av det doble belysningslysarket.På den andre siden av strålesplitteren plasserer du speil fem slik at speilplanet er på 45 grader til strålen som slippes ut i en fremoverretning. Bruk strålen som sendes ut fra speil fem for å danne den andre siden av det doble belysningslysarket. Deretter setter du opp det dobbeltsidige belysningssystemet på systemisk måte.Plasser sylindrisk linse to 150 millimeter unna speil tre, og en annen identisk sylindrisk linse 150 millimeter unna speil fem, på den andre siden av det doble belysningsoppsettet. Deretter plasserer du to speil, hver i tråd med sylindriske linser, i avstander på 50 millimeter for å reflektere strålen ved 90 grader. På begge sider av lysarket danner du en achromatic doublet fra et par linser, med den første linsen plassert 100 millimeter fra forrige speil, og har en en tomme diameter og en brennvidde på 100 millimeter.Den andre linsen skal plasseres 160 millimeter fra linse en, med en diameter på en tomme, og en brennvidde på 60 millimeter. Deretter plasserer du belysningsmålene en og to 150 millimeter fra de tidligere achromatic doublets, slik at de er i tråd med strålen. Strålen som slippes ut fra målene danner lysarket for avbildning av prøvene.Først forberede fluorescerende perle prøve ved å fortynne en 0,53 mikrometer perle løsning, en til 150,000, i en forvarmet brytning indeks matchende løsning, med en prosent lav smelte peker agarose. Deretter bruker du et stykke borosilikatglassrør med en indre diameter på 12 millimeter, og en ytre diameter på 18 millimeter, til en lengde på 30 millimeter. Pipette perlen agarose løsning i borosilikat rør og la agarose å stivne ved romtemperatur.Fyll nå et 3D-trykt ABS-kammer med en 99,5 prosent gylcerolløsning. Plasser borosilikatglassslangen, som inneholder perlene, inne i kammeret. Fest en 3D motorisert translasjonell fase til borosilikatglassrørene for å kontrollere bevegelsen og orienteringen til prøven i ABS-kammeret.Deretter bruker du spesialdesignet programvare til å skaffe bilder ved hjelp av sCMOS-kameraet, med en hastighet på 30 bilder per sekund. Bruk motorkontrolleren til å flytte prøven én millimeter i sideretningen, og få bilder ved hver trinn på én millimeter, til hele prøven er avbildet. Stable de oppkjøpte bildene ved hjelp av en visualiseringsprogramvare, og mål poengspredningsfunksjonen til systemet ved hjelp av disse perlebildene.Bruk en brytningsindeks matchende løsning, med en pH på 7,5, og oppløs en prosent agarose i matchende løsning. Plasser en ryddet voksen mus hjerte prøve i brytningsindeks matchende løsning, med en prosent agarose oppløst. Deretter setter du prøven inn i borosilikatglassrør, og la agarose å stivne ved romtemperatur.Fest en 3D motorisert translasjonell fase til borosilikatglassrørene for å kontrollere bevegelsen og retningen på prøven i et 3D-trykt ABS-kammer. Plasser prøven slik at den er i midten av den gaussiske strålen, skapt av det doble belysningssystemet. Deretter setter du oppkjøpshastigheten til sCMOS-kameraet til 30 bilder per sekund.Nå, ved hjelp av motorkontrolleren, flytte prøven en millimeter i aksial retning og skaffe bilder på hver en millimeter økning. Fortsett til hele prøven er avbildet. Stable de kjøpte bildene ved hjelp av en visualiseringsprogramvare.Utvikle 3D-bildene ved hjelp av disse bildestakkene. For å oppnå dette, dekonvolve poengspredningsfunksjonen som er bestemt tidligere, og bruk den for de kjøpte bildestakkene. Til slutt angir du en pikselterskelintensitetsverdi for å observere hjertets konturer, og legger til pseudofarge til bildene, basert på denne gråskalaintensiteten.På dag én postnatal kan man visualisere ventilene, atrium, ventrikkel, brystmuskulatur og trabekulasjon, blant andre funksjoner. På dag sju postnatal er funksjonene enda mer definert. Atrium, ventrikkel, ventrikulær hulrom dimensjoner, og ventrikkel veggtykkelse er alle tydelig.For å studere kardiomyocytt differensiering i hjertet, ble heterozygous knock-in mus cre-merket for å vise kardiomyocytter. De merkede cellene vises her, i hver planretning. De tre flyene ble slått sammen, for å skape en 3D-gjengivelse av hjertet.Den røde sirkelen i innfelt viser to gjennomsiktige musehjerter etter å ha gjennomgått klarhetsteknikken og satt inn i borosilikatglassslangen. I tillegg til å studere postnatale mus, kan bildesystemet også brukes til å studere det voksne musehjertet. Her vises GFP-merkede ROMK-kanaler på 7,5 måneder.Disse ble hovedsakelig funnet i ventrikulær vegg. Når mestret, kan denne teknikken gjøres i ett til to minutter. Når du prøver denne prosedyren, er det viktig å fjerne alle boblene rundt prøven i røret.Andre metoder, for eksempel automatisk segmentering av bildestablene, kan utføres for å svare på flere spørsmål knyttet til kardiovaskulær skade og regenerering. Etter utviklingen ble denne teknikken brukt av forskere til å studere hjertearkitekturen i postnatale og voksne utviklingsstadier hos mus.

light-sheet-fluorescence-microscopy-for-the-study-of-the-murine-heart

Research

JoVE Journal

Bioengineering

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Opprette Lim og Løselig overgangar for Imaging Cell Migration med fluorescens mikroskopi

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3D Orbital Tracking i en modifisert to-foton Microscope: en søknad til sporing av intracellulær Blemmer

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

Lys arks fluorescens mikroskopi å ta 4-dimensjonale bilder av effektene av modulerende skjæring Stress på utvikle sebrafisk hjertet

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

Biaxial mekanisk Characterizations av atrioventrikulær hjertet ventiler

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

Blodstrømavbildning med ultrarask Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

In vivo Avbildning av biologisk vev med kombinert to-foton fluorescens og stimulert ramanspredningsmikroskopi

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

Analyse av sebrafisk hjertesammentrekning med virtuell virkelighetsinteraksjon

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for gaining insights into structural and functional changes in response to injuries. However, obtaining high-resolution and high-speed 4-dimensional (4D, 3D spatial + 1D temporal) images of the zebrafish heart to assess cardiac architecture and contractility remains challenging. In this context, an in-house light-sheet microscope (LSM) and customized computational analysis are&#160;used to overcome these technical limitations. This strategy, involving LSM system construction, retrospective synchronization, single cell tracking, and user-directed analysis, enables one to investigate the micro-structure and contractile function across the entire heart at the single-cell resolution in the transgenic Tg(myl7:nucGFP) zebrafish larvae. Additionally, we are able to further incorporate microinjection of small molecule compounds to induce cardiac injury in a precise and controlled manner. Overall, this framework allows one to track physiological and pathophysiological changes, as well as the regional mechanics at the single-cell level during cardiac morphogenesis and regeneration.

Forfatter Spotlight: Høyoppløselig 4D-lysarkavbildning og virtuell virkelighet i sebrafisk for encellet analyse av hjertefunksjon

This protocol utilizes light-sheet imaging to investigate cardiac contractile function in zebrafish larvae and gain insights into cardiac mechanics through cell tracking and interactive analysis.

4D lysarkavbildning av sebrafiskhjertekontraksjon

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for ...