The authors thank Prof. Ralph Weissleder, Dr. Pat Zanzonico, and Dr. NagaVaraKishore Pillarsetty for helpful conversations and the NIH for funding (BMZ: 1K99CA178205-01A1)

윤리 선언문 : 설명 생체 내 동물 실험의 모든 승인 된 프로토콜과 기념 슬로안 케터링 암 센터의 기관 동물 관리 및 사용위원회 (IACUC)의 윤리적 지침에 따라 따라 수행 하였다. 의 Tz-BN-NOTA 1. 합성 <ol><li> 작은 반응 용기에 600 μL의 NaHCO3 완충액 (0.1 M, pH를 8.1) 7 MG-NH 2 -Bn NOTA (1.25 X 10-2 밀리몰)을 용해. 용액의 pH를 확인합니다. 필요한 경우, CO 3 0.1 M 나트륨이 적은 분취 량을 사용하여 8.1로 용액의 pH를 조정한다. </li><li> 1.7 ml의 microcentrifuge 관에 0.5 MG의 Tz-NHS (1.25 × 10 -3 밀리몰)에 NH 2 -Bn-NOTA 솔루션을 추가합니다. 참고 : (Tz)를-NHS는 건식 칭량 또는 건조 DMF 또는 DMSO (&lt;50 μL)의 원액에서 추가 할 수 있습니다. </li><li> 생성 된 반응 용액을 RT에서 30 분 동안 반응하도록 허용가벼운 교반. </li><li> 30 분 후, 반응이 -Bn NH-NOTA을 제거하는 역상 C 18 HPLC 크로마토 그래피를 사용하여 생성물을 정제. 의 Tz-NHS-BN과의 Tz-NOTA 가장 525 nm의 파장에서 모니터하면서 2 -Bn NH-NOTA는 254 nm의 파장에서 모니터 할 수있다. 참고 : 체류 시간은 분명히 각 실험실 (펌프, 열, 튜브 등)의 HPLC 장비 설치에 크게 의존한다. 그러나, 예를 제시하는 경우 0의 기울기 : (100)를 MeCN이 / H 2 O는 100 (모두 0.1 % TFA와 함께) : 0의 MeCN / H 2 O를 통해 25 분 및 분석 4.6 × 250mm의 C (18) 열 사용 ,의 Tz-BN-NOTA, Tz 일-NHS 및 NH (2)의 체류 시간은 -Bn-NOTA 각각 약 15 분, 16.5 분, 10 분, 것이다. 생성물 일층 또는 반 제조용 또는 C 18 분 취용 HPLC 컬럼을 사용하여 여러 개의 실행 중의 다른 성분의 반응으로부터 정제 할 수있다. 1 H-NMR, 항문ytical HPLC 및 ESI-MS는 완료의 Tz-BN-NOTA 전구체의 순도를 확인하는 데 사용할 수있는 모든 방법이있다. (11) </li><li> 액체 질소를 이용하여 수집 용 HPLC 용출액을 동결. </li><li> 불투명 한 알루미늄 호일에 얼어 붙은 수집 튜브를 감싸. </li><li> 동결 건조 선박 O에 얼어 붙은 포집 관을 배치 / N은 ​​HPLC 이동상을 제거합니다. </li><li> -80 ° C에서 어둠 속에서 정제 된 제품 (고체 밝은 분홍색)을 저장합니다. 참고 :이 절차의 허용 정지 점이다. 완료의 Tz-BN-NOTA 전구체는 이러한 조건 하에서 1 년 이상 안정하다. </li></ol> huA33-TCO 면역 접합체 2. 준비 <ol><li> 1.7 ml의 microcentrifuge 관에서, 1 ㎎ / ㎖ 건조 DMF에 TCO-NHS의 (2.7 mM)을 용액을 제조. </li><li> 1.7 ml의 마이크로 원심 분리 튜브에서, 2 ㎎ / ㎖의 인산 완충 생리 식염수를 1ml의 huA33 (13.3 μM) 용액의 pH 7.4 (0.01 M PO 4 3- 준비 </sub&gt; 0.154 M의 NaCl). </li><li> 작은 분취 량 0.1 M 나 2 CO 3 (&lt;5 μL)을 사용하여, 8.8-9.0로 항체 용액의 pH를 조정한다. pH를 모니터링하는 미세 전극으로 pH 종이 또는 pH 측정기를 사용하고, pH가 9.0 이상 오르지 않도록주의하십시오. </li><li> 항체 용액은 정확한 pH에서되면, 활성화 된 에스터 8 몰 당량에 대응하는 TCO-NHS 용액의 볼륨. 