NIH R01 HL085369 verir.

1. Gelişmiş stok çözümler hazırlanması <ol><li> Tyrode çözümü (16x) iki stok çözelti deiyonize su önceden hazırlayın ve 4 saklayın ° C: <ol class="lalpha"><li> Menkul Kıymetler (119,872 g / L NaCl, 3.056 g / L CaCl 2 (2H 2 O), 5,6 g / L KCl, 2,6274 g / L NaH 2 PO 4, 3,408 g / L MgCl 2 (6H 2 O), ( Fisher Bilimsel, Fair Lawn, NJ)); </li><li> Stok II (26,88 gr / L NaHCO3 (Fisher Scientific, Fair Lawn, NJ)). </li></ol></li><li> Floresan boyalar stok çözümleri hazırlayın. Tekrarlanan dondurma ve çözme önlemek için, -20 ° C, bir deney için yeterli hem de boyaların 30 ul alikotları depolamak: <ol class="lalpha"><li> Gerilim duyarlı boya RH237 (Invitrogen, Carlsbad, CA) stok solüsyonu, 1.25 mg / ml dimetil sülfoksit (DMSO, Sigma, St Louis, MO); </li><li> Kalsiyum göstergesi Rhod 02:00 (Invitrogen, Carlsbad, CA) stok solüsyonu, DMSO içinde 1 mg / ml. </li></ol></li><li> Önceden eksitasyon-kasılma uncoupler blebbistatin stok solüsyonu hazırlayın (Bioscience Tocris, St Louis, MO, DMSO içinde 2 mg / ml) ve 4 çözünmüş blebbistatin saklamak ° C </li></ol> 2. Perfüzyon çözümleri ve deney düzeneği 7 hazırlayın <ol start="4"><li> Taze 2L Tyrode çözüm hazırlamak (128.2mM NaCl, 1.3mm CaCl 2 (2H 2 O), 4.7mM KCl, 1.05mM MgCl 2 (6H 2 O), 1.19mM NaH 2 PO 4, 20mm NaHCO 3 11.1mM D-glikoz deiyonize su, pH = 7.35 ± 0.05). Stok solüsyonu Tyrode çözümü Stok I Stok II, 125 ml, 125 ml (bir deney için yeterli) 1750 mL deiyonize su eklenir ve karıştırılır ve glikoz 4g 2L yapmak için kullanılan ediliyor. </li><li> Perfüzyon sistemleri iki pompa açın. 40 ml / dak retrograd perfüzyon için kullanılan peristaltik pompa (Peri-Star, TEFE, Sarasota, Amerika Birleşik Devletleri) ayarlayın. Superfusion için kullanılan diğer bir peristaltik pompa (Cole-Parmer Masterflex Peristalic Pompa L / S, Cole-Parmer Instrument Company, Vernon Hills, Illinois) ayarlayın ve holding rezervuar 80 ml / dk &#39;ya perfüzat geri dönmek için. </li><li> Perfüzyon sistemi 2L deiyonize su ile, sonra 30 dakika ve% 70 etanol ile yıkayın. </li><li> Tyrode çözümü tüm deiyonize su sirkülasyonunu ve 5 mikron filtre (Millipore, Billerica, MA, ABD) üzerinden geçmek, oda tahliye. +37 Için perfüzat Sıcak ° C O 2 / CO 2 (% 95 /% 5) gaz köpüren bir çözüm içine bir su ceketi ve sirkülatör (ThermoNESLAB EX7, Newtown, ABD) ve oksijen perfüzat. PH metre (Oakton Instruments, Vernon Hills, IL) ile çözeltinin pH Monitör ve 7.35 ± 0,05 pH tutmak için O 2 / CO 2 köpüren hızını ayarlayabilirsiniz. Deney sırasında izleme pH ve sıcaklık devam edin. </li><li> Çift optik eşleme cihaz yüksek uzaysal (100x100 piksel, 230 ± piksel başına 20 mikron) ve temporal (1,000-3,000 kare / sn) çözünürlük iki MICAM Ultima-L CMOS kameralar (SciMedia, Costa Mesa, CA) oluşur. Fix kalsiyum görüntüleme kameranın önünde bir band geçiren filtre (590 ± 15 nm, Thorlabs, Newton, NJ); süre, uzun bir-pass filtre (&gt; 700 nm, Thorlabs, Newton, NJ) yerleştirilmiş olması gerekir belirlenen gerilim görüntüleme kamera önünde. Kameralar dikroik ayna (635 nm kesme, Omega Optik, Brattleboro, VT) içeren bir tutucu, birbirlerine dik olarak düzenlenir. Çift kamera sahibi hemen altında kalp dikroik ayna üzerine gelen emisyon ışığı odaklanan bir lens (Nikon Nikkor 