Name: mesoscopic optical imaging of whole mouse heart
Uploaded: 2021-10-14T14:15:00
Description: Watch this Scientific Journal Video about Mesoscopic Optical Imaging of Whole Mouse Heart at JoVE.com

Dette projekt har modtaget finansiering fra EU's Horizon 2020 forsknings- og innovationsprogram under tilskudsaftale nr. 952166 (REPAIR), MUR under FISR-programmet, projekt FISR2019_00320 og Regione Toscana, Bando Ricerca Salute 2018, PERCARE-projektet.

Alle dyrehåndtering og -procedurer blev udført i overensstemmelse med retningslinjerne i Europa-Parlamentets direktiv 2010/63/EU om beskyttelse af dyr, der anvendes til videnskabelige formål, og i overensstemmelse med det italienske sundhedsministeriums principper og forskrifter. Forsøgsprotokollen blev godkendt af det italienske sundhedsministerium (protokolnummer 647/2015-PR). Alle dyrene blev leveret af ENVIGO, Italien. Til disse forsøg blev der anvendt 5 C57BL/6J-hanmus på 6 måneder.
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clearing+of+fixed+tissue:+a+review+from+a+microscopist's+perspective.">Clearing of fixed tissue: a review from a microscopist's perspective.</a> Journal of Biomedical Optics. 21, 081205 (2016).</li><li>Chung, 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=Structural+and+molecular+interrogation+of+intact+biological+systems.">Structural and molecular interrogation of intact biological systems.</a> Nature. 497, 332-337 (2013).</li><li>Park, Y. 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(139), e57769 (2018).</li><li>Olianti, C., 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=3D+imaging+and+morphometry+of+the+heart+capillary+system+in+spontaneously+hypertensive+rats+and+normotensive+controls.">3D imaging and morphometry of the heart capillary system in spontaneously hypertensive rats and normotensive controls.</a> Scientific Reports. 10, 1-9 (2020).</li><li>Pianca, N., 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=Cardiac+sympathetic+innervation+network+shapes+the+myocardium+by+locally+controlling+cardiomyocyte+size+through+the+cellular+proteolytic+machinery.">Cardiac sympathetic innervation network shapes the myocardium by locally controlling cardiomyocyte size through the cellular proteolytic machinery.</a> The Journal of Physiology. 597, 3639-3656 (2019).</li><li>Di Bona, A., Vita, V., Costantini, I., Zaglia, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+clearer+view+of+sympathetic+innervation+of+cardiac+and+skeletal+muscles.">Towards a clearer view of sympathetic innervation of cardiac and skeletal muscles.</a> Progress in Biophysics and Molecular Biology. 154, 80-93 (2020).</li><li>Voigt, F. 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I dette arbejde blev der introduceret en vellykket tilgang til at rydde, plette og afbilde et helt musehjerte i høj opløsning. For det første blev en vævstransformationsprotokol (CLARITY) optimeret og udført, lidt modificeret til dens anvendelse på hjertevævet. For at opnå en effektiv rekonstruktion i 3D af et helt hjerte er det faktisk vigtigt at forhindre fænomenet lysspredning. CLARITY-metoden giver os mulighed for at opnå et meget gennemsigtigt intakt hjerte, men det kræver lange inkubationstider, når det...

