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 vil gerne udtrykke taknemmelighed over for Thao Nguyen og Atsushi Nakano fra UCLA for at give eksemplet mus til billede. Denne undersøgelse blev støttet af tilskud 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 System stjerner finansiering (til Juhyun Lee).

Alle de procedurer, der involverer brug af dyr er blevet godkendt af institutionelle anmeldelse udvalg (IACUC) på University of California, Los Angeles, Californien.
1. imaging System Setup
Bemærk: Se figur 1 og figur 2.
<ol><li>Hente en kontinuerlig bølge (CW) laser med 3 bølgelængde: 405 nm, 473 nm og 532 nm. Sted 2 spejle (M1 og M2) 150 mm fra hinanden og bringe dem på linje med deres spejl fly p...

<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 system og teknik beskrevet her udnytter en dobbelt-sidet belysning stråle kombineret med en optisk clearing til billede mus hjertet på efter fødslen og voksen stadier af dets udvikling. En traditionel enkelt belysning stråle lider photon spredning og absorption gennem tykkere og større væv prøver1,3. De dobbelt-sidet bjælker giver en mere jævn belysning af den prøve, derved at minimere effekten af striping og andre artefakter, der ofte ses i billed...

Lys-ark Fluorescens mikroskopi var en teknik, først udviklet i 1903 og bruges i dag som en metode til at studere genekspression og også til at fremstille 3D- eller 4-D modeller af væv prøver1,2,3. Denne billedbehandling metode bruger en tynd plade af lys til at belyse et enkelt fly af en prøve, således at kun at flyet er fanget af detektoren. Prøven kan derefter flyttes i aksial retning at fange hvert lag, en sektion ad gangen, og gøre en 3D-model efter efterbehandling af erhvervede billeder4. Men på grund af absorption og spredning af fotoner, LSFM har været begrænset til prøver, der er enten et par mikron tyk eller optisk gennemsigtig1.
Begrænsninger i LSFM har ført til omfattende undersøgelser af organismer, der har væv, der er optisk gennemsigtige, såsom zebrafisk. Undersøgelser, hvor hjertets udvikling og differentiering er ofte udført på zebrafisk siden der er bevarede gener mellem mennesker og zebrafisk5,6. Selv om disse undersøgelser har ført til fremskridt i hjertets forskning relateret til cardiomyopatier6,7, er der stadig behov for at gennemføre lignende forskning på højere niveau organismer såsom pattedyr.
Hjerte pattedyrvæv præsenterer en udfordring på grund af tykkelsen og opacitet af væv, absorption på grund af hæmoglobin i røde blodlegemer, og den opdeling, der opstår på grund af et enkeltsidet belysning af prøven under traditionelle LSFM metoder1 , 8. for at kompensere for disse begrænsninger, vi har foreslået at bruge dobbelt-sidet belysning og en forenklet version af klarhed teknik9 kombineret med et brydningsindeks matchende løsning (fælge). Derfor, dette system giver mulighed for billeddannelse af en stikprøve, der er større end 10 x 10 x 10 mm3 mens opretholde en god kvalitet opløsning i den aksiale og laterale fly8.
Dette system blev først kalibreret ved hjælp af fluorescerende perler arrangeret i forskellige konfigurationer i glas slangen. Derefter, at systemet blev brugt til at billede efter fødslen og voksen murine hjerter. Først, den postnatale mus hjerte blev afbildet på 7 dage (P7) til at afsløre den ventrikulære hulrum, tykkelsen af den ventrikulære væg, ventil strukturer og tilstedeværelsen af trabeculation. For det andet blev en undersøgelse udført for at identificere celler, der vil differentiere i cardiomyocytes ved hjælp af et postnatal mus hjerte på 1 dag (P1) med Cre-mærket cardiomyocytes og gul fluorescerende proteiner (YFP). Endelig blev voksen mus på 7,5 måneder afbildet for at konstatere tilstedeværelsen af renal ydre medullær kalium (ROMK) kanaler efter gen 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-ark Fluorescens mikroskopi har været meget anvendt til hurtig billede erhvervelse med en aksial højopløselige fra mikrometer til millimeter skala. Traditionelle lys-ark teknikker indebærer brug af en enkelt belysning stråle rettet vinkelret på vævsprøve. Billeder af store prøver, der er fremstillet ved hjælp af en enkelt belysning stråle indeholder striber eller artefakter og lider af en reduceret opløsning på grund af spredning og absorption af lys ved væv. Denne undersøgelse anvender en dobbelt-sidet belysning stråle og en forenklet klarhed optisk clearing teknik for murine hjertet. Disse teknikker giver mulighed for dybere billeddannelse ved at fjerne lipider fra hjertet og producere et stort felt af imaging, større end 10 x 10 x 10 mm3. Som et resultat, denne strategi gør det muligt at kvantificere de ventrikulære dimensioner, spore hjerte slægt og lokalisere den geografiske fordeling af hjertestop-specifikke proteiner og ion-kanaler fra den postnatale til voksen mus hjerter med tilstrækkelig kontrast og opløsning.

