Name: observation and analysis of blinking surface-enhanced raman scattering
Uploaded: 2018-01-11T14:00:00.000+00:00
Duration: 5 min 52 s
Description: Watch this Scientific Journal Video about Observation and Analysis of Blinking Surface-enhanced Raman Scattering at JoVE.com

저자는이 작품의 그들의 유익한 토론에 대 한 교수부터 Y. 오자키 (간사이 학원 대학) 및 박사 T. 이토 (국립 연구소의 첨단 산업 과학 및 기술)을 감사합니다. 이 작품의 교육부, 문화, 스포츠, 과학, 및 기술 (No. 16 K 05671)에서 KAKENHI (선진적인 과학 연구 c)에 의해 지원 되었다.

1. 샘플 준비
<ol><li>
실버 콜 로이드 나노 입자의 준비 20<ol>
<li>조작 실버 콜 로이드 나노 입자, 실버 질산염의 그리고 150 mL 200 mL 둥근 바닥 플라스 크에 물이 수화물 trisodium 시트르산의 0.030 g 0.030 g을 분해.</li>
		<li>플라스 크를 결합 하 여 환류 (Dimroth) 콘덴서와 함께.</li>
		<li>마그네틱 교 반기 및 저 어 바와 플라스 크에 솔루션을 저 어. 그런 다음 60 분 동안 150 ° C에 기름 목욕에서 플라스 크에서 감동적인 솔루션 열. 참고: 솔루션은 노란색, 다음은 하 회색을 돌 것 이다.</li>
		<li>서 스 펜 션, 실내 온도에 냉각 하 고 냉장고에 알루미늄 호 일로 덮여 플라스 크에 정지를 유지. 참고: 프로토콜 수 수 일시 중지이 시점에서. 콜 로이드 나노 입자를 사용 하 여 1 개월 이내는 냉장고에 저장 후.</li>
	</ol>
</li>
	<li>
다중 색에 대 한 샘플의 준비 점멸 방출 11<ol>
<li>현미경 슬라이드를 준비, 유리 접시 비누를 손으로 세척 하 고 물으로 씻어.</li>
		<li>유리 접시에 0.1% 폴 리-L-리 신 수성 솔루션을 추가 하 고 솔루션을 제거 하는 송풍기와.</li>
		<li>유리 접시에 실버 콜 로이드 현 탁 액을 추가 하 고 송풍기와 현 탁 액을 제거.</li>
		<li>액체 차단 펜으로 유리 접시에 드롭 영역을 묶습니다.</li>
		<li>유리 접시에 증류수를 삭제 하 고 다른 유리 접시 현미경 슬라이드를 만들고 증발에서 물을 방지 하기를 다룹니다.</li>
	</ol>
</li>
	<li>
단조로운 색된 점멸 관련에 대 한 샘플의 준비 7 , 8 , 9 , 10<ol>
<li>현미경 슬라이드를 준비, 유리 접시 비누를 손으로 세척 하 고 물으로 씻어.</li>
		<li>Thiacyanine 또는 thiacarbocyanine 염료와 실버 콜 로이드 현 탁 액을 혼합 (25 또는 4 µ M, 각각)와 2의 볼륨 비율로 NaCl (10mm) 용액: 1:1.</li>
		<li>유리 접시에 샘플 중지를 삭제 하 고 송풍기와 현 탁 액을 제거.</li>
		<li>액체 차단 펜으로 유리 접시에 드롭 영역을 묶습니다.</li>
		<li>나노 입자를 고정을 유리 접시에 NaCl (1 M)의 수성 해결책을 삭제 하 고 다른 유리 접시 현미경 슬라이드 플레이트를 만들고 솔루션에서 증발 방지를 커버.</li>
	</ol>
</li>
</ol>2입니다. 점멸은 나노 입자의 관찰
<ol><li>
샘플의 조명
	<ol>
<li>1.2 또는 1.3 프로토콜을 사용 하 여 거꾸로 현미경의 스테이지 준비 샘플 유리 접시를 놓습니다.</li>
		<li>목표 렌즈 (60 X)를 사용 하 여 유리 접시에 다양 한 색된 반점 (, 녹색, 노란색, 파란색과 빨간색)에 어두운 필드 콘덴서와 통해 하얀 빛을 사용 하 여 샘플 유리 접시 켜.</li>