Strukturel ombygning forbundet med hjertesygdomme kan påvirke elektrisk ledning og indføre elektromekaniske dysfunktioner i organet 1,2. Nuværende tilgange, der bruges til at forudsige funktionelle ændringer, anvender almindeligvis MR og DT-MR for at opnå en samlet rekonstruktion af fibroseaflejring, vaskulær træ- og fiberfordeling af hjertet, og de bruges til at modellere præferenceaktionspotentialeudbredelse (APP) stier over organet 3,4. Disse strategier kan give et smukt overblik over hjerteorganisationen. Imidlertid er deres rumlige opløsning utilstrækkelig til at undersøge virkningen af strukturel ombygning på hjertefunktion på celleniveau.
Det er udfordrende at placere denne ramme i en anden størrelsesorden, hvor enkeltceller kan spille individuelle roller på handlingspotentialeudbredelse. Den største begrænsning er ineffektiviteten af standardbilleddannelsesmetoder til at udføre billeddannelse i høj opløsning (mikrometrisk opløsning) i massive (centimeterstore) væv. Faktisk er billeddannelse af biologiske væv i 3D ved høj opløsning meget kompliceret på grund af vævets uigennemsigtighed. Den mest almindelige tilgang til at udføre 3D-rekonstruktioner i hele organer er at forberede tynde sektioner. Præcis sektionering, samling og billeddannelse kræver dog en betydelig indsats og tid. En alternativ tilgang, der ikke kræver udskæring af prøven, er at generere et gennemsigtigt væv. I løbet af de sidste år er der foreslået flere metoder til afklaring af væv 5,6,7,8. Udfordringen med at producere massivt, gennemsigtigt og fluorescerende mærket væv er for nylig opnået ved at udvikle ægte vævstransformationsmetoder (CLARITY9, SHIELD10). Clarity-metoden er især baseret på omdannelse af et intakt væv til en nanoporøs, hydrogelhybridiseret, lipidfri form, der gør det muligt at give høj gennemsigtighed ved selektiv fjernelse af membranlipiddobbeltlag. Især har denne metode vist sig at være vellykket også i hjertepræparat11,12,13,14. Men da hjertet er for skrøbeligt til at være egnet til en aktiv rydning, skal det ryddes ved hjælp af den passive tilgang, som kræver lang tid at give fuldstændig gennemsigtighed.
I kombination med avancerede billeddannelsesteknikker som lysarkmikroskopi har CLARITY potentialet til at afbilde 3D massivt hjertevæv ved mikrometrisk opløsning. I lysarkmikroskopi udføres belysningen af prøven med et tyndt lysark, der er begrænset i detektionsmålets fokusplan. Fluorescensemissionen opsamles langs en akse vinkelret på belysningsplanet15. Detektionsarkitekturen ligner widefield-mikroskopi, hvilket gør erhvervelsen meget hurtigere end laserscanningsmikroskoper. Flytning af prøven gennem lysarket gør det muligt at opnå en komplet tomografi af store prøver, op til centimeterstore prøver. På grund af den gaussiske stråles iboende egenskaber er det imidlertid kun muligt at opnå et meget tyndt (i størrelsesordenen nogle få mikron) lysark til en begrænset rumlig udvidelse, hvilket drastisk begrænser synsfeltet (FoV). For nylig er en ny excitationsordning blevet introduceret for at overvinde denne begrænsning og anvendt til hjernebilleddannelse, hvilket tillader 3d-rekonstruktioner med isotrop opløsning16.
I dette dokument præsenteres en passiv clearingmetode, der muliggør en betydelig reduktion af den clearingtid, der er nødvendig i henhold til CLARITY-protokollen. Den metodologiske ramme, der er beskrevet her, gør det muligt at rekonstruere et helt musehjerte med mikrometrisk opløsning i en enkelt tomografisk scanning med en anskaffelsestid i rækkefølgen af minutter.

