NIH는 R01 HL085369을 부여합니다.

1. 재고 솔루션의 고급 준비 <ol><li> 탈이온수 사전에 Tyrode의 솔루션 (16X)의 두 재고 솔루션을 준비하고 4 저장할 ° C : <ol class="lalpha"><li> 증권 I (119.872 g / L NaCl, 3.056 g / L CaCl 2 (2 시간 2 O), 5.6 g / L KCl, 2.6274 g / L 아니 2 PO 4 3.408 g / L MgCl 2 (6H 2 O), (피셔 과학, 공정한 잔디, NJ)); </li><li> 주식 II (26.88 g / L NaHCO3 (피셔 과학, 공정한 잔디, NJ)). </li></ol></li><li> 형광 염료의 재고 솔루션을 준비합니다. 반복 동결 융해를 방지하기 위해, 우리는 -20 ° C, 한 실험에서 충분 두 염료의 30 μl aliquots를 저장할 : <ol class="lalpha"><li> 전압에 민감한 염료 RH237 (Invitrogen, Carlsbad, CA) 주식 솔루션, 디메틸 sulfoxide에서 1.25 MG / ML 솔루션 (DMSO, 시그마, 세인트 루이스, MO); </li><li> 칼슘 표시기로드 - 오전 2시 (Invitrogen, Carlsbad, CA) 주식 솔루션, DMSO 1 MG / ML 솔루션입니다. </li></ol></li><li> 사전에 (Bioscience Tocris, 세인트 루이스, MO, DMSO 2 MG / ML 솔루션) 여기 - 수축 uncoupler의 blebbistatin 주식 솔루션을 준비하고 4 해산 blebbistatin를 저장 ° C. </li></ol> 2. 재관류 솔루션 및 실험 설정 7 준비 <ol start="4"><li> 갓 (128.2mM NaCl, 1.3mM CaCl 2 (2 시간 2 O), 4.7mM KCl, 1.05mM MgCl 2 (6H 2 O), 1.19mM 아니 2 PO 4, 20mM NaHCO 3 11.1mM D - 포도당 2L Tyrode의 솔루션을 준비 탈이온수, 산도에 = 7.35 ± 0.05). 주식 솔루션은 Tyrode의 솔루션 (하나의 실험을위한 충분한) 주식 I, II 증권 125 ML 125 ML에 탈이온수 및 믹스의 1750 ML를하고, 포도당의 4g의 2L하기 위해 사용되는 경우. </li><li> 재관류 시스템의 두 펌프를 켭니다. 40 ML / 분 역행 재관류에 사용되는 연동 펌프 (사망 시에 스타, WPI, 사라소타, 미국)으로 설정합니다. superfusion에 사용되는 다른 연동 펌프 (콜 - Parmer Masterflex Peristalic 펌프 L / S, 콜 - Parmer 악기 회사, 버논 힐스, 일리노 이주) 설정 80 ML / 분 들고 저수지로 다시 perfusate을 반환합니다. </li><li> 2L 탈이온수과 함께 다음 30 분 및 70 %의 에탄올과 재관류 시스템을 씻으십시오. </li><li> 일단 모든 탈이온수은 Tyrode의 솔루션을 배포하고 5 μm의 필터 (Millipore, Billerica, MA, 미국)를 통해 통과, 챔버에서 철수합니다. 37에 perfusate을 따뜻하게 ° 솔루션으로 O 2 / CO 2 (95 % / 5 %) 가스를 품어하여 물 재킷 및 유통자 (ThermoNESLAB EX7, 뉴타운, 미국)와 산소 perfusate와 C. 산도 측정기 (Oakton 인 스트 루먼트, 버논 힐스, IL)과 솔루션의 산도를 모니터링하고 7.35 ± 0.05에서 산도를 유지하기 위해 O 2 / CO 2 버블링의 속도를 조정합니다. 실험 기간 동안 모니터링 산도와 온도를 계속합니다. </li><li> 듀얼 광학 매핑 장치는 높은 공간 (100x100 픽셀, 230 ± 픽셀 당 20 μm의)과 측두엽 (1,000-3,000 프레임 / 초) 해상도를 두 MiCAM 울티마 - L CMOS 카메라 (SciMedia, 코스타 메사, CA)로 구성되어 있습니다. 지정된 칼슘 이미징 카메라 앞에 대역 통과 필터 (590 ± 15 nm의, Thorlabs, 뉴턴, NJ) 수정, 동안, 긴 패스 필터 (&gt; 700 nm의, Thorlabs, 뉴턴, NJ)에 위치해야합니다 지정된 전압 이미징 카메라 앞에. 