		<li>밝게 샘플 표면에 상대적으로 30 °의 각도 다이오드 펌프 고체 (DPSS) 연속파 (cw) 레이저 간섭 필터를 통해 전달 감쇠 광속을 사용 하 여 샘플 유리 접시.</li>
		<li>사용 하 여 레이저 조명 같은 색 주변에 단조로운 색된 반점으로 실버 nanoaggregates 관찰, 보기의 센터에 레이저 조명 영역을 이동 하 고 z-방향으로 단계를 조정 하 여 유리 접시에 반점에 초점.</li>
	</ol>
</li>
	<li>
점멸의 관찰
	<ol>
<li>목표 렌즈 후 긴-패스 필터를 삽입 하 고 간섭 필터를 통해 샘플 표면에 상대적으로 30 °의 각도에서 전달 DPSS cw 레이저 광선을 사용 하 여 샘플 유리 접시를 조명.</li>
		<li>그림 1 에서 보듯이 명소 점멸 찾기 (참고 그림 S1 보충 자료에서) 무대에는 x-및 y-방향으로 이동 하 여.</li>
		<li>거꾸로 현미경, 냉각된 디지털 전 하 결합 소자 (CCD) 카메라, 61-120 ms의 시간 분해능이 20 분에 대 한 결합으로 깜박이 명소의 기록 영상.</li>
	</ol>
</li>
</ol>3입니다. 깜박이 SER의 분석
<ol><li>
비디오에서 시간 프로필의 유래
	<ol>
<li>CCD 카메라를 제어 하는 소프트웨어에서 비디오 파일을 엽니다.</li>
		<li>깜박이 관광 명소 및 어두운 영역을 선택 하려면 별도로 지역 명소 비디오 이미지 없이 커버 하는 영역을 드래그 합니다.</li>
		<li>깜박이 관광 명소와 비디오에서 어두운 영역에서 신호 강도 시간 프로필 파생, 분석, 임시 분석 을 선택 하 고 임시 분석 창에서 계산 을 클릭 합니다.</li>
		<li>텍스트 파일로 데이터를 저장.</li>
	</ol>
</li>
	<li>
시간 프로 파일의 분석
	<ol>
<li>어두운 영역 또는 그림 2A 와 2B와 같이 다항식 함수과 시간 프로필을 빼서 시간 프로필의 기준선을 평평.</li>
		<li>약 2000 포인트, 난기본, 그리고 초기 농도의 표준 편차 σ, 그림 2C 및 2D에서 같이 구성 된 평균된 초기 강도 평가 합니다.</li>
		<li>내가기본 + 3σ의 임계값 보다 큰 농도 사용 하 여 어두운 이벤트에서 밝은 이벤트를 구별 하 고 각 이벤트의 기간을 기록 합니다. 그림 3에서 예를 들어 0에서 3.5476 이벤트 기록 어두운 이벤트로 s ( t 기간 = 3.5476 s), 4.0981 3.5476에서 이벤트를 기록 하 고 밝은 이벤트로 s (기간 t = 0.5505 s). 표 1에 표시 된 것 처럼 절차를 반복 합니다.</li>
		<li>표 2의 첫 번째 및 두 번째 라인에 표현 각 기간에 대 한 밝고 어두운 이벤트 수를 계산.</li>
		<li>이벤트 기간 t보다 짧은 제외한 각 기간에 대 한 이벤트 수를 합계 하십시오. 표 2의 두 번째 및 세 번째 라인에 표현, 예를 들어 각 기간에 대 한 이벤트 수를 합계 ( t 에 대 한 이벤트를 제외 하 고 0.0612 = s) 41 + 18 + 9 +...; 결과 t 에 대 한 합계 = 0.1223 s, 즉, 103.</li>
		<li>각 기간별로 합계를 분할 하 고 그들을 정상화. 표 2에 표현, 예를 들면, 기간 t 에 대 한 변론 분할 0.0612 = s 기간 0.0612 s. 결과 3,351.5791입니다. 그런 다음 표 2에 네 번째 줄에서 결과의 총 결과 나눕니다. 확률 분포는 0.64494 것 파생 됩니다.</li>
		<li>밝은 이벤트 P(t)에 대 한 그들의 기간 t 로그-로그 그래프에 대 한 확률 분포를 플롯 및 로그10P에(t) 로그10에 의해 (<img alt="Equation 1" src="/files/ftp_upload/56729/56729eq1.jpg">)는 전원 법 지 수 α에에 깜박이 자리 특정 추론할. P에(t)는으로 장착 하는 경우 <img alt="Equation 1" src="/files/ftp_upload/56729/56729eq1.jpg"> </img>, 그림 4A에 점선으로 표시 된 것 처럼 장착된 라인 P(t)의 값이 작은 플롯에서 일탈.</li>