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			<td>2-2&#8217; Thiodiethanol</td>
			<td>Sigma-Aldrich</td>
			<td>166782</td>
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			<td>Acrylamide</td>
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			<td>61-0140</td>
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			<td>AV-044 Initiator</td>
			<td>Wako Chemicals</td>
			<td>AVP5874</td>
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			<td>Sigma-Aldrich</td>
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			<td>Camera</td>
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			<td>Camera software</td>
			<td>Hamamatsu</td>
			<td>HC Image</td>
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			<td>Collimating lens</td>
			<td>Thorlabs</td>
			<td>AC254-050-A-ML</td>
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			<td>Detection arm</td>
			<td>Integrated optics</td>
			<td>0638L-15A-NI-PT-NF</td>
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			<td>Excitation lens</td>
			<td>Nikon</td>
			<td>91863</td>
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			<td>Extera&#236;nal quartz cuvette</td>
			<td>Portmann Instruments</td>
			<td>UQ-753</td>
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			<td>Fold mirrors</td>
			<td>Thorlabs</td>
			<td>BBE1-E02</td>
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			<td>Galvanometric mirror</td>
			<td>Thorlabs</td>
			<td>GVS211/M</td>
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			<td>Glucose</td>
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			<td>G8270</td>
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			<td>HCImage Live</td>
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			<td>4.6.1.19</td>
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			<td>Sigma-Aldrich</td>
			<td>H3375</td>
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			<td>Internal quartz cuvette</td>
			<td>Portmann Instruments</td>
			<td>UQ-204</td>
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			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P4504</td>
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			<td>Laser source</td>
			<td>Integrated Optics</td>
			<td>0638L-15A-NI-PT-NF</td>
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			<td>Thorlabs</td>
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			<td>Thorlabs</td>
			<td>KB25/M</td>
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			<td>MgCl2</td>
			<td>Chem-Lab</td>
			<td>CI-1316-0250</td>
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			<td>Motorized traslator</td>
			<td>Physisk Instrument</td>
			<td>M-122.2DD</td>
			<td></td>
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			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>59888</td>
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			<td>Objective</td>
			<td>Thorlabs</td>
			<td>TL2X-SAP</td>
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			<td>Paraformaldehyde</td>
			<td>Agar Scientific</td>
			<td>R1018</td>
			<td></td>
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			<td>Phosphate Buffer Solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
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			<td>Polycap AS</td>
			<td>Whatman</td>
			<td>2606T</td>
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			<td>Relay lens</td>
			<td>Qioptiq</td>
			<td>G063200000</td>
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			<td>Sodium Dodecyl Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>L3771</td>
			<td></td>
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			<td>Tube lens</td>
			<td>Thorlabs</td>
			<td>ACT508-200-A-ML</td>
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			<td>Tunable lens</td>
			<td>Optotune</td>
			<td>EL-16-40-TC-VIS-5D-1-C</td>
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			<td>Vacuum pump</td>
			<td>KNF Neuberger Inc</td>
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			<td>Water bath</td>
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			<td>WTB</td>
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mesoscopic optical imaging of whole mouse heart

Både genetiske og ikke-genetiske hjertesygdomme kan forårsage alvorlige ombygningsprocesser i hjertet. Strukturel ombygning, såsom kollagenaflejring (fibrose) og cellulær forskydning, kan påvirke elektrisk ledning, introducere elektromekaniske dysfunktioner og i sidste ende føre til arytmi. Nuværende prædiktive modeller af disse funktionelle ændringer er baseret på ikke-integrerede strukturelle oplysninger med lav opløsning. At placere denne ramme i en anden størrelsesorden er udfordrende på grund af ineffektiviteten af standardbilleddannelsesmetoder til udførelse af billeddannelse i høj opløsning i massivt væv. I dette arbejde beskriver vi en metodologisk ramme, der tillader billeddannelse af hele musehjerter med mikrometrisk opløsning. Opnåelsen af dette mål har krævet en teknologisk indsats, hvor fremskridt inden for vævstransformation og billeddannelsesmetoder er blevet kombineret. For det første beskriver vi en optimeret CLARITY-protokol, der er i stand til at omdanne et intakt hjerte til en nanoporøs, hydrogelhybridiseret, lipidfri form, der tillader høj gennemsigtighed og dyb farvning. Derefter beskrives et fluorescenslysarkmikroskop, der hurtigt kan erhverve billeder af et mesoskopisk synsfelt (mm-skala) med mikronskalaopløsningen. Efter mesoSPIM-projektet tillader det udtænkte mikroskop rekonstruktion af hele musehjertet med mikrometrisk opløsning i en enkelt tomografisk scanning. Vi mener, at denne metodologiske ramme vil gøre det muligt at afklare inddragelsen af cytoarkitekturens uorden i de elektriske dysfunktioner og bane vejen for en omfattende model, der overvejer både de funktionelle og strukturelle data, hvilket muliggør en samlet undersøgelse af de strukturelle årsager, der fører til elektriske og mekaniske ændringer efter vævsombygningen.