카메라 이색성 거울 (635 나노미터 컷오프, 오메가 광학, Brattleboro, VT)를 포함하는 보유자에 의해 서로 수직 정렬됩니다. 즉시 듀얼 카메라 홀더 아래 이색성 거울에 심장에서 나오는 방출 빛을 초점 렌즈 (니콘 NIKKOR 55mm 1:1.4 235,052)이 있습니다. 작동 거리는 약 3cm이다. 여기 조명은 할로겐 램프 (뉴 포트 출창 인 스트 루먼트, 스트 랫 포드, CT, SciMedia, 코스타 메사, CA)에 의해 생성되며 열 필터, 셔터, 그리고 대역 통과 필터 (520 ± 45 nm의)를 통해 전달됩니다. 유연한 라이트 가이드는 준비에 대역 통과 필터 조명을 지휘하고, 셔터는 준비가 염료의 photobleaching 피하기 위해 이미지를 수집하는 동안에만 빛에 노출되도록하는 데 사용됩니다. </li><li> 사전에 서성 거려 및 감지에 대한 자세 / AgCl이 전극을 준비하고 전에 마음을 배치하는 챔버에서 그들을 설치합니다. 앰프 및 필터가 적절한 수준으로 조정되었는지 확인합니다. </li></ol> 3. , 마우스 마음을 수확 cannulate하고, Langendorff 재관류를 설정 <ol start="10"><li> 그리고 intraperitoneal 주사로 헤파린 (100 대), 마취제 / xylazine (xylazine, 10 MG / kg bodyweight ketamin, bodyweight를 80mg/kg)으로 마우스를 마취. 통증 반응의 부족으로 마취의 적절한 수준을 약속드립니다. </li><li> 중간 흉골 절개 후, 신속하게 마음을 제거하고 (95% O 2 5 % CO 2) 산소에 씻어, 일정한 온도 (37 ± 1 ° C) Tyrode의 솔루션입니다. </li><li> 해부 현미경을 사용하여, 빠르게 대동맥를 파악하고 올바른 쇄골하 동맥 아래의 상승 대동맥에 걸쳐 깨끗한 절단합니다. 대동맥의 짧은 부분이 다음 사용자 지정에 연결된 것은 flared 팁 21 - 게이지 정맥했다. 4-0 검은 꼰 실크 봉합사는 (외과 특산품 주식 회사, 읽기, PA) 정맥에 심장 문제를 해결하는 데 사용됩니다. cannulation 후, 마음 retrogradely perfused하고 Tyrode의 솔루션을 superfused. 브리지 앰프 TBM4M, WPI, 사라소타; 역행 재관류 율이 대동맥 압력 60 사이에 80 mmHg를 (압력 변환기, 세계 정밀 계기 Inc의 (WPI), 사라소타, 미국 유지 2-5 ML / 분 범위에서 조정됩니다 미국). </li><li> 심장이 cannulated 후, 폐, thymus, 그리고 지방 조직은 다음 해부 및 제거됩니다. </li><li> 분리된 마음은 스트림 유도된 운동을 방지하기 위해 재관류 챔버 하단 꼭대기 (Sylgard의 코팅)에서 (파인 과학 도구) 박혀있다. 오른쪽과 왼쪽 심방 appendages도 늘어과 심방의 광학적 측정 최대한의 면적을 제공하는 챔버의 바닥에 (파인 과학 도구, Inc의 포스터 시티, CA) 고정된 있습니다. 매우 중요하다! 작은 실리콘 튜브가 폐동맥 혈관을 통해 좌심실에 삽입하고 인근 결합 조직에 실크 봉합사로 고정됩니다. 이것은 (파트 4 단계 19 참조) 여기 - 수축과 심실 uncoupler의 수축이 억제 후 특히 중요합니다 좌심실에 갇힌 perfusate의 솔루션 정체 및 산성화 방지합니다. </li><li> 사용자 정의 만든 전극 마스터 8 (AMP 인 스트 루먼트 (주), 예루살렘, 이스라엘) 또는 PowerLab 26T (AD 인 스트 루먼트, 시드니, 호주)에 의해 생성된 서성 거려 stimulations를 수행하기 위해 심장의 표면에 배치됩니다. </li><li> 작은 커버 유리가 진동 솔루션에서 모션 유물을 줄이기 위해 심장을 통해 솔루션의 표면에 고정됩니다. </li><li> 마음에 여기 빛을 집중. 또한, 듀얼 카메라 장치와 최대 해상도를 얻을 수 있도록 마음 사이의 거리를 조정합니다. </li><li> 객실의 모든 조명을 끄고 PowerLab 26T를 사용하여 전기 녹음을 시작. </li></ol> 4. 