AP 인정 교수 T Venkatesan, 감독, 누스 Nanoscience 및 나노기술 이니셔티브의 사무실 대리인 대통령 (싱가포르 국립 대학) (R-398-000-084-646)의 지원 합니다. CLD 지원의 싱가포르 교육부의 건강 국립 의료 연구 위원회 그것의 임상 과학자 자금 계획, NMRC/체코/035/2012, 그리고 싱가포르 국립 대학에서 인정합니다. Funders 연구 설계, 데이터 수집 및 분석, 결정 게시 또는 원고의 준비에 전혀 역할을 했다.

1. 센서 및 데이터의 수집의 제조
<ol>
	<li>니켈 금형의 준비
	<ol>
		<li>코트의 부정적인 전자 빔 220 nm 두께 층 600 µ m 두께 4에서 실리콘 웨이퍼에 저항. 전자 빔 리소 그래피 시스템을 사용 하 여이 웨이퍼에 디자인 된 nanohole 배열을 작성 합니다.<ol>
			<li>가속 전자 빔 쓰기, 각 300 µ m 필드 크기에 대 한 20 k의 낮은 dotmap (N)와 패턴 작성 (A) (즉, 거기 각 300 µ m2 지역에 매핑된 0.4 십억 점 고 각 점 안 전자 빔에 의해 노출도 됩니다 패턴 디자인에 따라). 110 µ C c m−2 에 저항에 대 한 전자 빔 레지스트 노출 복용량을 설정 하 고 800의 전류 (I)에 쓸 아빠 참고: 전자 빔 글에 노출 복용량 (D)에 의해 제어 됩니다 각 점 (T점), 계산에 대 한 노출 시간 <img alt="Equation 1" src="/files/ftp_upload/55597/55597eq1.jpg"> </img>. 110 µ C cm−2에서 노출 복용량에 대 한 노출된 각 도트에 전자 빔 집 시간은 0.5 µ s12입니다. 배열 1.8 m m2의 영역을 캡처, 이후 300 µ m2 필드 영역 양식 1 큰, 골드 nanohole 배열에 함께 물 렸 다의 36 패치 총이 있습니다.</li>
		</ol>
		</li>
		<li>10 개발자 솔루션에 4 인치 실리콘 웨이퍼를 immersing 하 여는 저항을 개발 s 고 건조 한 공기에 웨이퍼.</li>
		<li>실리콘 웨이퍼에 금속, 니켈, 구리, 알루미늄, 등의 씨앗 층을 예금 한다.</li>
		<li>니켈 믹 목욕에서 도금 시스템에서 웨이퍼를 전기 도금. 두 단계에 전기를 실시 합니다. 첫 번째 단계, 지속적인 95 분에서에서 0.7 A dm−2;의 전류 밀도 사용 하 여 이 완전히 니켈으로는 nanopatterns를 채웁니다. 두 번째 단계에서 125 분, 지속 사용 12 A dm−2 로 최종 니켈 금형 두께 300 µ m에 도달 (20 nm). PH 값 3.5-3.8에이 고 온도 52-54 ° c.</li>
		<li>부드러운 기계적인 힘을 적용 하 여 니켈 형 실리콘 기판에서 분리 합니다. 전자 빔 레지스트에서 잔류물을 씻어 하룻밤 긍정적인 감광 제 제거 시의 약 100 mL의 니켈 몰드를 담근 다.</li>
		<li>3 h. 위해 100 ° C에서 플라즈마 에칭 시스템 10 sccm에서 O2 가스와 100 W 3 분에에서 그것을 청소 피드 니켈 형 오븐에 건조.</li>
	</ol>
	</li>
	<li>골드 nanostructure의 제조
	<ol>
		<li>Heptadecafluoro-1,1,2,2-tetrahydrodecyl trichlorosilane (FDTS)의 150 µ L에서 니켈 형에 코트는 80 ° c.에 자기 조립 단층 (SAM) 코팅 기계 참고:이 nanoimprinting 단계 완료 후 감광 제 ("demolding")에서 금형의 분리 하면는 방지 접착 층을 형성할 것 이다. 기화 시간 180 이어야 한다 s, 반응 시간 되어야 900 s.</li>
		<li>에 4-10 막대기의 압력 및 40 ° C의 온도에서 10 분에 대 한 나노-imprinter을 사용 하 여 사진 치료할 수 없음의 300 nm 두께 층으로 코팅 된 유리 웨이퍼 저항 nanopatterns 인쇄물</li>
		<li>전송 형, 감광 제, 및 유리 웨이퍼 자외선 경화 시스템 및 30에 대 한 자외선 노출의 75 mW cm-2 와 photocure에 s. 참고: 만약 모든 단계를 올바르게 따른, 니켈 형 한다 쉽게 될 demolded는 감광 제에서.</li>
		<li>반응성 이온 에칭 (RIE) 시스템 수행 빈 엣지는 감광의 유리 기판 50 W 2에서 10 sccm의 O2 가스 흐름에 들여쓰기 영역에 유리를 노출 하는 s.</li>
