The authors thank Prof. Jan Grimm and Mr. Travis Shaffer for their helpful discussions and Mr. David Gregory for editing the manuscript. Technical services provided by the MSK Animal Imaging Core Facility, supported in part by NIH Cancer Center Support Grant P30CA008748-48, are gratefully acknowledged. The authors thank the NIH (K25 EB016673 and R21 CA191679, T.R. and 4R00CA178205-02, B.M.Z.), the MSK Center for Molecular Imaging and Nanotechnology (T.R.), the Tow Foundation (B.C.), and the National Science Foundation Integrative Graduate Education and Research Traineeship (IGERT 0965983 at Hunter College for B.C. and T.M.S.) for their generous support. The research reported in this publication was supported by funding from the King Abdullah University of Science and Technology.

윤리 문 : 승인 된 프로토콜과 기념 슬로안 케터링 암 센터 (MSK) 기관 동물 관리 및 사용위원회 (IACUC)의 윤리적 지침에 따라 따라 수행 된 기술 된 생체 내 동물 실험의 모든. Nebulizing 장치 1. 건설 <ol><li> 두 개의 나사 (X 25mm 2 ~ 4)를 사용하여 파트 B (12.7 X 10.7 X 1.8 cm 3)의 중앙에 수직으로 나무 부 A (12.5 X 2.5 X 1.8 cm 3)를 연결합니다. 나무 부품 C를 연결합니다 (11 × 2.5 × 1.8 cm 3) 파트 C (11 X 2.5 × 1.8 cm 3) 여전히 이동 될 수 있도록 하나의 나사를 사용하여 부품 A (12.5 X 2.5 X 1.8 cm 3)의 중간에. 그림 1을 참조하십시오. </li><li> 두 개의 루프, 하나에 하나를 형성하기를 통해 두 개의 구멍 플라스틱 3온스 미니 스프레이 (D)의 스프레이 병 트리거의 하단 끝을 통해 드릴 및 스테인레스 스틸 막대를 밀어 (1/16 &quot;강 10cm) (E) 트리거의 측면. </l난&gt; <li> 오프 미끄러지는 케이블 타이를 방지하기 위해 덕트 테이프 (F)와 분 무병의 저면 부분을 감싸. 두 개의 플라스틱 케이블 타이를 이용하여 나무 파트 C (11 X 2.5 × 1.8 cm 3) (28cm) (G)에 스프레이 병을 연결합니다. </li><li> 011 서보 모터 (I)를 잘라 및 서보 제어 (H)의 느슨한 케이블을 다시 연결합니다. 