부하 전압과 칼슘에 민감한 염료 및 여기 - 수축 uncoupler <ol start="19"><li> blebbistatin의 따뜻한 - 최대 0.6 ML. 개최 저수지에서 perfusate와 blebbistatin 0.5 ML를 섞습니다. Tyrode의 솔루션 1 ML에 blebbistatin의 나머지 0.1 ML를 희석하여 천천히 정맥 부근 마약 포트를 통해 (20 분 동안)을 주사. blebbistatin이 점차 모션 유물을 줄여으로 관찰. </li><li> 1 ML Tyrode의 솔루션에서 전압에 민감한 염료 RH237 재고 솔루션을 30 μL를 희석하여 천천히 blebbistatin 같은 사출 포트에 5-7분 이상 주입. </li><li> 30 μL 칼슘 표시기로드 - 오전 2시 1 ML Tyrode의 용액에 희석하여 천천히 주사 같은 포트를 통해 50-10 분 이상 적용 후 Pluronic F127 (Invitrogen, Carlsbad, CA, DMSO의 20 % 용액)과 혼합하여 1시 1분입니다 . </li><li> blebbistatin과 세포막과 cytosol에 도달하는 염료에 대한 5-10분 나올 때까지 기다리는 것이 좋습니다. 모션은 완전 제거되는 프로토콜로 계속 진행합니다. </li><li> 지속적으로 모든 절차를 통해 모니터 ECG 녹음은 심장의 정상적인 전기 기능을 보장합니다. </li><li> SciMedia 맞춤 소프트웨어 (SciMedia, 코스타 메사, CA)를 사용하여 형광 신호 기록 하십시요. </li></ol> 5. 대표 결과 : <img alt="그림 1" src="/files/ftp_upload/3275/3275fig1.jpg" /> 그림 1. 재관류, 전기 녹음 및 광학 매핑을위한 실험 설정. EM은 = 방출, LP = longpass <img alt="그림 2" src="/files/ftp_upload/3275/3275fig2.jpg" /> 그림 2. 심실 서성 거려 동안 기록된 실험 준비 및 신호 예제. 왼쪽 : ECG 신호 자세 / AgCl이 디스크 전극 (상단)과 S1S1 자극 프로토콜의 예입니다 (아래)에서 표시됩니다 수집하고 있습니다. 센터 : Langendorff 마우스 심장 준비. 오른쪽 : 대표 광학 작업 잠재력과 심방 (위)과 심실 (아래)에서 칼슘을 과도 신호가 표시됩니다. 노란색 화살표 (위) 심방 기록에서 볼 수있는 심실에서 오는 형광 신호를 산란을 가리 킵니다. LV = 좌심실; RV = 우심실; LA = 좌심방, RA = 바로 안뜰 <img alt="그림 3" src="/files/ftp_upload/3275/3275fig3.jpg" /> 그림 3. V m과 야생 타입 마우스 심장의 심실에서 고양이의 대표 광학 녹음. 그의 광학 녹음 (C)에서 볼 수있는 검은 점들로 표시되어 균등하게 게재 위치의 배열과 대답 실험 준비. B. 예제 (상자가 나타 배열의 중앙 위치에서 V m과 고양이의 추적 IN (C)). 균등하게 게재 위치의 배열에서 C. V m (파란색) 및 CAT (빨간색). 신호는 3x3 binned되었습니다. <img alt="그림 4" src="/files/ftp_upload/3275/3275fig4.jpg" /> 그림 4. 활성화지도와 전도. 가로 (T)과 흰색 화살표로 표시 길이 (L) 방향과 야생 타입 마우스 마음에서 A. 예 활성화지도. B. V m 신호 (위) 및 본 세 지점 DV / DT (아래)에 해당 (T1, T2, T3, L1, L2, L3). <img alt="그림 5" src="/files/ftp_upload/3275/3275fig5.jpg" /> 그림 5. 액션 잠재력과 칼슘 과도 기간 분석. 80 % 휴식 (CaD80)에서 80 % repolarization (APD80)와 칼슘을 과도 기간에 A. 액션 잠재 기간이지도는 제어 조건 하에서 심장 (왼쪽)에서 30 나노미터 이소 프로 테레 놀 응용 프로그램 (오른쪽) 이후에 표시됩니다. 심실의 황색 / 녹색 색상 (오른쪽) 이소 프로 테레 놀은 APD80 및 CaD80을 단축 나타냅니다. 야생 타입 마우스 심실 (상단)과 심방 (아래)에서 APD80 및 CaD80의 B. 예제 tracings.