Den udviklede passive rydningsopsætning gør det muligt at opnå et ryddet voksenmusehjerte (med en dimension af ordren 10 mm x 6 mm x 6 mm) på cirka 3 måneder. Alle komponenter i opsætningen er monteret som vist i figur 1. Den ubetydelige temperaturgradient mellem hvert clearingkammer (i størrelsesordenen 3 °C) gør det muligt at opretholde temperaturen i et passende område på tværs af alle kamre.
<img alt="Figure 1" clas...

Watch this Scientific Journal Video about Mesoscopic Optical Imaging of Whole Mouse Heart at JoVE.com

Mesoscopic Optical Imaging of Whole Mouse Heart

Vi rapporterer en metode til mesoskopisk rekonstruktion af hele musehjertet ved at kombinere nye fremskridt inden for vævstransformation og farvning med udviklingen af et aksialt scannet lysarkmikroskop.

Mesoskopisk optisk billeddannelse af hele musehjertet

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition ...

Denne metode kombinerer forbedring af clearingprotokollen med øjets evne til optisk sektionering af Mesos PIM-teknologien, så vi kan udføre en meso scopic rekonstruktioner ved mikrometrisk opløsning. Det giver mulighed for at afbilde massivt hjertevæv eller hele musehjerter hele løsningen i en enkelt scanning uden at miste den oprindelige tredimensionelle vævsorganisation. Efter at hjertet er blevet kanyleret af proximal aorta vasket i tyroopløsning og fastgjort i 4%Paraform aldehyd i 0,01 molær PBS, er det klar til at starte clearingprotokollen.Vask hjertet tre gange i 0,01 molar PBS ved fire grader Celsius i 15 minutter. Efter dette trin kan hjertet opbevares i PBS og 0,01% natriumazid ved fire grader Celsius i flere måneder. Inkuber hjertet i 20 ml hydrogelopløsning i rystende tilstand ved 15 o / min i tre dage ved fire grader Celsius.Afgasning af prøven ved stuetemperatur ved hjælp af en tørretumbler, en vakuumpumpe og et rørsystem, der forbinder tørretumbleren med både pumpebåndet og nitrogenrørledningen. Anbring prøven i tørretumbleren, og åbn hætteglasset, mens hætten holdes på den. Luk tørretumbleren, og fjern ilten fra røret ved at åbne nitrogenrørledningen.Tænd for vakuumpumpen for at fjerne ilten fra tørretumbleren i 10 minutter. Sluk for pumpen, og åbn tørretumblerens knap til nitrogenrørledningen. Åbn derefter nitrogenhanen.Når trykket er lig med det atmosfæriske tryk, skal du forsigtigt åbne tørretumbleren og lukke hætteglasset hurtigt. Hold hjertet i den afgassede hydrogelopløsning ved 37 grader Celsius i ca. tre timer i ro. Når hydrogelen er korrekt polymeriseret og fremstår helt gelatinøs, skal du forsigtigt fjerne hjertet fra det og placere det i en rydningsopløsning i hætteglasset med rydningsopløsning.Skift rydningsopløsningen i hætteglasset en gang hver tredje dag for at fremskynde afklaringsproceduren. Når hjertet fremstår helt afklaret, fjern det fra hætteglasset og vask det i 50 ml opvarmet PBS i 24 timer i rystende tilstand. Vask igen i 50 ml af en X PBS T i 24 timer i rystende tilstand.Inkubere prøven i 0,01 milligram pr. milliliter hvedekim aglutinin Alexa gulv 633 i tre milliliter af en X PBS T i rystende tilstand ved 50 o / min ved stuetemperatur i syv dage. Efter syv dages inkubation vaskes prøven i 50 ml af en X PBS T ved stuetemperatur ved omrystning i 24 timer. Prøven inkuberes i 4% PFA i 15 minutter, og vaskes derefter tre gange i 0,01 molær PBS i fem minutter hver.Inkuber hjertet successivt i 20 og 47% TDE i 0,01 molær PBS i otte timer hver. Og så endelig ved 68% TDE i 0,01 molar PBS for at give det krævede brydningsindeks på 1,46. Fyld forsigtigt ca. 80% af den eksterne kvarts quvet med brydningsindeksmediet.Fyld den