		<li>전자 빔 증 착 기계에 유리 웨이퍼에 크롬 (Cr) 금속 접착을 위한 5 nm 두께 층 및 plasmonic 센서에 대 한 금 (Au)의 100 nm 레이어를 입금. 1의 증 착 속도 사용 하 여 Cr 및 2 Å s− 1 Au Å s− 1 .</li>
		<li>O2 플라즈마 에칭 3 분 뒤에 아세톤에 15-s sonification 단계는 감광의 이륙을 수행 합니다.</li>
		<li>샘플 5 m m × 5 m m 칩으로 주사위. Nanohole 배열에 중앙 2 m m × 2 m m 칩의 차지할 것입니다.</li>
	</ol>
	</li>
	<li>데이터의 수집
	<ol>
		<li>송신기 광 섬유의 끝을 통해 종료 하는 흰 빛의 광선 조명을 90 °에 센서 표면 (nanohole 배열)에 사고는 광학 측정으로 장치를 설정 합니다. 참고: 라이트 온 nanohole 배열을 통해 전송 됩니다.</li>
		<li>섬유 광학 수신기와 전송 된 신호를 수집 하 고 자외선 보이는 분 광 계 300 1000의 범위 내에서 운영으로 기록 nm.</li>
		<li>소음 측정에서 낮은 최종 스펙트럼을 얻기 위해 20 양 평균 100 프레임에 각 프레임에 대 한 수집 시간을 설정 합니다.</li>
		<li>플로팅 소프트웨어를 사용 하 여 이전에 확인 된 전송 봉우리 (로렌츠 기반 메서드를 사용 하 여)에 따라 데이터를 분석.</li>
	</ol>
	</li>
</ol>
2. 센서 대량 감도 테스트
<ol>
	<li>표준 리 액체는 RI에서에서 다양 한 1.31 1.39와 액체 셀으로 입금.</li>
	<li>표준 리 액체에 센서 칩을 담가 그리고 하얀 빛의 광선으로 그것을 정렬. 전송 스펙트럼을 얻습니다.</li>
	<li>표면 활성 청소 시 약 각 측정 후 센서 칩을 청소 하 고 질소 가스로 건조.</li>
</ol>
3. 센서 표면 수정
<ol>
	<li>이전에 어떤 화학 수정 소 프로 파 놀, 아세톤, 그리고 이온된 수에 순차 침수로 센서 칩을 청소. 건조 질소 가스의 흐름에 실 온에서 건조.</li>
	<li>0.4 m m 및 1.6 m m 10-카-1-decanethiol 1-octanethiol 12 시간 실 온에서의 ethanolic 솔루션에 센서 칩을 품 어. 참고:이 것입니다 양식을 아민 반응 자기 조립 단층 (SAM).</li>
	<li>에탄올을 사용 하 여 철저 하 게 헹 구 고 실 온에서 건조.</li>
	<li>75mm sulfo-N-hydroxysuccinimide (sulfo NHS)와 15 m m 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)의 혼합물을 확인 합니다. 이 혼합물을 15 분 동안에 칩을 담가. 참고:이 carboxylic 그룹 SAM 활성화할 것 이다.</li>
	<li>센서 표면에 pH 4.5 아세테이트 버퍼에서 200 µ g/mL 안티 분 항 체 솔루션의 50 µ L를 자리 하 고 30 분 동안 품 어.</li>
	<li>15 분 동안 1 M HCl 방법 솔루션에 센서 칩을 immersing 하 여 unreacted 에스테 르를 비활성화 합니다.</li>
	<li>이온을 제거 된 물으로 칩을 헹 구 고 실 온에서 건조 질소 가스의 흐름에 건조.</li>
</ol>
4입니다. cTnI 분석 결과
<ol>
	<li>어떤 비 특정 바인딩 표면에 1% 소 혈 청 알 부 민 (BSA) 솔루션의 100 µ L를 탐지 하 여 차단 합니다.</li></ol>15 분 동안 품 어.
	<li>(PBS) 염 인산 염 버퍼에 센서 칩 3 번을 씻어. 기록 전송 스펙트럼 측정 셀에 칩을 삽입 합니다. 참고:이 참조 스펙트럼 이다.</li>
	<li>칩 표면에 표준 cTnI의 50 µ L를 자리 하 고 30 분 동안 습 한 환경에서 품 어.</li>
	<li>3 회 PBS 솔루션에 센서 칩을 씻어 고 전송 스펙트럼 기록 측정 셀에 삽입 합니다. 참고: 이것은 후 바인딩 스펙트럼입니다.</li>
	<li>50mm 글리신-HCl (pH 2) 1 분 동안에 칩을 잠수함 고에 씻어 PBS 솔루션 세 번 재생성 칩 표면에. 재생 단계의 성공을 확인 하는 PBS에 전송 스펙트럼을 측정 합니다.</li>