		<li>어두운 이벤트 P오프(t) 로그-로그 그래프에 그들의 시간 t 에 대 한 확률 분포를 플롯 및 로그10(로그10P오프(t) 맞는 <img alt="Equation 3" src="/files/ftp_upload/56729/56729eq3.jpg"> 는 전원 법 지 수 α오프 와는 잘림 시간 τ 동일한 점멸에서 자리를 추론할. P오프(t)는으로 장착 하는 경우 <img alt="Equation 3" src="/files/ftp_upload/56729/56729eq3.jpg"> </img>, 맞춤된 곡선 P오프(t)의 값이 작은 플롯에서 성적이 상자.</li>
		<li>3.2.1 3.2.8 비디오에서 다른 깜박이 관광 명소에 대 한 반복 합니다.</li>
	</ol>
</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+imaging+of+SERS+hot+spots.">Super-resolution imaging of SERS hot spots.</a> Chem. Soc. Rev. 43, 3854-3864 (2014).</li><li>Dieringer, J. A., Lettan, R. B., Scheidt, K. A., Van Duyne, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Frequency+Domain+Existence+Proof+of+Single-Molecule+Surface-Enhanced+Raman+Spectroscopy.">A Frequency Domain Existence Proof of Single-Molecule Surface-Enhanced Raman Spectroscopy.</a> J. Am. Chem. 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C., Meisel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+and+surface-enhanced+Raman+of+dyes+on+silver+and+gold+sols.">Adsorption and surface-enhanced Raman of dyes on silver and gold sols.</a> J. Phys. Chem. 86, 3391-3395 (1982).</li><li>Krichevsky, O., Bonnet, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+correlation+spectroscopy:+the+technique+and+its+applications.">Fluorescence correlation spectroscopy: the technique and its applications.</a> Rep. Prog. Phys. 65, 251-297 (2002).</li><li>Hess, S. T., Huang, S., Heikal, A. A., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+and+Chemical+Applications+of+Fluorescence+Correlation+Spectroscopy:+A+Review.">Biological and Chemical Applications of Fluorescence Correlation Spectroscopy: A Review.</a> Biochemistry. 41, 697-705 (2002).</li></ol>