55 mm 1:1.4 235.052), yoktur. Çalışma mesafesi yaklaşık 3cm. Uyarma ışık, bir halojen lamba (Newport Oriel Instruments, Stratford, CT; SciMedia, Costa Mesa, CA) tarafından oluşturulan ve ısı filtre, obtüratör ve bant geçiren filtre (520 ± 45 nm) geçirilir. Esnek bir ışık kılavuzu hazırlanması üzerine bant geçiren filtre ışık yönlendirir ve bir deklanşör hazırlık boyaların photobleaching önlemek için sadece görüntü alımı sırasında ışığa maruz olduğunu sağlamak için kullanılır. </li><li> Pacing ve algılama için önceden hazırlayın ve Ag / AgCl 2 elektrot odasında kalp yerleştirmeden önce bunları yükleyin. Amplifikatörler ve filtreler uygun seviyelere ayarlanmış olduğundan emin olun. </li></ol> 3. , Fare kalp Hasat cannulate ve Langendorff perfüzyon kurdu <ol start="10"><li> Ve intraperitoneal heparin (100 adet), ketamin / xylazine (ksilizin, 10 mg / kg ketamin, 80mg/kg vücut ağırlığı) ile fare anestezisi. Ağrı refleksi eksikliği ile uygun bir anestezi düzeyi sağlayın. </li><li> Orta sternal insizyon sonra, hızlı kalp kaldırmak ve (% 95 O 2,% 5 CO 2) oksijenli yıkayın, sabit sıcaklığı (37 ± 1 ° C) Tyrode çözümü. </li><li> Diseksiyon mikroskop kullanarakAort hızla belirlemek ve sağ subklavian arter altında çıkan aortun boyunca temiz bir kesim yapmak. Aort kısa bir bölümü daha sonra özel bir bağlı alevlendi bir ucu ile 21 gauge kanül yaptı. 4-0 siyah örgülü ipek sütür (Cerrahi Uzmanlıkları Corporation, Reading, PA) kanül üzerine kalbi düzeltmek için kullanılır. Kanülasyon sonra, kalp retrograd perfüze ve Tyrode çözümü ile superfused. Bridge Amplifikatör TBM4M, TEFE, Sarasota, retrograd perfüzyon oranı aort basıncı 60 ile 80 mmHg (Basınç detektörler, Dünya hassas aletler Inc (TEFE), Sarasota, Amerika Birleşik Devletleri tutmak için 2-5 ml / dak aralığında ayarlanır. ABD). </li><li> Sonra kalp kanüle, akciğer, timus ve yağ dokusu disseke ve kaldırılır. </li><li> Izole kalp akışı bağlı hareket etmesini önlemek için perfüzyon odasının alt apeks (Sylgard kaplamalı) (Güzel Bilim Araçları) tutturulmuş. Sağ ve sol atriyal uzantıları da gergin ve kulakçıklar optik ölçümler için maksimum yüzey alanı sağlar odası, alt (Güzel Bilim Araçlar, Inc, Foster City, CA) tutturulmuş. Çok önemli! Küçük bir silikon tüp pulmoner venler ile sol ventrikül içine yerleştirilir ve yakındaki bağ dokusu ipek sütür ile sabittir. Bu çözüm tıkanıklık ve bir eksitasyon-kasılma uncoupler ile ventriküler kasılmaları bastırılması sonra özellikle önemlidir (bkz. Bölüm 4 adımda 19) sol ventrikül, sıkışıp perfüzat asitlenme önler. </li><li> Master-8 (AMP Instruments Ltd, Kudüs, İsrail) veya PowerLab 26T (AD Instruments, Sidney, Avustralya) tarafından oluşturulan pacing uyarılara yapmak için yapılmış özel bir elektrod kalbin yüzeyine yerleştirilir. </li><li> Küçük bir kapak cam, titreşimli bir çözüm hareket artifakı azaltmak için kalp üzerinde çözüm yüzey üzerine monte edilmiştir. </li><li> Odak kalp ikaz ışığı. Buna ek olarak, çift kamera aparatı ve maksimum çözünürlük elde edilecek, böylece kalp arasındaki mesafeyi ayarlamak. </li><li> Odadaki tüm ışıkları kapatın ve elektrik kayıtları PowerLab 26T kullanarak başlamak. </li></ol> 4. Yük gerilimi ve kalsiyum duyarlı boyalar ve eksitasyon-kasılma uncoupler <ol