예를 들어, 2 ㎎ / ㎖ huA33 항체 용액 (1.33 × 10 -8 몰 huA33)의 1 ml의 1 ㎎ / ㎖ TCO-NHS 용액 (1.07 × 10 -7 몰 TCO-NHS)의 7.9 μl를 추가합니다. 용액의 부피 5 % DMF를 초과하지 마십시오. </li><li> 부드럽게 microcentrifuge 관을 여러 번 반전하여 솔루션을 섞는다. </li><li> 불투명 한 알루미늄 호일의 microcentrifuge 관을 감싸. </li><li> 이 솔루션은 온화한 교반과 함께 실온에서 1 시간 동안 배양 할 수 있습니다. </li><li> RT에서 1 시간 후, 사전 포장 된 일회용 SIZ를 사용하여 생성 된 면역 접합체를 정제전자 제외 탈염 열입니다. 보관시 컬럼에 존재하는 방부제를 제거하기 위해 공급자에 의해 설명 된 바와 같이 첫째, 크기 배제 열을 씻어. 그 후, 크기 배제 컬럼에 반응 혼합물을 추가 1.5 ㎖의 0.9 % 멸균 식염수와 열 린스,이어서 용 리제로서 0.9 % 멸균 식염수 2 ㎖를 사용하여 제품을 수집한다. 참고 :이 단계는 2 ml의 솔루션으로 완성 된 huA33-TCO를 얻을 것입니다. </li><li> 분광 광도계 UV-비스에서 얻어진 huA33-TCO의 농도를 측정한다. </li><li> 높은 농도가 요구되는 경우, 50,000 분자량 컷 - 오프로 원심 필터 유닛을 이용 huA33-TCO 용액을 농축시킨다. 주 : huA33 및 기타 잘 알려진 항체 (예를 들어, 베바 시주 맙, 트라 스투 주맙, 세툭시 맙 및 J591)의 다양한 집중되는 매우 관대 한 반면, 집계 및 침전이 다른 경우 농도에 따라 발생할 수 있음을 유의해야합니다. 연구진은이 proced 시도새로운 항체 올바는 문학 또는 항체를 집중할지 여부와 관련하여 문제가되는 항체의 자신의 지식을 신뢰해야합니다. </li><li> 어둠 속에서 4 ° C에서 완성 된 huA33-TCO의 면역을 저장합니다. 참고 :이 절차의 허용 정지 점이다. 완성 된 단클론 항체 접합체-TCO이 보관 조건에서 3 개월 이상 안정해야한다. </li></ol> 의 Tz-BN-NOTA 3. 64 구리의 방사선 참고 : 프로토콜의이 단계는 취급 및 방사능의 조작을 포함한다. 방사능과 다른 작업을 수행 - - 다음 단계를 수행하기 전에 연구자들은 자신의 집 기관의 방사선 안전 부서와상의해야합니다. 이온화 방사선에 대한 노출을 최소화하기 위해 가능한 모든 조치를 취합니다. <ol><li> 1.7 ml의 microcentrifuge 관에서 0.5 ㎎ / ㎖ (723 μM)의 Tz-BN-NOTA의 솔루션을 준비합니다. </li><li> 1.7 ML의는 MicroC에서entrifuge 튜브, 0.2 M NH 4 OAC의 pH 5.5 완충액 400 μL에의 Tz-BN-NOTA 용액 (5 μg의) 10 μl를 추가합니다. </li><li> 적절한 방사 화학 노트 유지의 관심에서 측정하기 전에 아래의 프로토콜 (3.4-3.8)에서 다음의 단계 후 투여 교정기를 사용하여 샘​​플에서 방사능의 양을 기록한다. 이 방사 화학적 수율의 정확한 결정에 도움이 될 것입니다. </li><li> 의 Tz-BN-NOTA 솔루션 (64) 구리의 2,000 μCi를 (74 MBq의)를 추가합니다. 주 : 일반적으로, [64의 Cu]의 CuCl 2는 0.1 N HCl을 소량 (&lt;30 μL)에 공급되고, 따라서 단지 소량은 (&lt;10 μL)이 스톡 용액은 방사성 표지 반응에 필요하다. [64의 Cu]의 CuCl 2 콘텐츠의 많은 양이 필요할 경우, 방사성 표지 반응은 전체 반응 부피를 증가 관대하다. 