indre kvarts quvet med det samme brydningsindeksmedium. Dyp prøven inde i den indvendige quvet. Flyt forsigtigt prøven til bunden af quvet ved hjælp af tynd pincet og arranger hjertet med sin længdeakse parallelt med quvetens hovedakse for at minimere excitationslysbanen over vævet under scanningen.Fastgør forsigtigt det skræddersyede stik over den indvendige quvet med to skruer. Monter prøven til mikroskoptrinnet ved hjælp af magneter. Oversæt det lodrette prøvetrin manuelt for at nedsænke den interne quvet i den eksterne.Tænd excitationslyskilden med en bølgelængde på 638 nanometer, og indstil den til lav effekt i størrelsesordenen tre milliwatt. Flyt prøven ved hjælp af den motoriserede oversætter for at belyse en indre plade af vævet. Tænd for HC image live-kamerasoftwaren, og indstil kameraudløseren i ekstern kantudløserlysarktilstand for at drive kameraets anskaffelsesudløser ved hjælp af den brugerdefinerede software, der styrer hele opsætningen.Aktivér automatisk lagring i kamerasoftwaren, og indstil outputmappen, hvor billederne skal gemmes. Juster prøvepositionen manuelt i X Y-planet med de lineære oversættere for at flytte prøven til midten af kamerasensorens synsfelt. Flyt prøven langs Z-aksen ved hjælp af den lineære motoriserede oversætter for at identificere hjertegrænser til tomografisk rekonstruktion.Forøg lasereffekten til ca. 20 milliwatt, før du begynder til billeddannelsessessionen. Start den tomografiske erhvervelse ved at klikke på startknappen i optagelsespanelet i billedbehandlingssoftwaren, og begynd samtidig at flytte prøven langs Z-aksen med en konstant hastighed på seks mikrometer i sekundet ved hjælp af den motoriserede oversætter. Kombinationen af klarhedsmetoden med TDE som brydningsindeksmedium ændrer ikke prøvens endelige volumen væsentligt og fører heller ikke til en isotrop deformation af prøven.Excitationsoptikken producerede et lysark med et minimum spild på ca. seks mikrometer, der divergerede op til 175 mikrometer ved kanten af synsfeltet. Synkroniseringen af kameraets rulleskodde med den aksiale scanning af lysarkaffaldet sikrede, at emissionssignalet kun blev opsamlet i prøvedelen, der var ophidset med spild af lysarket, hvilket resulterede i et gennemsnitligt F W H M på ca. 6,7 mikrometer langs hele synsfeltet. Mikroskopets Z-punktspredningsfunktion blev også estimeret ved en tomografisk rekonstruktion af den fluorescerende nanosfære, og en F W H M på 6,4 mikrometer blev estimeret af pasformen.Systemets potentiale til at løse enkelte cellulære membraner i tre dimensioner med et tilstrækkeligt signal til støjforhold i hele organet blev også bekræftet. Afgasningsproceduren er det mest kritiske trin i protokollen. Hvis det ikke udføres korrekt, kan vævet støde på skader, der indikerer under inkubationen i en rengøringsopløsning.Den præsenterede protokol kan kombineres med en multifarvningsprotokol for at opnå en hel organrekonstruktion, der integrerer forskellige biologiske strukturer og kan anvendes til at studere patologiske modeller.

mesoscopic-optical-imaging-of-whole-mouse-heart

Research

JoVE Journal

Biology

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Kultur Fremgangsmåde til Embryonale Mouse Heart

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

Optisk Kortlægning af Intra-reticulum Ca<sup&gt; 2+</sup&gt; Og transmembrane potentiale i Langendorff-perfunderet kanin Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

Drosophila Forberedelse og Longitudinal Imaging of Heart Funktion in vivo Brug Optisk Kohærens mikroskopi (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...