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+optical+mapping+of+intracellular+free+calcium+and+action+potentials+from+Langendorff+perfused+hearts.">Simultaneous optical mapping of intracellular free calcium and action potentials from Langendorff perfused hearts.</a> Curr Protoc Cytom. 12, 17-17 (2009).</li><li>Lou, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+panoramic+imaging+of+epicardial+electrical+activity.">Quantitative panoramic imaging of epicardial electrical activity.</a> Ann Biomed Eng. 36, 1649-1658 (2008).</li><li>Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+new,+long-wavelength,+voltage-sensitive+dyes+in+the+heart.">Properties of new, long-wavelength, voltage-sensitive dyes in the heart.</a> J Membr Biol. 208, 125-140 (2005).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이 실험에서 우리는 여기 - 수축과 심실 uncoupler의 수축이 억제 후 특히 중요합니다 작은 실리콘 튜브를 추가하여 Langendorff 재관류 방법을 수정했습니다. 실리콘 튜브는 솔루션 정체, 재관류 솔루션의 산성화 및 좌심실의 국소 빈혈의 개발을 방지하는 데 사용됩니다. 마우스 마음이 저체온증에 매우 민감합니다, 따라서 심장에 걸쳐 온도 변화는 행동 잠재력의 기간이에서 인공 차이가 발생할 수 있습니다. 따라서 난방 시스템은 실험 8 전체 기간 동안 37 ° C의 일정한 온도를 유지하기 위해 재관류 챔버에서 구현되었습니다. Langendorff 모델은 마음의 innervation을 유지하지 않기 때문에, 하나는 생리 동정심과 parasympathetic 톤 9 조사하기 위해 perfusate에 신경 전달 물질을 추가하는 것을 고려해야합니다. 역행 재관류 외에, 마음의 superfusion의 추가는 산도와 온도 등 적절한 환경 변수를 유지하는 데 도움이됩니다. 이 방법에서는 Langendorff perfused 마음이 수평으로 배치되었다. 수직 Langendorff 재관류 설정은 10도 사용할 수 있지만 약간 다른 심장 역학 11 발생할 수 있습니다. CMOS 카메라뿐만 아니라, 다른 메이커도 사용할 수 있으며,지도 V m 동시에 CAT 12 적용할 수 있습니다. 높은 spatio - 시간적 해상도의 CMOS 카메라 응용 프로그램은 레코딩의 정확성을 보장하지만, 광학 매핑 신호는 단일 세포에서되지 않습니다. 오히려 각각의 형광 신호가 광학 배율에 따라 세포의 수백 또는 수천에서 비롯됩니다. 더 큰 심실 형광 광 산란에 의해 심방 신호를 왜곡 수 있으므로 광학적으로 기록된 신호의 해석주의가 필요합니다. 마우스 준비의 또 다른 제한은 심장 13 작은 크기로 인해 표면의 곡률에 의해 유도된 신호 왜곡과 소음이다. 전도 속도 측정은뿐만 아니라 마우스 심장의 곡률에서뿐만 아니라 전극 극성과 가상 전극에서 변경할 수 있습니다. 전도 속도, 활성화 이방성 및 repolarization지도의 정확도를 달성하기 위해, 마음의 표면에 카메라의 초점을 수정하는 것은 필수적입니다. 이 방법에서는 실시간 ECG 녹음은 심장 전기 생리학의 광 조사를 보완할 수 있습니다. 전압에 민감한 염료 (RH237)과 칼슘 표시기 (로드 - 오전 2시)이 있기 때문에 자신의 빠른 응답 프로토콜 유사한 여기, 독특한 발광 스펙트럼 3,7에 사용됩니다. V m 고양이 RH237 이외 및로드 - 오전 2시 3 측정하는 데 사용할 수있는 염료의 다른 조합이 가능합니다. 대형 스토 크의 시프트 (&gt; 200nm)와 소설 전압에 민감한 염료, PGHI이 때문에 PGHI 및로드 - 오전 2시 14 층 사이의 방출 파장의 높은 분리의 더 나은 V의 m 고양이의 신호를 허용 발견되었습니다. 향후 개선 소설 형광 프로브, 새로운 영상 감지기의 개발, 향상된 이미지 처리 소프트웨어를 탐구에 초점을 수 있습니다. 높은 해상도와 3D 광학 매핑을위한 새로운 광학 이미징 modalities 또한 광학 매핑 5 중요한 미래의 방향입니다.