start="19"><li> Warm-up blebbistatin 0.6 ml. 0.5 mL holding rezervuar perfüzat blebbistatin karıştırın. Tyrode çözümü 1 mL blebbistatin kalan 0.1 mL seyreltilir ve kanül yakınında bulunan bir ilaç bağlantı noktası üzerinden yavaş yavaş (bir 20 dakikalık süre içinde) enjekte. Blebbistatin yavaş yavaş hareket artifakı azaltır gözlemleyin. </li><li> 1 ml Tyrode çözümü voltaj duyarlı boya RH237 stok solüsyonu 30 mcL sulandırınız ve yavaş yavaş blebbistatin aynı enjeksiyon portuna 5-7 dakika içinde enjekte. </li><li> 30 mcL kalsiyum göstergesi Rhod-02:00 1 ml Tyrode çözümü seyreltilir ve 5-10 dakika boyunca yavaş yavaş aynı enjeksiyon portu yoluyla uygulanan Pluronic F127 (Invitrogen, Carlsbad, CA, DMSO% 20 çözüm) ile karıştırılır ve 1:1 . </li><li> Blebbistatin ve hücre zarı ve sitoplazmada ulaşmak için boyalar için 5-10 dakika bekleyin. Hareket tamamen bastırıldı protokol ile devam edin. </li><li> Sürekli bütün prosedür üzerinden monitörü EKG kayıtları, kalbin normal elektrik fonksiyonunu sağlamak. </li><li> SciMedia özel yazılım (SciMedia, Costa Mesa, CA) kullanılarak floresan sinyal kayıtları başlar. </li></ol> 5. Temsilcisi sonuçları: <img alt="Şekil 1" src="/files/ftp_upload/3275/3275fig1.jpg" /> Şekil 1 perfüzyon, elektrik kayıtları ve optik haritalama için deneysel kurulum. EM = emisyon; Lp = longpass <img alt="Şekil 2" src="/files/ftp_upload/3275/3275fig2.jpg" /> Şekil 2 ventriküler pacing sırasında kaydedilen Deneysel hazırlama ve sinyal örnekleri. Sol: EKG sinyalleri 2 Ag / AgCl disk elektrotlar (En İyi) ve S1S1 stimülasyon protokolü bir örnek gösterilmiştir (Alt) toplanan. Merkezi: Langendorff fare kalp hazırlanması. Sağ: optik Temsilcisi aksiyon potansiyelleri ve kulakçıklar (Üst) ve ventriküllerin (Alt) kalsiyum geçici sinyalleri gösterilmiştir. Sarı ok (Üst), atriyal kayıtlarında görülmektedir ventriküller, gelen floresan sinyal saçılma noktaları. AG = sol ventrikül; RV = sağ ventrikül, LA = sol atriyum; RA = sağ atrium <img alt="Şekil 3" src="/files/ftp_upload/3275/3275fig3.jpg" /> Şekil 3 V m ve vahşi bir tür fare kalp ventriküller Cat Temsilcisi optik kayıtlar. A. optik kayıtları (C) görülebilmektedir siyah noktalar işaretlenmiş eşit aralıklı yerlerden bir dizi deney hazırlık. B. Örnek dizi merkezi bir konuma V m ve CAT (bkz. kutu izleme In (C)). C. V, eşit aralıklı yerlerden dizi m (mavi) ve CAT (kırmızı) . Sinyaller 3x3 binned edildi. <img alt="Şekil 4" src="/files/ftp_upload/3275/3275fig4.jpg" /> Şekil 4 Etkinleştirme harita ve iletim. A. enine (T) ve beyaz oklarla gösterilen boyuna (L) yönden vahşi bir tür fare kalp bir örnek aktivasyon haritası. B. V m sinyalleri (Üst) ve görülen üç puan dV / dt (Alt) karşılık gelen (T1, T2, T3, L1, L2, L3). <img alt="Şekil 5" src="/files/ftp_upload/3275/3275fig5.jpg" /> Şekil 5 Aksiyon potansiyeli ve kalsiyum geçici süre analizi. % 80% 80 gevşeme (CaD80) repolarizasyon (APD80) ve kalsiyum geçici süresi A. Aksiyon potansiyeli süresi haritalar kontrol koşulları altında bir kalp (Sol) ve 30 nM isoproterenol uygulama (Sağ) sonra gösterilmiştir. Ventriküller yeşil / sarı renk (Sağ) kısaltılmış isoproterenol APD80 ve CaD80 gösterir. Vahşi tip fare ventriküllerin (Üst) ve kulakçıklar (Alt) APD80 ve CaD80 B. Örnek iz.