그런 다음, 덕트 테이프를 사용하여 나무의 부품 A (12.5 X 2.5 X 1.8 cm 3)의 정상에 서보 모터를 연결합니다. </li><li> 종이 클립 (K)를 사용하여 서보 모터 레버 연필 (J)를 부착. 단단히 플라스틱 피복 꼬임 선 (L)를 사용하여 강철 막대의 연필 루프의 가장 바깥 쪽 부분을 연결 덕트 테이프로 연필 단부를 고정. </li><li> 서보 모터 제어부의 자기 케이블 커넥터 (M)을 절단하고, 스피커 케이블 (N)으로 다시 연결. 이어서, 목재 부 B (12.7 X 10.7 X 1.8 cm 3)에 서보 모터 제어부 테이프. 반 자기 커넥터가있는 W1 케이블을 잘라 구리의의 느슨한 끝으로 한 부분을 첨부peaker 케이블 (1m). 사용 가능한 W1 케이블과 9V 배터리의 (자기) I2 토글 스위치 및 P1 전원을 연결합니다. </li></ol> [방법] 2. 감도 결정 <ol><li> 1.5 ML의 microcentrifuge 관에서 260 μg의 (347 nmol의), 52 μg의 (69 nmol의), 26 μg의 (34 nmol의)의 양 역삼 투 물 (100 μL)의 [Ru로 (BPY) 3] CL 2의 솔루션을 준비 5 μg의 (6.9 nmol의) 3 μg의 (3.5 nmol의) 520 NG (694 pmol의) 260 NG (347 pmol의) 52 NG (69 pmol의) 26 NG (34 pmol의) 5 NG (6.9 pmol의) 3 NG (3.5 pmol의). </li><li> 각 100 μL 믹스 [(RU BPY)을 3] CL 암모늄 세륨 질산염 수용액 100 μL 2 용액 ((NH 4)이 세륨 (NO 3) 6) 현미경 슬라이드에 물 (25 mM)을한다. </li><li> 이미징 소프트웨어를 초기화하여 생물 발광 리더의 인수를 설정합니다. <ol><li> 사용자 프로필에 로그인하고 아퀴를 찾습니다sition 제어판. &quot;초기화&quot;악기가 준비가 될 때까지 기다려야을 클릭합니다. </li><li> 선택 해제 &quot;영상 모드&quot;를 찾아 &quot;발광&quot;과 &quot;사진&quot;이 선택되어 있는지 확인하고 &quot;형광&quot;. </li><li> 변경 &quot;노출 시간&quot;20 초에 &quot;발광&quot;에 대한 설정. &quot;비닝을&quot;다음과 같이 &quot;발광&quot;에 대한 나머지 설정을 중간; &quot;F / 정지&quot;: 1; 그리고 &quot;배출 필터&quot;를 엽니 다. 주 : 노출 시간은 계측 및 사용 실험 설정에 따라 적용해야 할 수 있습니다, 제시된 설정 다른 경우. </li><li> &quot;노출 시간&quot;:은 &quot;사진,&quot;다음 설정을 사용하여 자동; &quot;비닝&quot;: 중간; 및 &quot;F / 정지&quot;8. &quot;필드보기의&quot;드롭 다운 메뉴에 대한 target.Look 이미징에 따라 &quot;제목 높이&quot;를 조정합니다. 초기 설정은 &quot;C.&quot;입니다 는 C 사이의 &quot;B&quot;로 변경 (14cm 거리amera 및 샘플 단계). </li></ol></li><li> 산화제로부터 보호하기 위해 상기 촬상 챔버의 바닥에 검은 건설 용지에 현미경 슬라이드를 배치하여 분무기를 설정한다. 