그러나, 방사성 표지 반응 용액의 pH를 보장하기 위해주의 깊게 모니터링되어야이는 pH가 4.0 이하로 떨어지지 않습니다. </li><li> 이 솔루션은 온화한 교반과 함께 실온에서 10 분 동안 품어하도록 허용합니다. </li><li> 인큐베이션 10 분 후, 역상 C 18 HPLC 크로마토 그래피를 사용하여 생성물을 정제. 체류 시간은 분명히 각 실험실 (펌프, 열, 튜브 등)의 HPLC 장비 설치에 크게 의존한다. 그러나, 예를 제공하는 경우의 구배 5:95의 MeCN / H 2 O 95 (모두 0.1 % TFA 사용) : 5의 MeCN / H 2 O 15 분 동안 사용하는, 64 CU-Tz-의 체류 시간 무료, 복합체를 형성하지 못한 64 Cu를 약 2-4 분에서 용매 전면으로 용출 반면 BN-NOTA은 약 9.8 분이어야합니다. </li><li> 회전 증발기를 사용하여, HPLC 용 출제를 제거. </li><li> 0.9 % 멸균 식염수에 64 CU-의 Tz-BN-NOTA 생성물을 재용. 주 : (64) 구리의 12.7 시간의 물리적 반감기를 고려할 때,이 절차의 허용 정지 점 없습니다. 64의 합성 CU-의 Tz-BN을-NOT 수행방사성 리간드의 주입 직전에, 스텝 4.5 즉시 3.7 단계에 따르십시오. </li></ol> 생체 Pretargeted PET 영상 4. 참고 : 프로토콜 제 3 절에서와 같이, 프로토콜의이 단계는 취급 및 방사능의 조작을 포함한다. 다음 단계를 수행하기 전에 연구자들은 자신의 집 기관의 방사선 안전 부서와상의해야합니다. 이온화 방사선에 대한 노출을 최소화하기 위해 가능한 모든 조치를 취합니다. <ol><li> 그리고 여자를 누드 마우스 피하 이식 1 × 6 SW1222 대장 암 세포에서하는 것은 이러한 (9~12일 접종 후) 100-150mm 3 이종 이식으로 성장 할 수 있습니다. (11) </li><li> 0.9 % 멸균 식염수에 0.5 ㎎ / ㎖의 농도로 제 2 프로토콜로부터 huA33-TCO 용액의 분취 액을 희석한다. </li><li> 이종 - 함유 마우스의 꼬리 정맥 내로 huA33-TCO 용액 200 μL (100 μg의)을 주입. </li><li> 마우스의 종양에서 huA33-TCO의 축적을위한 24 시간을 허용합니다. </li><li> 0.9 % 멸균 식염수에서 1.5 mCi의 / ml의 농도로 64 CU-의 Tz-BN-NOTA 방사성 리간드를 희석. </li><li> 의 꼬리 정맥 내로 (6.7 MBq의 / nmol의 특정 활성을 가정하면, 64 CU-의 Tz-BN-NOTA 1.6 nmol의; 11.1 MBq 내지 300 μCi를) 64 CU-의 Tz-BN-NOTA 방사성 리간드 용액 200 μl를 주입 이종 이식 베어링 쥐. </li><li> 촬상 원하는 시점에서 (예를 들어, 2, 6, 12, 또는 24 시간 후 분사), 2 % 이소 플루 란 마취 마우스 : 산소 가스 혼합물. </li><li> 작은 동물 PET 스캐너의 침대에 마우스를 놓습니다. 산소 가스 혼합물 : 1 % 이소 플루 란을 이용하여 스캔하는 동안 마취를 유지한다. 스캐너 침대에 동물을 배치하기 전에, 토​​우 - 핀치 방법을 사용하여 마취를 확인하고 마취 동안 건조를 방지하기 위해 마우스의 눈 연고 수의학 적용. </li><li> 정적 통해 마우스 PET 데이터를 획득350-700 keV의 에너지의 창 6 나노초의 일치 타이밍 윈도우를 사용하여 2000 만 일치 이벤트 최소 스캔. 이미지의 인수를 완료 한 후, 무인 마우스를 벗어나지 않아이 의식을 회복 할 때까지 다른 마우스와 케이지로 변경하지 마십시오. </li></ol>