저자는 공개 상관이 있다.

실버 nanoaggregate 접합에서 SER는 발생 합니다. 따라서, 우리는 시트르산 음이온으로 덮여 있다 콜 로이드 나노 입자, 보다는 오히려 nanoaggregates를 준비 해야 합니다. 실버 집계 효과 폴 리-L-리 신,-NH3+ 이며는 SER의 기원의 추가 의해 만들어진 소금에서 형성 된다 또는 NaCl, 보충 자료의 그림 s 2 와 같이에서 Na+ 양이온. 또한, 넓은 지역에 많은 관광 명소를 밝히는, ?...

표면 강화 라만 산란 (SERS) 고귀한 금속 표면에서 고감도 라만 분광학 이다. 금속 표면에 단일 분자의 정보를 조사 수 이후 기능적인 그룹 분자에서 진동 모드를 통해 날카로운 피크 위치에 따라 분자 구조에 대 한 자세한 정보를 제공 하는 라만 스펙트럼 관련1,2,3을 사용 하 여. 단일 분자 수준에서 흡착과 실버 nanoaggregate에서 깜박이 신호 관찰1,2,3,,45,6, 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16, 그리고 스펙트럼 변동1,2,3,,45,6,7,8, 9,10,11,12,,1314. 깜박임 무작위로 나노미터 크기의 nanoaggregate 접속점에 향상 된 전자기 (EM) 필드 밖으로 이동 하는 단일 분자에 의해 유도 될 수 있다. 따라서, 점멸 SERS 농도 및 bi 분석2,,317의 포아송 분포를 사용 하는 기술에 비해 단일 분자 검출에 대 한 간단한 증거를 간주 됩니다. 그러나, 강하게 Ag 표면에 분자 행동에 달려 있습니다, 깜박이 변동 스펙트럼의 상세한 메커니즘은 아직도 논란이 있습니다.
이전 학문에서는, 점멸 관련 분석 된 고 확산 계수는 향상 된 EM 필드12,13,14 밖으로 이동 하는 분자의 농도 계산할 수 있습니다 자기 상관 함수를 사용 하 여 . 또한, 불안정 총 강도 나타내는 정규화 된 표준 편차 점수는 신호15시간 프로필에서 파생 되었습니다. 그러나, 이러한 분석 방법 몇 가지 분자의 동작에 따라 수 있습니다. 반면, 깜박이 SER의 슈퍼 고해상도 이미징, 향상 된 EM 필드에 단일 분자 동작 확인 된16될 수 있습니다. 그러나, 이러한 기술은 향상 된 EM 필드에만 이러한 매개 변수를 얻을 수 있습니다. (예를 들어, 깜박이 SERS)에서 넓은 범위 내 단일 분자의 임의의 동작 전원 법 보다는 평균4,,56,7,8로 대표 될 수 있다 ,,910,11, 단일 반도체 양자 점 (QD)18,19의 점멸 형광 비슷합니다. 전원 법 분석4,5,6,7,,89,10,11, 분자 동작을 사용 하 여 (향상 된 EM 필드)에 밝은 상태와 어두운 상태10;에서 예상할 수 있는 즉, 전체 실버 표면 분자의 행동은 추정 될 수 있습니다.
이 기술에 대 한 실버 콜 로이드 nanoaggregates는 사용된4,5,6,7,,89,,1011. 이러한 nanoaggregates 그들은 특정 한 파장에 흥분 하는 때 강력 하 게 향상 된 전자기 필드를 영향을 미칠 다양 한 지역화 된 표면 플라스몬 공명 (LSPR) 밴드를 표시 합니다. 따라서, 관련 액티브 실버 나노 콜 로이드 서 스 펜 션, 그리고 일부 데이터에 존재 하는 즉시 얻을 수 있습니다. 특정 크기, 모양, 그리고 준비는 간단한 nanostructures의 경우 SER 점멸의 LSPR 의존 다른 의존7;을 숨길 수 있는 즉, LSPR에 좋은 소식이 나 나쁜 nanostructure 사용 하는 경우 매개 변수 상수, 되며 다른 의존 따라서 숨겨져 있을 것 이다. 전원 법 분석 실버 콜 로이드 nanoaggregates4,,56,7,8, 에서 깜박이 관련의 다양 한 종속성을 발견 하는 데 사용 되었습니다. 9 , 10 , 11.