의 100 μLdroplet 믹스 [Ru로 (BPY)을 3] CL 2 -solution 수용액 100 μL (NH 4) 2 세륨과 (NO 3) 6. 녹색 상자의 십자선을 준수하십시오. <ol><li> 흑색 도화지에 촬상 대상을 배치, 관심 영역은 녹색광 박스 십자선의 중심이되도록 시료 스테이지 상에 디스플레이. 나무 지원에서 플라스틱 스프레이 병을 분리하여 분무기를 준비합니다. 그 플라스틱 저장 용기에 (물 / 에탄올 (3) 1)과 나무 지원에 다시 연결 트리 에틸 아민 용액을 채 웁니다. </li><li> 생물 발광 리더 내부의 분무기를 놓고 전원 코드가 분무기 코드에서 분리되어 있는지 확인하십시오. 확인이 전원 스위치에이고, 토글 스위치가 꺼져 있고 빨간색 LED가 켜집니다 분무 노즐 헤드에 의해 촬상 대상을 향해 카메라 뷰 방해를 최소화하면서 분무 흐름 촬상 주제에 관심의 영역을 향해 가리키는되도록 분무기를 놓는다. </li><li> 스프레이에서 그들을 보호하기 위해 (현미경 슬라이드 또는 주사 부위에 예를 들어, 흰색 마크) 잠재적 인 핫스팟을 통해 건설 용지의 작은 검은 조각을 놓습니다. 장소는 촬상 피사체 분무기 또는 자성 도어 래치를 방해하지 않도록, 촬상 챔버 내부 분무기 리모컨 코드의 적어도 40cm. 이미징 시스템 덮개를 닫습니다. 참고 : 십자선에서 설정 &quot;보기의 필드&quot;에 따라 크기를 변경할 것 &quot;이미지 생활;&quot; 이것은 &quot;B&quot;로 설정되어 있는지 확인합니다. </li></ol></li><li> 상기 영상 시퀀스를 시작하여 화상을 취득. 에서 &quot;획득&quot;을 클릭 &quot;취득 제어판.&quot; 제 1 이미징에 sequ줬어, 원하는 (권장) 경우 자동 저장을 활성화하고 데이터 폴더를 선택합니다. 순서가 끝날 때까지 &quot;편집 영상 레이블&quot;대화 상자를 무시합니다. 주 : 제어 소프트웨어는 기기의 동작을 단계별로 실시간으로 표시됩니다. 측정 준비 및 오른쪽 위치에 시료 스테이지를 이동 한 후에는 카메라 셔터를 열고, 측정 시간을 카운트한다. 셔터 개구는 기계에 의해 생성되는 클릭 소리로 들릴 수있다. </li><li> 화학 발광을 생성하는 전환 스위치를 세 번 전환 : 셔터가 열리면, 트리 에틸 아민의 용액을 세 버스트 (물 / 에탄올 (3) 스프레이 버스트 당 0.24 ± 0.04 mL의 1)을 스프레이. 참고 : 샘플 단계는 측정하는 동안 이동합니다. 이 수 있도록 기기 내부에 충분한 (최소 40cm) 케이블을 둡니다. 이 솔루션은 상승 관에 의해 열망 할 수있는 분무기로 살포 할 것을 파이프에 기포가 없는지 확인합니다. 몇 가지 예비 BATTERIE이필요한 경우 준비 분무기에 대한의. </li></ol> 생체 이미징 3. 전신 정맥 주입 후 <ol><li> 1.5 ML의 microcentrifuge 튜브에서 [Ru로 (BPY) 3] 2 nmol의 CL 8 내지 33를 함유하는 인산 완충 식염수 (PBS) 용액을 100 μL를 준비한다. 