Den teknik beskrevet brugt her en dobbelt-sidet belysning stråle kombineret med en optisk clearing af vævsprøver mus hjertet for at opnå en dybere imaging dybde og en større imaging volumen med tilstrækkelig imaging opløsning (figur 1). Hvis du vil kalibrere systemet, blev fluorescerende perler placeret inde glas slangen og inden for billedbehandling system. Perlerne blev derefter afbildet og visualiseret i x-y plane, y-z planet, og i x-z planet, for a...

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 undersøgelse anvender en dobbelt-sidet belysning lys-ark Fluorescens mikroskopi (LSFM) teknik kombineret med optisk clearing for at studere murine hjertet.

Lys-ark Fluorescens mikroskopi for studiet af 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 fordel ved denne teknologi er, at det giver mulighed for hurtig billeddannelse af enten musens hjerte. Og det kan bruges til at observere tilstedeværelsen af luftkanaler efter genterapi. Selv om denne metode kan give indsigt i udvikling kardiologi, det kan også anvendes til andre systemer, såsom neurologi og pulmonologi.Generelt vil enkeltpersoner kæmpe med denne teknik, fordi montering af en billeddannelse af den voksne mus hjerte kan være svært, og det er forskelligt fra andre, traditionelle teknikker, såsom konfokale og omvendt mikroskopi. Placer en kontinuerlig bølge laser med tre bølgelængder af 405 nanometer, 473 nanometer, og 532 nanometer. Derefter anbringe spejl en og tilpasse det med spejlet flyet ved 45 grader til strålen.Dette vil lede laseren mod strålesplitteren, som danner den dual-sidede belysningsopsætning. Dernæst passerer strålen gennem en 50 millimeter diameter neutral tæthed filter, en stråle expander, og en 25 millimeter diameter pin hul, alle placeret 150 millimeter fra hinanden. Pass strålen gennem en 50-50 stråle splitter, placeret 150 millimeter fra pinhole.Dernæst skal du placere spejl to 150 millimeter fra bjælken splitter, ved 90 grader til den forreste stråle, og tilpasse den, så dens spejl plan er i en 45 graders vinkel til strålen. Derefter sted spejl tre 100 millimeter fra spejl to, og tilpasse det, så det spejl plan er i en 45 graders vinkel til strålen reflekteres fra spejl to. Brug denne reflekterede stråle til at danne den ene side af det dobbelte belysningslysark.På den anden side af bjælken splitter, sted spejl fem, så det spejl plan er på 45 grader til den stråle, der udsendes i en fremadrettet retning. Brug den stråle, der udsendes fra spejl fem, til at danne den anden side af det dobbelte lysark. Dernæst skal du oprette det dobbeltsidede belysningssystem på systemisk vis.Placer cylindrisk linse to 150 millimeter væk fra spejl tre, og en anden identisk cylindrisk linse 150 millimeter væk fra spejl fem, på den anden side af den dobbelte belysning setup. Dernæst placere to spejle, hver i overensstemmelse med de cylindriske linser, på afstande på 50 millimeter for at afspejle strålen ved 90 grader. På begge sider af lysarket danner en akromatisk doublet fra et par linser, hvor den første linse placeres 100 millimeter fra det tidligere spejl og har en diameter på en tomme og en brændvidde på 100 millimeter.Den anden linse skal placeres 160 millimeter fra linse en, med en diameter på en tomme, og en brændvidde på 60 millimeter. Derefter placere belysning mål en og to 150 millimeter fra de tidligere akromatisk doublets, så de er i overensstemmelse med strålen. Den stråle, der udsendes fra målene, danner lysarket til billeddannelse af prøverne.Først forberede fluorescerende perle prøve ved at fortynde en 0,53 mikrometer perle opløsning, en til 150, 000, i en præ-opvarmet brydningsindeks matchende opløsning, med en procent lav smelte peger agarose. Brug derefter et stykke borosilikatglasrør med en indre diameter på 12 millimeter og en ydre diameter på 18 millimeter til en længde på 30 millimeter. Pipette perle agarose opløsning i borosilikat slange og lad agarose at størkne ved stuetemperatur.Nu fylde en 3-D trykt ABS kammer med en 99,5 procent gylcerol løsning. Placer borosilikat glasrør, der indeholder perlerne, inde i kammeret. Fastgør en 3D motoriseret translationel fase til borosilikatglasslangen for at styre prøvens bevægelse og orientering i ABS-kammeret.Brug derefter specialdesignet software til at erhverve billeder ved