<table><tbody><tr><td>Silver nitrate, 99.8%</td><td>Wako</td><td>194-00832</td><td></td></tr><tr><td>Trisodium citrate dihydrate, 99. %</td><td>Wako</td><td>191-01785</td><td></td></tr><tr><td>Poly-L-lysine aqueous solution, 0.1%</td><td>Sigma-Aldrich</td><td>P8920</td><td></td></tr><tr><td>3,3&#039;-disulfopropylthiacyanine triethylamine</td><td>Hayashibara Biochemical Laboratories</td><td>NK-2703</td><td>a kind of thiacyanine dyes</td></tr><tr><td>3,3&#039;-diethyl-5,5&#039;-dichloro-9-methylthiacarbocyanine iodine salt</td><td>Hayashibara Biochemical Laboratories</td><td>SMP-9</td><td>a kind of thiacarobocyanine dyes</td></tr><tr><td>Sodium chloride, 99.5%</td><td>Wako</td><td>191-01665</td><td></td></tr><tr><td>Dimroth condenser</td><td>Iwaki</td><td>61-9722-22</td><td>perchased from AS ONE</td></tr><tr><td>Magnetic stirrer</td><td>Corning</td><td>DC-420D</td><td></td></tr><tr><td>Oil bath</td><td>Advantech</td><td>OS-220</td><td></td></tr><tr><td>Glass plate</td><td>Matsunami</td><td>S-1112</td><td>Microscope slide</td></tr><tr><td>Blower</td><td>Hozan</td><td>Z-288</td><td>Air duster</td></tr><tr><td>Liquid blocker pen</td><td>Daido Sangyo</td><td></td><td>LIQUID BLOCKER (Super Pap Pen). Ready-to-use hydrophobic barrier pen designed for immunohistochemistry applications</td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>IX-70</td><td></td></tr><tr><td>Objective lens</td><td>Olympus</td><td>LCPlanFl 60&amp;times;</td><td>NA 0.7</td></tr><tr><td>Dark field condenser</td><td>Olympus</td><td>U-DCD</td><td>NA 0.8&amp;ndash;0.92</td></tr><tr><td>Cooled digital CCD camera</td><td>Hamamatsu</td><td>ORCA-AG</td><td>controlled by software Aqua Cosmos</td></tr><tr><td>Software for the cooled digital CCD camera</td><td>Hamamatsu</td><td>AquaCosmos</td><td>used for also derivation of the time-profiles from the blinking spots in the video&amp;nbsp;</td></tr><tr><td>Color CCD camera</td><td>ELMO</td><td>TNC-C920</td><td>not used for analysis</td></tr><tr><td>DPSS laser</td><td>RGB laser system</td><td>NovaPro532-75</td><td>&amp;lambda; = 532 nm;&lt;br/&gt; 60 mW (corresponds to a power density of 600 W/cm2)</td></tr><tr><td>Interference filter</td><td>Semrock</td><td>LL01-532-12.5</td><td></td></tr><tr><td>Long pass filter</td><td>Semrock</td><td>BLP01-532R-25</td><td></td></tr><tr><td>Software for the distinguishment and counting of the bright/dark events</td><td>home-maid</td><td></td><td>programmed by C++</td></tr><tr><td>Software for the fitting by a power law</td><td>LightStone</td><td>Origin6.1</td><td></td></tr></tbody></table>

observation and analysis of blinking surface-enhanced raman scattering

nanoaggregate 접속점에 단일 분자에서 관찰 표면 강화 된 라만 산란 (SERS) 점멸 합니다. 여기, 프로토콜 관련 활동을 준비 하는 방법에 선물 된다 nanoaggregate 실버, 미세한 이미지에서 특정 깜박이 명소의 비디오를 기록 하 고 깜박이 통계 분석. 이 분석에서 전원 법률 그들의 지속 시간에 상대적인 밝은 이벤트에 대 한 확률 분포를 재현합니다. 어두운 이벤트에 대 한 확률 분포는 지 수 함수 전원 법으로 적합 하다. 전원 법의 매개 변수는 모두 밝고 어두운 상태에서 분자 동작을 나타냅니다. 랜덤 워크 모델 및 전체 실버 표면에서 분자의 속도 추정 될 수 있습니다. 평균, 자기 상관 함수 및 슈퍼 해상도 관련 이미지를 사용 하는 경우에 추정 하는 것이 어렵습니다. 점멸의 기원만이 분석 방법에 의해 확인할 수 없습니다 때문에 미래에 전원 법 분석 스펙트럼 이미징, 함께 결합 되어야 합니다.