수용액 (NH 4)이 준비 세륨 (NO 3)과 동시에 물 (25 mM)을 6. </li><li> 정맥 건강한 마우스의 꼬리 정맥에 100 μL [(RU BPY) 3] Cl2를 주입 (N = 5). </li><li> CO 2 질식 통해 주사 후, 마우스를 안락사 10 분. <ol><li> 다음 심장과 폐를 노출하는 U 자형의 늑골 아치를 제거, 몸통에서 Y 컷과 피부를 제거합니다. 좌심실 (18)에 24 게이지 바늘을 통해 PBS 20 mL로 우심방에 배출구를 절단 주사하여 마우스를 관류. 조심스럽게 배를 잘라피부와 신장과 간을 노출. 눈에 보이는 컷을 만드는 기관을 통해 길이 방향으로 잘라. </li></ol></li><li> 다음과 같이 변경하여, 단계 2.3-2.6에 설명 된대로 인수를 설정합니다. <ol><li> 철저하게 분무기 플라스틱 저장 용기를 세척 한 후, 대신에 트리 에틸 아민의 물 (NH 4) 2 용액 세륨 (NO 3) (6) (25 밀리미터)로 채운다. 참고 : 그것은 철저하게 결정화 때문에, 모든 사용 후 분무기 노즐을 세척하는 것이 중요하다 (NH 4) 2 세륨 (NO 3) 6 여러 사용 후 스프레이 노즐을 파괴 할 수있다. </li></ol></li><li> 이미지에 대한 전체 동물 또는 기관 샘플을 사용합니다. <ol><li> 전체 복부 이미징 카메라와 악기의이면 가리키는 헤드 대향 개방 복부와 마우스 커스 위치. 기관을 중심으로하는 녹색 빛 상자 십자선에 (예를 들어, 간 또는 신장)을 이미지화한다. </li><li> 에프또는 기관 이미징 및 정량 각각의 촬상 기기에서 마우스를 제거하고 아래에 핀. 이미 개방 체강부터 내부 기관 (예, 신장, 간, 폐, 근육, 비장, 뇌 및 심장)을 절제. 근육 조직을 절제하기 위해 뒷다리 피부를 잘라. 조심스럽게 뇌를 절제하기 위해 메스로 두개골을 엽니 다. <ol><li> 관심있는 기관은 간, 신장, 비장 인 경우, 길이 방향으로 절반의 모든 장기를 잘라 배양 접시 또는 검은 건설 용지의 조각에 각 기관을 배치합니다. </li></ol></li><li> 단일 기관에 대한 화학 발광 추적의 상대적 방출을 설정하는 단계 2.3-2.6에 설명 된 절차를 따르십시오. </li></ol></li></ol> 림프절 4. 생체 내 이미징 <ol><li> [(RU BPY) 3] Cl2를 80 nmol의 PBS를 함유하는 용액 10 μL를 준비한다. 의 수용액 (NH 4) 2 세륨을 준비 (NO 3) 6 워싱턴에서동시에 터 (25 mm)이다. </li><li> subdermally 건강한 쥐의 뒷발에 용액 10 μL를 주입 (N = 5). 음성 대조군으로, 순수한 PBS 10 μL와 반대쪽의 발을 주입. 주입 후 CO 2 질식을 통해 15 분 생쥐를 희생. 오금 림프절까지 림프 운하를 노출 모두 뒷 다리에 피부를 제거합니다. </li><li> 단계 3.4에 설명 된대로 인수를 설정합니다. </li><li> 양쪽 뒷다리에서 오금 림프절을 제거 절반을 절단하고 정량화하기위한 목적으로, (단계 3.5.3) 이전에 설명한 바와 같이, 페트리 접시에서 산화제로 스프레이. </li></ol>