hjælp af sCMOS-kameraet med en hastighed på 30 billeder pr. sekund. Brug motorcontrolleren til at flytte prøven en millimeter i sideretningen og hente billeder ved hvert enkelt millimeterinterval, indtil hele prøven er afbildet. Stak de erhvervede billeder ved hjælp af en visualisering software, og måle punkt spredning funktion af systemet ved hjælp af disse perle billeder.Brug en brydningsindeks matchende opløsning, med en pH-værdi på 7,5, og opløse en procent agarose i matchende opløsning. Placer en ryddet voksen mus hjerte prøve i brydningsindeks matchende løsning, med en procent agarose opløst. Derefter indsættes prøven i borosilikatglasrør, og lad agarose at størkne ved stuetemperatur.Fastgør en 3D motoriseret translationel fase til borosilikatglasslangen for at styre prøvens bevægelse og orientering i et 3D-printet ABS-kammer. Placer prøven, så den er i midten af gaussiske stråle, skabt af det dobbelte belysningssystem. Derefter indstille erhvervelse sats af sCMOS kamera til 30 billeder i sekundet.Nu, ved hjælp af motor controller, flytte prøven en millimeter i aksial retning og erhverve billeder på hver en millimeter interval. Fortsæt, indtil hele eksemplet er afbildet. Stable de erhvervede billeder ved hjælp af en visualisering software.Udvikt 3D-billederne ved hjælp af disse billedstakke. For at opnå dette, dekonvolve punkt spredning funktion bestemmes tidligere, og udnytte det for de erhvervede billede stakke. Endelig skal du indstille en pixelgrænseintensitetsværdi for at observere hjertets konturer og føje pseudofarve til billederne baseret på denne gråskalaintensitet.På dag ét post-natal, kan man visualisere ventiler, atrium, hjertekammer, pektinate muskel, og trabeculation, blandt andre funktioner. På dag syv efter fødslen, er funktionerne endnu mere defineret. Atrium, hjertekammer, ventrikelhule dimensioner, og ventrikel vægtykkelse er alle indlysende.At studere cardiomyocyte differentiering i hjertet, heterozygode mus blev cre-mærket til at vise kardiomyocytter. De mærkede celler vises her i hver planretning. De tre fly blev slået sammen for at skabe en 3D-gengivelse af hjertet.Den røde cirkel region i indsat viser to gennemskinnelige mus hjerter efter at have gennemgået klarhed teknik og blive indsat i borosilikat glasrør. Ud over at studere postnatale mus kan billeddannelsessystemet også bruges til at studere det voksne musehjerte. Her vises GFP-mærkede ROMK-kanaler efter 7,5 måneder.Disse blev hovedsageligt fundet i ventrikelvæggen. Når mestrer, kan denne teknik gøres i et til to minutter. Når du forsøger denne procedure, er det vigtigt at fjerne alle boblerne omkring prøven i røret.Andre metoder, såsom automatisk segmentering af billedet stakke, kan udføres for at besvare yderligere spørgsmål i forbindelse med hjerte-kar-skade og regenerering. Efter dens udvikling, denne teknik blev brugt af forskere til at studere hjertearkitektur i postnatal og voksen udviklingsmæssige stadier i 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.

Oprettelse Lim og Opløseligt Gradienter for Imaging Cell Migration med 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 ...

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 modificeret to-foton mikroskop: en ansøgning til sporing af intracellulære vesikler

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-ark Fluorescens mikroskopi til fange 4-dimensionelle billeder af virkningerne af modulerende Shear Stress på udvikle zebrafisk 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.

Biaksiale mekaniske beskrivelser af atrioventrikulær hjerteklapper

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).

Blodgennemstrømning Imaging med Ultrafast 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 Billeddannelse af biologisk væv med kombineret to-fotonfluorescens og stimuleret Raman-spredningsmikroskopi

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

Analyse af zebrafiskhjertesammentrækning med Virtual Reality-interaktion

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: Højopløselig 4D Light-Sheet Imaging og Virtual Reality i zebrafisk til enkeltcelleanalyse af hjertefunktion

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 lys-ark billeddannelse af zebrafisk hjertekontraktion

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