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Çıkar çatışması ilan etti.

Bu deneyde bir eksitasyon-kasılma uncoupler ile ventriküler kontraksiyonlar bastırılması sonra çok önemli olan küçük bir silikon tüp ekleyerek Langendorff perfüzyon yöntemi değiştirilebilir. Silikon tüp çözüm tıkanıklığı, perfüzyon çözüm asitlenme, ve sol ventrikül iskemi gelişimini önlemek için kullanılır. Fare kalp hipotermi çok duyarlı, bu nedenle, kalp boyunca sıcaklık değişimleri aksiyon potansiyeli süresi suni farklılıklar neden olacaktır. Sonuç olarak, ısıtma sistemi, deney 8 tamamının sırasında 37 ° C sabit sıcaklıkta korumak için perfüzyon odasında hayata geçirildi . Langendorff modeli kalp innervasyon korumak olmadığından, fizyolojik, sempatik ve parasempatik 9 araştırmak için perfüzat nörotransmitterlerin ekleyerek dikkate almak gerekiyor . Retrograd perfüzyon yanı sıra, kalp superfusion ek, pH ve sıcaklık gibi uygun çevresel parametrelerin korumaya yardımcı olur. Bu yöntemde, Langendorff perfüze kalp yatay olarak yerleştirilmiştir. Dikey Langendorff perfüzyon kurulum 10 de kullanılıyor olabilir, ancak biraz daha farklı bir kardiyak mekaniği 11 neden olabilir. CMOS kameraların yanı sıra, alternatif dedektörler de mevcuttur ve haritası V m ve aynı anda CAT 12 uygulanabilir. Uygulama yüksek ayıklanmasıdır çözünürlüklü CMOS kameraların kayıtları doğruluğu garanti eder; ancak, optik haritalama sinyalleri tek bir hücre değildir. Aksine, her floresan sinyal optik büyütme bağlı olarak, yüzlerce ya da binlerce hücre geliyor. Çok daha büyük bir ventrikül floresan optik saçılma atriyal sinyallerini bozabilir, bu nedenle optik kaydedilen sinyalleri dikkatli yorumlanması gereklidir. Fare preparat başka bir sınırlama kalp 13 küçük boyutu nedeniyle yüzey eğriliği ile indüklenen sinyal bozulma ve gürültü. Iletim hızı ölçümleri sadece fare kalp eğriliği değil, aynı zamanda elektrot polarite ve sanal elektrotlar değişmiş olabilir. Iletim hızı, aktivasyon anizotropi ve repolarizasyon haritalar doğruluk elde etmek için, kalp yüzeyinde kamera odaklama düzeltmek şarttır. Bu yöntemde, gerçek zamanlı EKG kayıtları, kardiyak elektrofizyoloji optik soruşturma tamamlayabilir. Gerilim duyarlı boya (RH237) ve kalsiyum göstergesi (Rhod 02:00) hızlı yanıt nedeniyle protokol, benzer uyarma, ve farklı emisyon spektrumları 3,7 kullanılır. V m ve CAT RH237 dışında ve Rhod-02:00 3 ölçmek için kullanılan boyalar alternatif kombinasyonları vardır. Büyük bir Stoke vardiya (&gt; 200 nm) ile bir gerilim romanı duyarlı boya, PGHI, çünkü PGHI ve Rhod-02:00 14 arasında emisyon dalga boylarında büyük bir ayrılık daha iyi V m ve CAT sinyalleri izin bulundu. Gelecek gelişmeler yeni flüoresan millerini, yeni görüntüleme dedektörleri gelişimi, ve geliştirilmiş görüntü işleme yazılımı üzerine odaklanabilir. Yüksek çözünürlük ve 3 boyutlu optik haritalama için yeni optik görüntüleme yöntemleri de optik haritalama 5 gelecekte önemli yön vardır .

<table border="1"><tbody><tr><td> Kimyasal </td><td> Şirket </td><td> Katalog Numarası </td></tr><tr><td> NaCl </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> S271-1 </td></tr><tr><td> CaCl 2 (2H 2 O) </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> C79-500 </td></tr><tr><td> KCl </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> S217-500 </td></tr><tr><td> MgCl 2 (6H 2 O) </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> M33-500 </td></tr><tr><td> NaH 2 PO 4 (H 2 O) </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> S369-500 </td></tr><tr><td> NaHCO 3 </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> S233-3 </td></tr><tr><td> D-glikoz </td><td> Fisher Scientific, Fair Lawn, NJ </td><td> D16-1 </td></tr><tr><td> Blebbistatin </td><td> Bioscience Tocris Ellisville, MO </td><td> 1760 </td></tr><tr><td> RH237 </td><td> Invitrogen, Carlsbad, CA </td><td> S1109 </td></tr><tr><td> Rhod-02:00 </td><td> Invitrogen, Carlsbad, CA </td><td> R1244 </td></tr><tr><td> Pluronic F127 </td><td> Invitrogen, Carlsbad, CA </td><td> P3000MP </td></tr><tr><td> Dimetil sulphoxide (DMSO) </td><td> Sigma, St Louis, MO </td><td> D2650 </td></tr></tbody></table>