5. 표면 플라스몬 공명 (SPR) 측정
<ol>
	<li>PBS-T 버퍼 SPR 시스템에서 다중화 된 SPR 센서 칩을 실행 합니다. 참고: PBS T 버퍼의 구성 이며 20 mM 나 인산, 150 mM NaCl, 0.05% 트윈 20. PH 7.4입니다.</li>
	<li>CTnI 표준 및 항 체을 사용 하 여 4 단계에서 설명한 대로.</li>
	<li>EDC (0.2 M) 및 sulfo NHS 5 분 수행 50 µ g/mL 항 체 560의 5 분 주입 (0.05 M)의 혼합물 및 5 분 주입 1 M HCl 방법 솔루션의 6 사용 가능한 채널 3을 활성화 합니다.</li>
	<li>센서 칩으로 90도 회전 하 고 다른 농도에서 cTnI 표준 주입 (75, 30, 7.5, 그리고 2.5 ng/mL).</li>
	<li>관찰의 SPR 판독을 통해 실시간으로에서 칩에 상호 작용에서 항 체를 활용 합니다.</li>
	<li>50mm 글리신-HCl (pH 2) 1 분을 주입 하 여 칩을 다시 생성 합니다.</li>
</ol>

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저자 아무 경쟁 금융 관심사 있다.

사건과 nanostructures 빛 사이 상호 작용을 시뮬레이션 그 변화는 분석의 농도의 기능으로 기록 될 수 있습니다 (전송 스펙트럼)에 적절 한 피크를 확인할 수 있습니다. 그 변화는 분석을 추적할 수 있습니다 오른쪽 밴드의 선택에 중요 한 센서의 구조에 대해 밴드의 지역화는 중요 하다. 시각화는 시뮬레이션을 통해 달성 될 수 있다. 이것은 또한 analytes의 바이오 센 싱을 수 있도록 하는 최적의 구조 ?...