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The authors have nothing to disclose.

이 PET pretargeted 촬상 전략의 주요 장점은 직접 표지 된 항체에 의해 생성 된 배경 방사선 량의 단지 일부만 타겟 간 배경 화상의 콘트라스트를 종양의 윤곽을 할 수 있다는 것이다. 예를 들어, 여기에 설명 된 대장 암 이미징 시스템에서, 급성 생체 분포 실험의 데이터는 표지 된 64 CU-NOTA-huA33 89 ZR-DFO-huA33 함께 64 Cu- 기 pretargeting 전략 선량 계산을 수행하는 데 사용 하였다. 임...

지난 30 년 동안, 양전자 방출 단층 촬영 (PET)는 암의 진단 및 관리에 필수적인 임상 도구가되었다. 항체는 긴 암 바이오 마커에 ​​대한 그들의 절묘한 친화력과 특이성으로 인해 종양 양전자 방출 방사성 동위 원소의 전달을위한 유망한 벡터를 고려하고있다. 항체의 1, 2 단, 상대적으로 느린 생체 내 약물 동태는 여러 날에 방사성 동위 원소의 사용을 의무화 물리적 반감기. 부분 바디 CT 스캔 달리 - - 조합은 환자의 비 - 표적 기관, radioimmunoconjugates 정맥 따라서 주입되기 때문에 특히 임상 적 의의이다 중요한 합병증 높은 방사선 량을 수득 할 결과를 흡입 투여로 신체의 모든 부분에서, 심문 조직에 관계없이. 이 문제를 우회하기 위해 상당한 노력을 develo의 전념하고있다동시에 내재 약동학 적 한계를 둘러싸하면서함으로써 항체의 유리한 특성을 활용, 방사성 동위 원소 및 표적 부분을 분리 PET 영상 전략의 pment. 이러한 전략은 - 가장 자주 pretargeting 또는 다단계 타겟팅라고 - 전형적으로 네 단계를 채택한다 : (1) 항원 및 방사성 리간드를 모두 결합 할 수있는 항체의 투여; (2) 표적 조직 및 혈액에서 그 간극에서 항체의 축적; (3) 작은 분자 방사성 리간드의 투여; 및 (4)를 초과하는 방사성 리간드의 급속한 냄. 일부 경우에 3-8 이어 항체에 방사성 리간드의 생체 결찰은 추가적인 소거 에이전트는 항체의 배설을 촉진시키기 위해 단계 (2)와 (3) 사이에 주입된다 그 종양을 결합 아직 혈액에 남아 있습니다. (5) 대체로, TW를 말하기pretargeting 전략의 O 유형은 문학에서 가장 널리 있습니다. 모두 전임상 모델에서 성공적으로 검증되었지만, 그들은 또한 임상 적용을 방해 한 키 제한을 갖는다. 첫 번째 전략은 스트렙 타비 딘 - 복합 항체와 비오틴 - 수정 방사능 표지 사이의 높은 친화력에 의존; 그러나, 스트렙 타비 딘 - 수정 항체의 면역 원성은 번역과 관련하여 우려되는 문제로 입증되었습니다. 5,6,9,10 두 번째 전략은, 대조적으로, 암을 모두 결합하는 유전자 조작 된 특이 적 항체를 사용 후자의 경로가 확실히 창조적 인 반면 바이오 마커 항원과 작은 분자 방사성 동위 원소 표지 합텐. 3,11-14은 광범위한 적용은 시스템의 모듈 방식의 복잡성, 비용 및 부족에 의해 제한됩니다. 최근에는 개발 (역 전자 수요 딜스 - 알더 I에 기초를 pretargeted PET 이미징 방법을 게시EDDA) 트랜스 -cyclooctene (TCO) 및 테트라 (Tz 일 사이의 고리 화 반응;.에 의해 예시 그림 1) (11) 반응 자체가 수십 년 동안 알려져있다 동안은 IEDDA 화학은 클릭 화학 바이오 콘쥬 게이션 기술로 최근 몇 년 동안 르네상스를 경험하고있다 랄프 Weissleder, 조셉 폭스, 펩티드, 항체 및 나노 입자의 형광 이미지 등을 포함한 12-15 IEDDA의 고리가 설정의 넓은 범위에서 적용되었다. 