<table border="1"><tbody><tr><td> 화학 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> NaCl </td><td> 피셔 과학, 공정한 잔디, NJ </td><td> S271 - 1 </td></tr><tr><td> CaCl 2 (2 시간 2 O) </td><td> 피셔 과학, 공정한 잔디, NJ </td><td> C79 - 500 </td></tr><tr><td> KCl </td><td> 피셔 과학, 공정한 잔디, NJ </td><td> S217 - 500 </td></tr><tr><td> MgCl 2 (6H 2 O) </td><td> 피셔 과학, 공정한 잔디, NJ </td><td> M33 - 500 </td></tr><tr><td> 아니 2 PO 4 (H 2 O) </td><td> 피셔 과학, 공정한 잔디, NJ </td><td> S369 - 500 </td></tr><tr><td> NaHCO 3 </td><td> 피셔 과학, 공정한 잔디, NJ </td><td> S233 - 3 </td></tr><tr><td> D - 포도당 </td><td> 피셔 과학, 공정한 잔디, 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> 로드 - 오전 2시 </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> 디메틸 sulphoxide (DMSO) </td><td> 시그마, 세인트 루이스, MO </td><td> D2650 </td></tr></tbody></table>

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

마우스 마음이 수종의 유전 공학을위한 저비용 기술의 존재로 인해 심혈관 연구 인기있는 모델입니다. 마우스 심장의 심장 혈관 생리 phenotyping 쉽게 transmembrane 잠재력에 대한 다양한 프로브 (V M), 칼슘 과도 (CAT), 및 기타 매개 변수를 고용하는 형광 이미징을 사용하여 수행할 수 있습니다. 여기 - 수축 결합은 행동 잠재력과 세포 내 칼슘 역학의 특징이므로, 심장 1-4에서 같은 위치에서 동시에 V m 고양이 모두를지도에 매우 중요하다. Langendorff에서 동시 광학 매핑은 마우스 마음은 심장 마비, arrhythmias, 대사 질환 및 기타 심장 질환을 기본 메커니즘을 명료하게하다 수있는 잠재력이있다 perfused. 정품 인증을 시각화는 전도 속도, 작업 잠재 기간, 그리고 사이트의 수많은에 다른 매개 변수는 세포 수준 조사에서 얻을 수없는뿐만 광학 매핑 1,5,6에 의해 해결되었다. 본 논문에서 우리는 사용하는 최첨단 CMOS 이미징 기술.을 높은 spatio - 시간적 해상도 마우스 마음에 V m와 고양이의 동시 광학 매핑에 대한 계측 설치 및 실험 조건을 제시 이 방법으로 얻은 일관된 광학 녹음 Langendorff perfused 마우스 마음의 동시 광학 매핑이 가능하고 신뢰할 수있는 모두이다 보여줍니다.

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

그대로 고립 Langendorff에 transmembrane 잠재력 (VM)과 일시적인 세포 내 칼슘 (CAT)의 광학 매핑하는 동안이 종이 자세히 해부 절차를, 수단 설치, 그리고 실험 조건 마우스 마음을 perfused.

마우스 중심에 액션 후보와 칼슘 과도의 광학 매핑

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

27.0K 조회수.  Washington University in St. Louis. 그대로 고립 Langendorff에 transmembrane 잠재력 (VM)과 일시적인 세포 내 칼슘 (CAT)의 광학 매핑하는 동안이 종이 자세히 해부 절차를, 수단 설치, 그리고 실험 조건 마우스 마음을 perfused.

비디오: 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.

입체 광학 해상도 Photoacoustic 현미경

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

연구

생체공학

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.

Langendorff - perfused 토끼 하트의 Multiparametric 광 매핑

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.

세포 융합의 탐지를위한 Cre - 훈제 연어 P 재조합 접근법 VIVO에서</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.

기본, 섬유 긴 간격 및 Segmental 긴 간격 콜라겐의 체외 합성에

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).

의 광학 검출<em&gt; E. 대장균</em메조 포러스 실리콘 바이오 센서에 의해&gt; 박테리아

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.

입자 추적 Microrheology에 의한 바이오 필름의 기계적 성질의 제자리 매핑에서

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 상호 작용의 연구에 대한 3D 콜라겐 젤과 마이크로 채널 준비<em&gt; 생체</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.

개발 Zebrafish 마음에 전단 응력을 조절의 효과의 4 차원 이미지를 캡처 빛 시트 형광 현미경 검사 법

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.

Murine 심장 연구에 대 한 빛-시트 형광 현미경 검사 법

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.

Biaxial 기계 Characterizations 것 심장 밸브의

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