그림 111에서 같이 폴 리-L-리 신 1.2 프로토콜에 의해 준비와 함께 실버 nanoaggregates에서 관련 및 표면 강화 된 형광 색된 점멸 명소 관찰 된다. 반면에, SERS에서 단조로운 색된 점멸 명소 프로토콜 1.37,8,,910에 의해 준비 된 염료 분자와 실?...

Watch this Scientific Journal Video about Observation and Analysis of Blinking Surface-enhanced Raman Scattering at JoVE.com

Observation and Analysis of Blinking Surface-enhanced Raman Scattering

표면 강화 라만 점멸의 분석이이 프로토콜에 설명 합니다 전원 법을 사용 하 여 실버 표면에 단일 분자의 랜덤 워크 인 비 산.

관찰 및 깜박이 표면 강화 된 라만 산란의 분석

From a single molecule at a silver nanoaggregate junction, blinking surface-enhanced Raman scattering (SERS) is observed. Here, a protocol is presented on ...

observation-and-analysis-of-blinking-surface-enhanced-raman-scattering

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7.3K 조회수.  Kwansei Gakuin University. 표면 강화 라만 점멸의 분석이이 프로토콜에 설명 합니다 전원 법을 사용 하 여 실버 표면에 단일 분자의 랜덤 워크 인 비 산.

비디오: Observation and Analysis of Blinking Surface-enhanced Raman Scattering

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ultrafast pulses, a system that can temporally separate spectrally overlapping signals on a picosecond timescale can isolate promptly arriving Raman scattered light from late-arriving fluorescence light. Here we discuss the construction and operation of a complex nonlinear optical system that uses all-optical switching in the form of a low-power optical Kerr gate to isolate Raman and fluorescence signals. A single 808 nm laser with 2.4 W of average power and 80 MHz repetition rate is split, with approximately 200 mW of 808 nm light being converted to &lt; 5 mW of 404 nm light sent to the sample to excite Raman scattering. The remaining unconverted 808 nm light is then sent to a nonlinear medium where it acts as the pump for the all-optical shutter. The shutter opens and closes in 800 fs with a peak efficiency of approximately 5%. Using this system we are able to successfully separate Raman and fluorescence signals at an 80 MHz repetition rate using pulse energies and average powers that remain biologically safe. Because the system has no spare capacity in terms of optical power, we detail several design and alignment considerations that aid in maximizing the throughput of the system. We also discuss our protocol for obtaining the spatial and temporal overlap of the signal and pump beams within the Kerr medium, as well as a detailed protocol for spectral acquisition. Finally, we report a few representative results of Raman spectra obtained in the presence of strong fluorescence using our time-gating system.

We discuss the construction and operation of a complex nonlinear optical
system that uses ultrafast all-optical switching to isolate Raman from fluorescence
signals. Using this system we are able to successfully separate Raman and fluorescence
signals utilizing pulse energies and average powers that remain biologically safe.

공명과 자연 래맨 Microspectroscopy에 형광 배경 거부

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ...

연구

생체공학

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.

We describe the fabrication and characterization of nano-biological systems interfacing nanostructured substrates with immobilized proteins and aptamers. The relevant experimental steps involving lithographic fabrication of nanostructured substrates, bio-functionalization, and surface-enhanced Raman spectroscopy (SERS) characterization, are reported. SERS detection of surface-immobilized proteins, and probing of protein-ligand and aptamer-ligand binding is demonstrated.

표면 EBL 날조 된 나노 구조 기판을 사용하여 생체 분자의 라만 분광학 감지 강화

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules ...