다른 중에서 피터 콘티의 기의 매혹적인 작품뿐만 아니라 핵 이미징 .. 27 bioorthogonal 클릭 화학의 종류의 숫자 동안 - 비판적 - radiohalogens 및 radiometals 모두 16-26 결찰은 항복 높은 깨끗하고 빠른 (K 1&gt; 30,000 M -1 초 -1), 선택하고, - 구리 촉매 아 지드 - 알킨 cycloadditions, 변형 승진 아 지드 - 알킨 cycloadditions, 그리고 슈타 우 딩거 LIG 손해 포함관리 포인트 -. 그것은 전체 유기체에서 응용 프로그램을 pretargeting에 매우 적합 IEDDA 화학을하게 빠른 반응 속도와 bioorthogonality의 고유 한 조합뿐만 아니라 bioorthogonal있는이 라인을 따라 28, 29, 그것의 최근 보고서 것이 중요합니다 우리의 실험실 pretargeting에 IEDDA 화학을 첫 적용되지 않았습니다. IEDDA와 pretargeted 영상의 첫 번째 보고서는 Rossin의 작업, 등에서 일어나 (111)에 표지 테트라를 사용하는 SPECT 방법론을 기능을 갖춘 30. 우리는 상술 한 바와 같이, pretargeting 방법은 네 가지 매우 간단한 단계 (그림 2). 손 프로토콜, 64 CU-NOTA 표지 된 방사성 리간드 및 테트라 huA33 항체의 TCO 변성 공액을 채용 대장 암 PET 이미징 pretargeted 전략을 설명한다. 그러나,이 방법의 궁극적 모듈화는 GR 중 하나입니다트랜스 -cyclooctene 잔기로서 eatest 자산은 비 내재화 항체에 추가 될 수 있고, 테트라 방사성 기자의 다양한 부착 될 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="565">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrazine NHS Ester</td>
			<td>Sigma-Aldrich</td>
			<td>764701</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trans-cyclooctene NHS Ester</td>
			<td>Sigma-Aldrich</td>
			<td>764523</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p-NH2-Bn-NOTA</td>
			<td>Macrocyclics</td>
			<td>B-601</td>
			<td>Store at -80 &#176;C</td>
		</tr>
	</tbody>
</table>

harnessing the bioorthogonal inverse electron demand diels-alder cycloaddition for pretargeted pet imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a 64Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with 64Cu or 89Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the trans-cyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes.