optical mapping of action potentials and calcium transients in the mouse heart

Fare kalbi, bu türün genetik mühendisliği için düşük maliyetli teknoloji varlığı nedeniyle kardiyovasküler çalışmalar için popüler bir model. Fare kalp Kardiyovasküler fizyolojik fenotipleme transmembran potansiyeli için çeşitli sondalar (V m), kalsiyum transientler (CAT) ve diğer parametreler istihdam floresan görüntüleme kullanarak kolayca yapılabilir. Tahrik olma-daralma kaplin, aksiyon potansiyeli ve hücre içi kalsiyum dinamikleri ile karakterize edilir; bu nedenle, kalp 1-4 aynı yerden aynı anda V m ve kedi Her iki harita derece önemli. Langendorff eş zamanlı optik haritalama perfüze fare kalpleri, kalp yetmezliği, aritmiler, metabolik hastalık ve diğer kalp hastalıklarının altında yatan mekanizmaları aydınlatmak için bir potansiyele sahiptir. Aktivasyon Görselleştirme, iletim hızı, aksiyon potansiyeli süresi ve sitelerin sayısız diğer parametreler hücresel düzeyde soruşturma elde edemez ama iyi optik haritalama 1,5,6 tarafından çözüldü. Bu yazıda, state-of-the-art CMOS görüntüleme teknolojisi kullanarak yüksek alansal ve zamansal çözünürlüğe sahip fare kalplerinde V m ve kedi aynı anda optik haritalama için enstrümantasyon kurulum ve deneysel koşullar mevcut Bu yöntem ile elde edilen tutarlı optik kayıtları Langendorff perfüze fare kalpleri eşzamanlı optik haritalama uygulanabilir ve güvenilir hem de göstermektedir.

Watch this Scientific Journal Video about Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart at JoVE.com

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

Bu kağıt sağlam izole Langendorff transmembran potansiyeli (Vm) ve geçici hücre içi kalsiyum (CAT) optik haritalama sırasında diseksiyon prosedürü, enstrümantal kurulum ve deneysel koşullar fare kalpleri perfüze.

Fare Kalp Aksiyon Potansiyelleri ve Kalsiyum Geçici Optik Haritalama

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

optical-mapping-action-potentials-calcium-transients-mouse

Research

JoVE Journal

Bioengineering

27.1K Görüntüleme.  Washington University in St. Louis. Bu kağıt sağlam izole Langendorff transmembran potansiyeli (Vm) ve geçici hücre içi kalsiyum (CAT) optik haritalama sırasında diseksiyon prosedürü, enstrümantal kurulum ve deneysel koşullar fare kalpleri perfüze.

Video: Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Üç boyutlu optik çözünürlük foto akustik Mikroskopi

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Biyomühendislik

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Çok parametreli Langendorff perfüze Tavşan Kalp Optik Haritalama

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

Hücre Fusion Tespit Cre-ETOX P Rekombinasyon Yaklaşım In Vivo</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

Yerli, Fibröz Uzun Aralığı ve Bölümlere Uzun Aralığı Kollajen in vitro sentezi olarak

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

Optik Algılama<em&gt; E. coli</emMezopor Silikon Biyosensörlerin tarafından&gt; Bakteri

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Parçacık izleme Microrheology tarafından Biyofilmler Mekanik Özelliklerinin Yerinde Haritalama

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D Etkileşimleri Çalışmaları 3D Kollajen jeller ve Mikrokanallarda Hazırlanması<em&gt; İn Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

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.

Yamultma stres gelişmekte olan zebra balığı kalp oransal etkileri 4 boyutlu görüntülerini yakalamak için ışık sayfalık floresans mikroskobu

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

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

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

Fare kalp çalışma için ışık sayfalık floresans mikroskobu

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

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.

Atriyoventriküler kalp kapakları biaxial mekanik karakterizasyonu

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

Methods Article

Fare Kalp Aksiyon Potansiyelleri ve Kalsiyum Geçici Optik Haritalama