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The authors have nothing to disclose.

여기서는 광학적 화학 발광 리포터에 의해 생성 된 광자의 방출을 통해 조직을 서술 할 수있는 기술을 제안 하였다. 다른 대조적으로, 더 많은 화학 발광이 리포터 시스템은 비 방사성 매우 높은 민감도 수준에서 검출을 용이하게하는 이미징 프로브를 이용하는, 기술, 4, 5, 6, 7, <...

최근 수십 년 동안, 이미징 기술은 의사들이 질병을 진단하고 모니터하는 방식을 혁명을 일으켰다. 이러한 영상 기술은, 그러나, 양전자 방출 단층 촬영 (PET), 단일 광자 방출 전산화 단층 촬영 (SPECT), 컴퓨터 단층 촬영 (CT) 및 자기 공명 영상 (MRI) 등의 전신 이미징 시스템에 크게 제한되어왔다. 특별한주의를 암에 지불 된, 기술 이미징 혁신은 크게이 질병 진단 및 치료하는 방식을 개선했습니다. 수술실 이러한 발전에도 불구하고, 이러한 영상 기술 단지에 맞지 않는 한 곳이있다. 전신 이미징 기술 수술 계획에 도움을 줄 수 있지만, 그들은 일반적으로 종양 조직을 모두 제거되었거나 잔류 종양 조직을 절제 한 숨겨져 남아 있는지 의사 실시간으로 판별 할 충분히 높은 공간 해상도가 부족하다. 더 침윤성는 것을 확인종양 마진이 남긴 가장 중요한 수술의 목표 중 하나이며, 외과 의사는 엄격하고 신중 조직 절제술 사이에 꽉 밧줄을 걸어 가야한다. 지나치게 제거하면, 환자에게 부작용이 악화된다; 너무 작은이 제거 된 경우, 재발률은 3이 증가한다. 따라서, 정확한 종양 마진을 묘사하는 것이 중요하며, 우리는 화학 발광 수술 촬상 다르게 설정 기술에 들키지 않고 남아 있었다 악성 조직의 시각화 외과 도와서 종양 마진의 확인의 정확성을 개선하는 데 도움이 될 수 있다고 믿는다. 현재 수술 이미징 시스템으로서의 가능한 유틸리티 조사되고 많은 영상 기술이있다. 이러한 β- 및 γ 방사선 방출 프로브 침 (4), 광 형광 5, 6 라만 분광법을 포함 </sup&gt;, 7, Cherenkov 발광 8,9. 그러나 현재까지 이들 중 어느 것도 표준 임상 도구로 설정되지되고있다. 광 형광 이미징 지금까지 이들 기술 중 가장 유망한 것으로 입증 가장 탐구 그러므로있다. 이미 많은 애플리케이션을위한 유용한 도구로 도시되었지만, 한계가없는 것은 아니다. 실제로, 그 주요한 결점은 본질적 autofluorescent 생체 조직에 의해 생성되는 배경 형광이다. 이 배경 autofluorescent 신호는 형광 신호의 발생을 위해 필요한 외부 광원에 의해 상기 형광 물질 외에, 주변 조직의 여기의 산물이다. 실용적인 관점에서 볼 때,이 형광도 잠재적으로 수술실에서이 기술의 유용성을 제한 할 수있는 낮은 신호 대 잡음비를 초래할 수있다. 교장형광 이미징 위에 화학 발광 이미징의 장점은 여기 광이 필요 없다는 것이다. 그 결과, 배경 형광도 없다. 화학 발광 이미지에서, 여기 에너지 대신에 화학적으로 생성된다. 이 프로세스는 높은 신호대 잡음비가 발생할 수 있으므로 의도하지 않은 배경 신호를 생성하지 않고. 이것은 궁극적으로 절제의보다 정밀하고 정확한 검출 될 수 있습니다. 다소 놀랍게도, 수술 중 영상 기술로이 방법의 유용성은 10 미개척 남아있다. 사실,이 기술에 가장 가까운 예는 마우스 (11), (12, 13)에 마이 엘 로퍼 옥시 다제에 의한 루미놀의 산화이다. 하이와 로우 백그라운드 신호 발생 (1) 최소 자기 형광 : 화학 발광 바이오 메디컬 이미징은 다음과 같은 장점을 제공 할 수있는 연구 오히려 비경 영역 그러므로gher 신호대 잡음비; (2) 표시 상태에서 근적외선 범위 화학 발광 배출량 가변 파장; 및 (3), 링커 기술과 함께 이미 존재하는 생체 분자를 표적으로하는 경우 표적 분자 이미징 프로브 (14)의 전체 라이브러리에 대한 액세스를 제공 functionalizable 화학 발광 착체. 이 원리 증명 연구는 루테늄 계 조영제를 이용한 생의학 속에서 화학 발광 이미징의 전위 유틸리티를 나타낸다. 이 화합물의 화학 발광 특성은 물론 1960 년대 중반 15 거슬러 올라가는 조사와 연구한다. 화학 활성화되면, 에이전트는 의료 촬상 목적에 적합 약 600 nm의 빛 (16)을 생성한다. 활성화 에너지는 여기 상태 물 17 -foll 650 NS의 수명이 리드 산화 환원 반응에 의해 제공된다이 여기 상태의 완화에 광자의 생성에 의해 빚진. 특수하게 설계된 원격 분무기를 사용하여, 우리는 두 화합물이 생체 외 및 생체 내에서 검출 할 수 있었다. 초기 실험 결과는이 기술의 추가 조사를 제안 매우 유망하다.