특별 한 광학 전송 (EOT)에서 첫 번째 보고서 Ebbesen 외. 19981에 의해 출판 되었음 이후 화학 센서 nanohole 배열에 따라 수많은 조사의 주제 되었습니다. 빛 nanohole 구조의 서브 파장 크기의 주기적인 배열에 impinges, 향상 된 전송 특정 파장에서 발생 합니다. 이 사건 빛 블로흐 파 표면 polariton (BW-SPP) 또는 지역화 된 표면 plasmons (LSP)2커플 때 발생 합니다.
기본 물리 법칙 같은 정기적인 배열 바이오 센 싱은 간단 때 악용. 분자 또는 금속의 인터페이스 근처의 흡착 스펙트럼에서 전송 밴드의 위치를 이동 하는 차례로 금속 접촉 매체의 유전율을 변경 합니다. 나노-엔지니어링 모양, 크기, 및 분리 거리3,,45스펙트럼 자체를 조정할 수 있습니다. 디자인, 센서 EOT에 따라 분자 바인딩 이벤트의 조사 기간 동안 특정 할당6,,78 을 용이 하 게 그들의 스펙트럼에서 특성 밴드 있다. 이것은 중요 한 이점을 상용 표면 플라스몬 공명 (SPR) 플랫폼 이다.
센서 EOT를 사용 하 여 일반적으로 광학 조명을된 빔 감지 표면에 입사 되도록 정렬 광원을 포함 한다. 기술 공동 폴리머 템플릿 및 간섭 및 나노 리소 그래피, 등 대형 nanohole 서피스를 생성 하는 빈약한 재현성9. 정확 하 게 날조 EOT 현상 보여 큰 표면에 이러한 제한으로 인해 광학 현미경 광원 및 검출기를 올바르게 배치 하는 데 필요한 했다. 단순화 기술, 높은 품질 nanoimprinting 리소 그래피 (NIL)10 고용 되었다. 이 칩에 감지 표면에 대 한 보고 현미경에 대 한 필요성을 제거 대형 센서 표면11 (mm-규모)의 생산을 사용할 수 있습니다. 대신,이 센서는 표준 광섬유 케이블을 쉽게 인터페이싱 될 수 있습니다.
이 nanohole 배열에 대 한 전송 봉우리 근처-적외선 영역 (NIR)을 볼 수에 포함 됩니다, 그것은 완벽 하 게 수성 환경에서 생체에 대 한 바인딩 이벤트 감지에 적합 합니다. Nanohole 배열의 예상된 광 동작 시뮬레이션 했다. 결과 다음 표준 굴절 인덱스 (RI)의 액체와 연구를 통해 확인 됩니다. 이 배열 심장 분의 농도 측정 하는 데 사용 했다 나 (cTnI) 인간의 혈 청의 복잡 한 배경에서. cTnI는 급성 심근 경색의 진단 위한 임상 골드 표준입니다.
이 센서를 사용 하 여 감지 하 여 탐지 (LOD)의 0.55 ng/mL의 제한에서 인간의 혈 청에 cTnI는 임상 관련 계량 가능 하다. 검출은 가장 일반적으로 사용 되는 기술이 도메인, 효소 연결 된 immunosorbent 분석 결과 (ELISA) 보다 훨씬 더 빠른. 또한, 감지 표면 수 쉽게 생성 및 따라서 재사용. 따라서,이 작품 복잡 한 biofluids 내에서 바이오 센 싱에 대 한 가능한 포인트의 케어 (POC) 기술로 nanohole 배열의 약속을 보여 줍니다.

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using extraordinary optical transmission to quantify cardiac biomarkers in human serum

분석 결과 감도, 재현성, 및 인간 혈 청의 배경에 대해 analytes를 안정적으로 모니터링 하는 기능 포인트의 케어 (POC) 설정에서 임상 관련성을 바이오 센 싱 플랫폼에 대 한 중요 한 있습니다.
Nanoimprinting 리소 그래피 (NIL) 감지 영역 1.5 m m x 1.5 m m로 저렴 한 비용, 조작에 사용 되었다. 감지 표면 nanoholes, 약 140 nm2의 지역에 각각의 고 충실도 배열 되었다. 전무의 좋은 재현성 12 개별적으로 제조 된 표면에, 최소한의 칩 변형을 한 칩, 한 측정 전략을 가능 하 게. 이러한 지역화 된 nanoimprinted 표면 플라스몬 공명 (LSPR) 칩 안정적으로 bioanalyte에서에서 다양 한 2.5 biofluid에서 복잡 한 배경 가운데 75 ng/mL이 케이스, 인간 혈 청 농도 측정 하는 그들의 능력에 광범위 하 게 테스트 되었습니다. 없음 사용 차례로 필요가 현미경으로이 바이오 센서는 일반적으로 사용 가능한 실험실 광원 쉽게 인터페이스 될 수 있습니다 큰 감지 영역의 세대의 높은 정밀도. 이 바이오 센서에서에서 검색할 수 심장 분 혈 청 0.55 ng/ml, 탐지 (LOD)의 한도에서 높은 감도 가진 임상 관련입니다. 그들은 또한 낮은 칩 변화 (때문에 제조 프로세스의 높은 품질) 표시. 결과 널리 사용 되는 효소 연결 된 immunosorbent 분석 결과 (ELISA)와 commensurable-분석 실험을 기반으로 하지만 기술을 유지 한 LSPR 기반 감지 플랫폼 (즉, 적중이 소형화 및 멀티플렉싱, 그것의 장점 더 가능한 POC 응용 프로그램에 대 한).