공학

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

라만 분광학을 사용하여 세포와 세포외 기질의 비 접촉, 레이블이없는 모니터링

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

High-speed Continuous-wave Stimulated Brillouin Scattering Spectrometer for Material Analysis

Recent years have witnessed a significant increase in the use of spontaneous Brillouin spectrometers for non-contact analysis of soft matter, such as aqueous solutions and biomaterials, with fast acquisition times. Here, we discuss the assembly and operation of a Brillouin spectrometer that uses stimulated Brillouin scattering (SBS) to measure stimulated Brillouin gain (SBG) spectra of water and lipid emulsion-based tissue-like samples in transmission mode with &lt;10 MHz spectral-resolution and &lt;35 MHz Brillouin-shift measurement precision at &lt;100 ms. The spectrometer consists of two nearly counter-propagating continuous-wave (CW) narrow-linewidth lasers at 780 nm whose frequency detuning is scanned through the material Brillouin shift. By using an ultra-narrowband hot rubidium-85 vapor notch filter and a phase-sensitive detector, the signal-to-noise-ratio of the SBG signal is significantly enhanced compared to that obtained with existing CW-SBS spectrometers. This improvement enables measurement of SBG spectra with up to 100-fold faster acquisition times, thereby facilitating high spectral-resolution and high-precision Brillouin analysis of soft materials at high speed.

We describe the construction of a rapid continuous-wave-stimulated-Brillouin-scattering (CW-SBS) spectrometer. The spectrometer employs single-frequency diode-lasers and an atomic vapor notch-filter to acquire transmission spectra of turbid/non-turbid samples with high spectral-resolution at speeds up to 100-fold faster than those of existing CW-SBS spectrometers. This improvement enables high-speed Brillouin material analysis.

소재 분석에 대 한 고속 연속파 자극된 브 릴 루 앵 산란 분 광 계

Recent years have witnessed a significant increase in the use of spontaneous Brillouin spectrometers for non-contact analysis of soft matter, such as ...

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. SRS imaging modality can be obtained using commercial laser-scanning microscopes by equipping with a non-descanned forward detector with proper bandpass filters and lock-in amplifier (LIA) detection scheme. A schematic layout of a typical SRS microscope includes the following: two pulsed laser beams, (i.e., the pump and probe directed in a scanning microscope), which must be overlapped in both space and time at the image plane, then focused by a microscope objective into the sample through two scanning mirrors (SMs), which raster the focal spot across an x-y plane. After interaction with the sample, transmitted output pulses are collected by an upper objective and measured by a forward detection system inserted in an inverted microscope. Pump pulses are removed by a stack of optical filters, whereas the probe pulses that are the result of the SRS process occurring in the focal volume of the specimen are measured by a photodiode (PD). The readout of the PD is demodulated by the LIA to extract the modulation depth. A two-dimensional (2D) image is obtained by synchronizing the forward detection unit with the microscope scanning unit. In this paper, the implementation of an SRS microscope is described and successfully demonstrated, as well as the reporting of label-free images of polystyrene beads with diameters of 3 &#181;m. It is worth noting that SRS microscopes are not commercially available, so in order to take advantage of these characteristics, the homemade construction is the only option. Since SRS microscopy is becoming popular in many fields, it is believed that this careful description of the SRS microscope implementation can be very useful for the scientific community.

In this manuscript, the implementation of a stimulated Raman scattering (SRS) microscope, obtained by the integration of an SRS experimental set-up with a laser scanning microscope, is described. The SRS microscope is based on two femtosecond (fs) laser sources, a Ti-Sapphire (Ti:Sa) and synchronized optical parametric oscillator (OPO).

자극된 라만 산란을 기반으로 한 비선형 현미경 구현

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. ...