<table><tbody><tr><td>Wood part A (12.5 &amp;times; 2.5 &amp;times; 1.8 cm)</td><td>&amp;nbsp;Woodcraft</td><td>131404</td><td>Cut from a 3/4&amp;rdquo; x 24&amp;rdquo; x 30&amp;rdquo; birch plywood sheet</td></tr><tr><td>Wood part B (12.7 &amp;times; 10.7 &amp;times; 1.8 cm)</td><td>Woodcraft</td><td>131404</td><td>Cut from a 3/4&amp;rdquo; x 24&amp;rdquo; x 30&amp;rdquo; birch plywood sheet</td></tr><tr><td>Wood part C (11 &amp;times; 2.5 &amp;times; 1.8 cm)</td><td>Woodcraft</td><td>131404</td><td>Cut from a 3/4&amp;rdquo; x 24&amp;rdquo; x 30&amp;rdquo; birch plywood sheet</td></tr><tr><td>Screws (4 &amp;times; 25 mm)</td><td>Screwfix</td><td>79939</td><td></td></tr><tr><td>Harmon Face Values 3 oz mini sprayer</td><td>Bed, Bath and Beyond</td><td></td><td></td></tr><tr><td>stainless steel rod (10 cm of 1/16&amp;rdquo; steel)</td><td>Metals Depot Int. Inc.</td><td>2192</td><td></td></tr><tr><td>Pencil Classic HB</td><td>Papermate</td><td>58592</td><td></td></tr><tr><td>Paper clip</td><td>Office Depot</td><td>221720</td><td></td></tr><tr><td>speaker cable</td><td>RCA Inc.</td><td>AH1650SN</td><td></td></tr><tr><td>Energizer 9V alkaline battery</td><td>Energizer Holdings Inc.</td><td>EN22</td><td></td></tr><tr><td>Hitech HS-82MG Micro Servo Motor, 3.4 kg/cm output torque @ 6V</td><td>Hitech RCD USA Inc.</td><td>32082S</td><td></td></tr><tr><td>Name</td><td>Company</td><td>Catalog Number</td><td>Comments</td></tr><tr><td>28 cm plastic cable ties</td><td>General Electric Inc.</td><td>50725</td><td></td></tr><tr><td>Duct tape</td><td>3M Inc.</td><td>3939</td><td></td></tr><tr><td>littleBits w1 wire</td><td>littleBits Inc.</td><td>w1 wire</td><td></td></tr><tr><td>littleBits p1 power</td><td>littleBits Inc.</td><td>p1 power</td><td></td></tr><tr><td>littleBits i2 toggle switch</td><td>littleBits Inc.</td><td>i2 toggle switch</td><td></td></tr><tr><td>littleBits 011 servo</td><td>littleBits Inc.</td><td>011 servo</td><td></td></tr><tr><td>20 cm plastic covered wire twist ties</td><td>Four Star Plastics</td><td>71TIE8000</td><td></td></tr><tr><td>Tris(2,2&amp;prime;-bipyridyl)dichlororuthenium(II) hexahydrate</td><td>Sigma-Aldrich Inc.</td><td>224758</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Ammonium cerium(IV) nitrate</td><td>Sigma-Aldrich Inc.</td><td>22249</td><td></td></tr><tr><td>Isofluorane</td><td>Baxter Healthcare</td><td>1001936060</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>PBS1</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>2854</td><td></td></tr><tr><td>Triethylamine</td><td>Sigma-Aldrich Inc.</td><td>T0886</td><td></td></tr><tr><td>Water</td><td></td><td></td><td>Water was purified using a Milipore Mili-Q (R &amp;ge; 18 M&amp;Omega;)</td></tr><tr><td>Female nude (outbred) mice</td><td>Jackson Laboratories</td><td>1929</td><td>age 5 - 6 weeks</td></tr><tr><td>Strain C57BL/6J&amp;nbsp;&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>NU/J male mice at&amp;nbsp;</td><td>Jackson Laboratories</td><td>2019</td><td>age 6 &amp;ndash; 8 weeks</td></tr><tr><td>IVIS 200 bioluminescence reader</td><td>Caliper Live Science</td><td></td><td></td></tr><tr><td>Live Image 4.2 software</td><td>Perkin-Elmer</td><td>128165</td><td></td></tr><tr><td>Microscope slides</td><td>ThermoScientific</td><td>4951PLUS4</td><td></td></tr></tbody></table>

a novel technique for generating and observing chemiluminescence in a biological setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

프로토콜 제 1 절에서 설명 된 분무기 시스템을 저비용으로 용이하게 이용 가능한 재료로 구성 될 수있다. 원격 트리거 생물 발광 판독기 내부에 환원 / 산화제의 분사 (그림 1)에 대한 삽입 될 것입니다. 우리의 디자인은 렌즈로부터 14cm 거리에서 생물 발광 리더 내에서 분무기의 안전한 작동 할 수 있습니다. 렌즈의 김서림이나 흐려짐이 작동 동안 관찰되지 ?...