실험의 처음 세 단계 -의 Tz-BN-NOTA, huA33에 TCO의 접합의 합성과의 Tz-BN-NOTA의 방사성 표지는 (도 3 및도 4) 구성 - 매우 안정적. 상기 절차의 경우의 Tz-BN-NOTA 구조체는 고 수율 및 고순도로 합성 하였다. ~ huA33 항체는 4.2 ± 0.6 TCO / 단클론 항체로 수정하고, Tz 일-BN-NOTA은&gt; 85 % 붕괴 보정 수율, 그리고 특정 활동&gt; 99 % 방사 화학적 순도 정제 된 방사성 리간드를 얻을 수 (64) 구리와 방...

Watch this Scientific Journal Video about Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging at JoVE.com

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.

Pretargeted PET 영상에 대한 Bioorthogonal 역 전자 수요 딜스 - 알더 고리 모으고

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for ...

harnessing-bioorthogonal-inverse-electron-demand-diels-alder

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JoVE Journal

Bioengineering

8.7K 조회수.  Memorial Sloan Kettering Cancer Center. The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand. 

비디오: Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

의 주문형 정량 분석​​을위한 미세 유체 기반 Electrotaxis<em&gt; Caenorhabditis의 엘레</em&gt; &#39;로코

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

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생체공학

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

형광 현미경과 영상 셀 마이그레이션을위한 접착제 및 가용성식이 그라디언트를 만들기

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

위한 도구로 리포좀 캡슐화 된 근적외선 형광의 형광 담금질<em&gt; 생체</em&gt; 광학 이미징

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

빠른 스캔 전자 스핀 공명 이미징 생리 학적으로 중요 매개 변수에 대한 새로운 길을 엽니 다<em&gt; 생체</em

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

상호 빛 전자 현미경 검사 법 (클레 멘 타인) 추적 하 고 바이러스 성 단백질을 이미징 관련 Cryo 움직일 셀 구조

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various factors affect the relaxation rates, such as buffer composition, solution pH, temperature, and magnetic field. In this last regard, the spin-lattice relaxation time can be measured at clinical field strengths, but at lower fields, where these compounds are dispensed from the polarizer and transported to the MRI, the relaxation is even faster and difficult to measure. To have a better understanding of the amount of magnetization lost during transport, we used fast field-cycling relaxometry, with magnetic resonance detection of 13C nuclei at ~0.75 T, to measure the nuclear magnetic resonance dispersion of the spin-lattice relaxation time of hyperpolarized [1-13C]pyruvate. Dissolution dynamic nuclear polarization was used to produce hyperpolarized samples of pyruvate at a concentration of 80 mmol/L and physiological pH (~7.8). These solutions were rapidly transferred to a fast field-cycling relaxometer so that relaxation of the sample magnetization could be measured as a function of time using a calibrated small flip angle (3&#176;-5&#176;). To map the T1 dispersion of the C-1 of pyruvate, we recorded data for different relaxation fields ranging between 0.237 mT and 0.705 T. With this information, we determined an empirical equation to estimate the spin-lattice relaxation of the hyperpolarized substrate within the mentioned range of magnetic fields. These results can be used to predict the amount of magnetization lost during transport and to improve experimental designs to minimize signal loss.

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates.