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps at high speed, and high spatial resolution of thin samples by directly interrogating their molecular vibrations. In its standard implementation, SRS microscopy is narrowband and forms images with only a single vibrational frequency at a time. However, this approach not only hinders the chemical specificity of SRS but also neglects the wealth of information encoded within vibrational spectra. 
These limitations can be overcome by broadband SRS, an implementation capable of extracting a vibrational spectrum per pixel of the image in parallel. This delivers hyperspectral data that, when coupled with chemometric analysis, maximizes the amount of information retrieved from the specimen. Thus, broadband SRS improves the chemical specificity of the system, allowing the quantitative determination of the concentration of the different constituents of a sample. Here, we report a protocol for chemical imaging with broadband SRS microscopy, based on a home-built SRS microscope operating with a custom differential multichannel-lock-in amplifier detection. It discusses the sample preparation, alignment of the SRS apparatus, and chemometric analysis. By acquiring vibrational Raman spectra, the protocol illustrates how to identify different chemical species within a mixture, determining their relative concentrations.

We present a protocol to acquire chemical images with broadband stimulated Raman scattering (SRS) microscopy. Based on an SRS microscope that operates with differential multichannel-lock-in detection, the protocol describes the sample preparation, adjustment of the SRS apparatus, and chemometrics to disentangle different constituents of chemically heterogeneous samples.

광대역 자극 라만 산란 현미경을 기반으로 한 멀티플렉스 화학 이미징

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps ...

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging technologies. Hyperspectral SRS and CARS imaging offer Raman spectral information at every pixel, which enables better separation of different chemical compositions. Although both techniques require two excitation lasers, their signal detection schemes and spectral properties are quite different. The goal of this protocol is to perform both hyperspectral SRS and CARS imaging on a single platform and compare the two microscopy techniques for imaging different biological samples. The spectral focusing method is employed to acquire spectral information using femtosecond lasers. By using standard chemical samples, the sensitivity, spatial resolution, and spectral resolution of SRS and CARS in the same excitation conditions (i.e., power at the sample, pixel dwell time, objective lens, pulse energy) are compared. The imaging contrasts of CARS and SRS for biological samples are juxtaposed and compared. The direct comparison of CARS and SRS performances would allow for optimal selection of the modality for chemical imaging.

This paper directly compares the resolution, sensitivity, and imaging contrasts of stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) integrated into the same microscope platform. The results show that CARS has a better spatial resolution, SRS gives better contrasts and spectral resolution, and both methods have similar sensitivity.

화학 이미징을 위한 하이퍼스펙트럼 자극 라만 산란 및 일관된 안티스토크스 라만 산란 현미경의 직접 비교

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging ...

화학

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

생체 내 결합 된 이광자 형광 및 자극 된 라만 산란 현미경을 사용한 생물학적 조직 이미징

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has been shown to be able to provide hematoxylin and eosin (H&amp;E)-equivalent images that allow fast and reliable diagnosis of brain cancer. Such capability has enabled exciting intraoperative cancer diagnosis applications. Two-color SRS imaging of tissue can be done with either a picosecond or femtosecond laser source. Femtosecond lasers have the advantage of enabling flexible imaging modes, including fast hyperspectral imaging and real-time, two-color SRS imaging. A spectral-focusing approach with chirped laser pulses is typically used with femtosecond lasers to achieve high spectral resolution. 
Two-color SRS acquisition can be realized with orthogonal modulation and lock-in detection. The complexity of pulse chirping, modulation, and characterization is a bottleneck for the widespread adoption of this method. This article provides a detailed protocol to demonstrate the implementation and optimization of spectral-focusing SRS and real-time, two-color imaging of mouse brain tissue in the epi-mode. This protocol can be used for a broad range of SRS imaging applications that leverage the high speed and spectroscopic imaging capability of SRS.

Stimulated Raman scattering (SRS) microscopy is a powerful, nondestructive, and label-free imaging technique. One emerging application is stimulated Raman histology, where two-color SRS imaging at the protein and lipid Raman transitions are used to generate pseudo-hematoxylin and eosin images. Here, we demonstrate a protocol for real-time, two-color SRS imaging for tissue diagnosis.

조직 진단을 위한 마우스 뇌의 실시간 두 가지 색상 자극 라만 산란 영상

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.