Watch this Scientific Journal Video about A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting at JoVE.com

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

생물학적 설정에서 생성 및 관측 화학 발광위한 새로운 기술

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

a-novel-technique-for-generating-observing-chemiluminescence

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

8.4K 조회수.  Memorial Sloan Kettering Cancer Center. This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

비디오: A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

마우스의 이미지 세균 감염에 루시페라제를 사용하여

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

연구

면역학 및 감염병학

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

제어 하 고 셀 상호 작용에서 유전자 표현 패턴을 분석 하는 Optogenetic 메서드

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

유전학

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

생체 듀얼 기판 발광 생물 이미징에

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

의학

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

제자리에서 측정 및 셀 밀도의 상관 관계 및 발광 박테리아의 발광

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

생화학

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

발광 생물 세균 이미징<em&gt; 생체 내</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast enhancement in vitro and in vivo. FUEL shares many of the same underlying principles as Bioluminescence Resonance Energy Transfer (BRET), yet greatly differs in the acceptable working distances between the luminescent source and the fluorescent entity. While BRET is effectively limited to a maximum of 2 times the F&ouml;rster radius, commonly less than 14 nm, FUEL can occur at distances up to &micro;m or even cm in the absence of an optical absorber. Here we expand upon the foundation and applicability of FUEL by reviewing the relevant principles behind the phenomenon and demonstrate its compatibility with a wide variety of fluorophores and fluorescent nanoparticles. Further, the utility of antibody-targeted FUEL is explored. The examples shown here provide evidence that FUEL can be utilized for applications where BRET is not possible, filling the spatial void that exists between BRET and traditional whole animal imaging.

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence FUEL

Expanding the foundation and applicability of Fluorescence by Unbound Excitation from Luminescence (FUEL) by surveying the relevant principles and demonstrating its compatibility with a multitude of fluorophores and antibody-targeted conditions.

브레 너머 단계 : 언 바운드 흥분에 의한 형광 발광에서 (연료)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast ...

생체공학

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate light-gated ion channels and pumps in neurons. While this bioluminescent optogenetics (BL-OG) approach confers a chemogenetic component to optogenetic tools, it is not limited to use in neuroscience. Rather, bioluminescence can be harnessed to activate any photosensory protein, thus enabling the manipulation of a multitude of light-mediated functions in cells. A variety of luciferase-luciferin pairs can be matched with photosensory proteins requiring different wavelengths of light and light intensities.
Depending on the specific application, efficient light delivery can be achieved by using luciferase-photoreceptor fusion proteins or by simple co-transfection. Photosensory proteins based on light-dependent dimerization or conformational changes can be driven by bioluminescence to effect cellular processes from protein localization, regulation of intracellular signaling pathways to transcription. The protocol below details the experimental execution of bioluminescence activation in cells and organisms and describes the results using bioluminescence-driven recombinases and transcription factors. The protocol provides investigators with the basic procedures for carrying out bioluminescent optogenetics in vitro and in vivo. The described approaches can be further extended and individualized to a multitude of different experimental paradigms.

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence-light emitted by a luciferase enzyme oxidizing a small-molecule substrate, a luciferin-can be harnessed to activate photosensory proteins, thereby adding another dimension to light stimulation and enabling the manipulation of a multitude of light-mediated functions in cells across temporal and spatial scales.

생체 발광 광유전학 2.0 : 생체 발광을 활용하여 체외 및 생체 내에서 광감각 단백질을 활성화 합니다.

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

루시 페라 제 Bioluminescence 기자를 사용하여 유전자 발현의 셀 자치 Circadian 시계 리듬을 모니터링

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

생물학

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

세포 독성의 지속적인 실시간 모니터링을위한 자율적으로 바이오 루미 네 센트 포유 동물 세포

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

생물학적 설정에서 생성 및 관측 화학 발광위한 새로운 기술

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