과분극[1-13C] 피루바테의 스핀 격자 이완 자기장 의존도 측정

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different species. The octopus is one of these exceptional animals that has given scientists new insights into the fields of neuroscience, robotics, regenerative medicine, and prosthetics. Research with this species of cephalopods requires the set-up of complex facilities and intensive care for both the octopus and its ecosystem that is critical for the project&#39;s success. This system requires multiple mechanical and biological filtering systems to provide a safe and clean environment for the animal. Along with the control system, specialized routine maintenance and cleaning are required to effectively keep the facility operating long term. It is advised to provide an enriched environment to these intelligent animals by changing the tank&#39;s landscape, incorporating a variety of prey, and introducing challenging tasks for them to work through. Our results include MRI and a whole-body autofluorescence imaging as well as behavioral studies to better understand their nervous system. Octopuses possess unique physiology that can impact many areas of biomedical research. Providing them with a sustainable ecosystem is the first crucial step in uncovering their distinct capabilities.

Understanding the unique physiological and anatomical structures of octopuses can greatly impact biomedical research. This guide demonstrates how to set-up and maintain a marine environment to accommodate this species and includes state-of-the-art imaging and analytical approaches to visualize octopus' nervous system anatomy and function.

생물 의학 및 생물 공학 연구를 위한 문어 생태계 구축

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different ...

Bioorthogonal Chemical Imaging of Cell Metabolism Regulated by Aromatic Amino Acids

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, an abundant supply of AAAs is important for cancer cells to maintain their rapid growth and division. With this, there is a rising demand for a highly specific, noninvasive imaging approach with minimal sample preparation to directly visualize how cells harness AAAs for their metabolism in situ. Here, we develop an optical imaging platform that combines deuterium oxide (D2O) probing with stimulated Raman scattering (DO-SRS) and integrates DO-SRS with two-photon excitation fluorescence (2PEF) into a single microscope to directly visualize the metabolic activities of HeLa cells under AAA regulation. Collectively, the DO-SRS platform provides high spatial resolution and specificity of newly synthesized proteins and lipids in single HeLa cell units. In addition, the 2PEF modality can detect autofluorescence signals of nicotinamide adenine dinucleotide (NADH) and Flavin in a label-free manner. The imaging system described here is compatible with both in vitro and in vivo models, which is flexible for various experiments. The general workflow of this protocol includes cell culture, culture media preparation, cell synchronization, cell fixation, and sample imaging with DO-SRS and 2PEF modalities.

We present a protocol to directly visualize metabolic activities in cells regulated by amino acids using deuterium-oxide (heavy water D2O) probed stimulated Raman scattering (DO-SRS) microscopy, which is integrated with two-photon excitation fluorescence microscopy (2PEF).

방향족 아미노산에 의해 조절되는 세포 대사의 생체 직교 화학 이미징

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, ...

가상 현실 상호 작용을 통한 제브라피시 심장 수축 분석

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for gaining insights into structural and functional changes in response to injuries. However, obtaining high-resolution and high-speed 4-dimensional (4D, 3D spatial + 1D temporal) images of the zebrafish heart to assess cardiac architecture and contractility remains challenging. In this context, an in-house light-sheet microscope (LSM) and customized computational analysis are&#160;used to overcome these technical limitations. This strategy, involving LSM system construction, retrospective synchronization, single cell tracking, and user-directed analysis, enables one to investigate the micro-structure and contractile function across the entire heart at the single-cell resolution in the transgenic Tg(myl7:nucGFP) zebrafish larvae. Additionally, we are able to further incorporate microinjection of small molecule compounds to induce cardiac injury in a precise and controlled manner. Overall, this framework allows one to track physiological and pathophysiological changes, as well as the regional mechanics at the single-cell level during cardiac morphogenesis and regeneration.

저자 스포트라이트: 심장 기능의 단일 세포 분석을 위한 제브라피시의 고해상도 4D 광시트 이미징 및 가상 현실

This protocol utilizes light-sheet imaging to investigate cardiac contractile function in zebrafish larvae and gain insights into cardiac mechanics through cell tracking and interactive analysis.

제브라피시 심장 수축의 4D 광시트 이미징

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

Methods Article

Pretargeted PET 영상에 대한 Bioorthogonal 역 전자 수요 딜스 - 알더 고리 모으고