측정을 위한 광 설치 그림 1A에 표시 됩니다. 실제 nanohole 배열의 이미지 그림 1B에서 제공 됩니다. 감지 프로세스를 운전 하는 물리학을 이해 하기 콤솔 시뮬레이션 소프트웨어는 수성 환경에서 plasmonic 필드의 분포를 시뮬레이션 하기 위해 사용 되었다. 시뮬레이션 결과 다음 실제 측정에 관련 되었다. 이전에 게시 연구 만든 가?...

Watch this Scientific Journal Video about Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum at JoVE.com

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

이 작품에서는 특별 한 광학 전송의 원리에 의해 작동 하는 높은-품질 감지 배열을 조작 하는 nanoimprinting 리소 그래피 방법을 설명 합니다. 바이오 센서 낮은-비용, 강력 하 고 사용 하기 쉬운, 그리고 심장 분을 검출할 수 있다 임상 관련 농도 (99번째 백분위 수 차단 ∼10-400 pg/mL, 분석 결과 따라)에서 혈 청에서 나.

특별 한 광학 전송 인간의 혈 청에 심장 Biomarkers 척도를 사용 하 여

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

using-extraordinary-optical-transmission-to-quantify-cardiac

Research

JoVE Journal

Bioengineering

6.2K 조회수.  National University of Singapore. 이 작품에서는 특별 한 광학 전송의 원리에 의해 작동 하는 높은-품질 감지 배열을 조작 하는 nanoimprinting 리소 그래피 방법을 설명 합니다. 바이오 센서 낮은-비용, 강력 하 고 사용 하기 쉬운, 그리고 심장 분을 검출할 수 있다 임상 관련 농도 (99번째 백분위 수 차단 ∼10-400 pg/mL, 분석 결과 따라)에서 혈 청에서 나.

비디오: Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

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 ...

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13 Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2 Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

실란 커플링 에이전트를 사용하여 실리카 광 Biosensors에 생물 학적 프로브를 부착

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

콘트라스트 계산하기 관류 고정 시편의 단층 촬영을 이용하여 인간의 심장 정맥 시스템의 해부학 적 재건

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

정의 인간 공학 심장 조직의 건설 심장 세포 치료의 메커니즘을 연구하는

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the light distortion during the propagation within the fiber. Our methodology is based on digital optical phase conjugation employing only a single spatial light modulator, where the optical wavefront is individually modulated at different regions of the modulator, one region per light signal. Digital optical phase conjugation approaches are considered to be faster than other wavefront shaping approaches, where (for example) a complete determination of the wave propagation behavior of the fiber is performed. In contrast, the presented approach is time-efficient since it only requires one calibration per light signal. The proposed method is potentially appropriate for spatial division multiplexing in communications engineering. Further application fields are endoscopic light delivery in biophotonics, especially in optogenetics, where single cells in biological tissue have to be selectively illuminated with high spatial and temporal resolution.

We demonstrate the transmission of multiple independent signals through a multimode fiber using wavefront shaping employing a single spatial light modulator. By modulating the wavefront for each signal individually, spatially separated foci are transmitted. Potential applications are multiplexed data transfer in communications engineering and endoscopic light delivery in biophotonics.

파면 정형을 이용하여 광섬유를 통해 다중 신호 전송

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

고립 된 뮤린 심실 근세포의 광학 이미징

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a key hemodynamic parameter related to cerebral oxygen supply. Resting state low frequency fluctuations based on oxygenation contrast have been shown to provide correlations between functionally connected regions. The presented protocol uses optical diffuse correlation spectroscopy (DCS) to assess blood flow-based resting state functional connectivity (RSFC) in the human brain. Results of CBF-based RSFC in human frontal cortex indicate that intra-regional RSFC is significantly higher in the left and right cortices compared to inter-regional RSFC in both cortices. This protocol should be of interest to researchers who employ multi-modal imaging techniques to study human brain function, especially in the pediatric population.

This protocol demonstrates how to measure resting state functional connectivity in the human prefrontal cortex using a custom-made diffuse correlation spectroscopy instrument. The report also discuss practical aspects of the experiment as well as detailed steps for analyzing the data.

광학 확산 상관 분광법을 사용하여 인간의 뇌의 대뇌 혈류 기반 휴식 상태 기능 적 연결

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

인간 조직형 심장 슬라이스의 이중 전압 및 칼슘 광학 매핑에 의한 전임상 심장 전극생리학 평가

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

인간 iPSC 유래 심근세포, 심장 섬유아세포 및 내피 세포를 사용하여 3D 심장 미세 조직 배열의 제조

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...