이 연구는 일본 교육문화체육관광부(18K04843)에서 YST ERATO(JPMJER1502)에서 N. 노무라까지 과학연구보조금으로 부분적으로 지원되었다.

1. 샘플 준비
<ol><li>유리 슬라이드에 우물이 있는 1mm 두께의 실리콘 개스킷을 놓습니다.</li>
	<li>실리콘 개스킷의 우물에 1mm 두께0.8%(w/v) 아가로즈 슬래브를 놓습니다.</li>
	<li>임의미생물세포배양주의 세포밀도를 600nm(OD660) = 1.0에서 광학 밀도로 희석한다.</li>
	<li>아가로즈 슬래브에 세포 현탁액의 5 μL 알리쿼트를 놓습니다.</li>
	<li>유리 커버슬립으로 부드럽게 덮습니다.</li>
</ol>2. 현미경의 설정
참고: CRIF 기술은 공초점 반사 현미경 검사법(CRM)과 멀티채널 공초점 현미경 분석법을 결합합니다. CRM은 세포 선천성 형광과는 무관한 세포 형태학 및 공간 국소화를 위한 정보의 근원 역할을 합니다. 멀티채널 공초점 현미경 검사는 세포 선천성 형광의 스펙트럼 정보를 제공합니다. 다음 프로토콜에서, CRM 또는 공초점 형광 현미경 분석기로 취득된 모든 심상은 CRM 영상 또는 다중 채널 공초점 현미경 분광법 심상으로, 각각 지칭된다.
<ol><li>소멸된 스펙트럼 채널과 공초점 현미경을 광증 튜브(PMT) 또는 GaAsP 검출기에 연결합니다. 참고: 이러한 설정은 여러 제조업체에서 사용할 수 있습니다.</li>
	<li>현미경에 높은 숫자 조리개 (NA) 목표를 적절한 배율로 장착하십시오. 참고: NA > 1.4를 가진 63x 목표는 세균성 세포를 분석하기 위해 권장됩니다.</li>
	<li>현미경에 반 반사 거울(예: NT 80/20)을 장착하여 사고 광의 세포 분산에 의존하여 세포 형태를 시각화하는 CRM을 수용하십시오.</li>
	<li>멀티채널 공초점 현미경 검사법의 경우 현미경에 이색 거울을 장착하십시오. 예를 들어, MBS InVis405, MBS458, MBS458/514, MBS488/543, 또는 MBS 488/543/633 빔 스플리터 405, 458, 488, 514, 543, 또는 633 nm, excitationly를 사용합니다.</li>
	<li>레이저 파워 미터를 사용하여 각 여기 파장에 대한 조명 강도를 조정합니다. 여기 파장을 통해 현미경 의 밑에 출력을 일정하게 유지하십시오 (예를 들어, 63x 목표와 함께 50 μW를 사용하십시오).</li>
</ol>3. 이미지 수집
<ol><li>현미경 소프트웨어를 사용하여 핀홀 크기를 1AU로 설정합니다.</li>
	<li>각 여기 파장에 대해 픽셀 거주 시간(예: 스캔 속도)을 설정합니다. 참고: 픽셀 이너무 긴 픽셀 거주 시간은 세포를 손상시킬 수 있습니다. 셀의 광피해를 최소화하기 위해 픽셀이 지나치게 긴 시간을 피하십시오. 세균성 시료의 경우, 픽셀 거주 시간 <55.6 μs/μm2 (현미경의 절개 출력이 ~17 μW/cm2인 경우)는 일반적으로 성장 억제를 피하기에 적합하다. 이 매개 변수는 유기체 및 실험 설정에 따라 달라질 수 있습니다.</li>
	<li>스캔 해상도를 설정합니다. 박테리아와 같은 작은 세포의 경우 1,024 x 1,024의 스캐닝 영역을 사용하십시오.</li>
	<li>관심 영역이 다루어지되도록 Z 스캐닝 범위를 설정합니다. 참고: 아가로즈 슬래브에 분포된 세균 및 효모 세포 샘플의 경우, -15 μm의 Z 스캐닝 범위는 일반적으로 충분합니다.</li>
	<li>가시 파장 범위(예: 416-691 nm)를 캡처하기 위해 탈세 검출기를 설정합니다. 8-10 nm의 스펙트럼 창을 사용합니다.</li>
	<li>다중 채널 공초점 현미경 이미지를 가장 긴 에서 최단 흥분 파장에서 순서로 획득하여 형광 이미지의 Z 스택을 만듭니다.</li>
	<li>CRM 이미지를 획득합니다.</li>
	<li>획득한 이미지를 16비트 티프 파일로 디렉토리에 저장합니다. XXX가 흥분 파장인 명명 규칙 XXXcYzZZ.tif를 사용하여 파일의 이름을 지정하고, YY는 검출기 채널 번호이고, ZZ는 Z 슬라이스 번호이며, "c"와 "z"는 각각 검출기 번호와 Z 슬라이스 번호의 접두사입니다.<ol>
<li>예를 들어, 다중 채널 공초점 마이크로스펙트로스크 이미지가 405nm, 검출기 어레이의 제1 검출기 채널의 내분 파장으로 촬영되고, Z-스택의 5번째 슬라이스인 경우, "405c01z05.tif"라고 지칭한다. CRM 이미지의 경우 XXX 대신 문자열 "CRM"을 사용합니다(예: "CRMc01z05.tif"). 참고: 2D 또는 3D 세분화가 이미지 데이터에 가장 적합한지 여부를 결정합니다. 작은 세포가 2D 평면에 제약을 받는 상황에서 2D 세분화 방법을 사용합니다(예: 유리 표면에 어항을 인고하는 세균 모집단). 세포 집단이 3차원 공간(예를 들어, 생물막 및 조직 샘플)에 분포되는 상황에서 3D 세분화 방법을 사용하거나 세포 크기가 광학 슬라이스의 두께(예를 들어, 효모 세포, 포유류 세포)의 두께보다 크다. 2D 세분화의 경우 섹션 4를 참조합니다. 3D 세분화의 경우 섹션 5를 참조하십시오.</li>
	</ol>
</li>
</ol>4. 2D 이미지 분석
<ol><li>워크스테이션에 이미지 분석 소프트웨어(예: MATLAB)를 장착합니다.</li>
	<li>단세포 선천적 형광 서명의 세포 세분화 및 재구성을 수행합니다.<ol>
<li>이미지 분석 소프트웨어를 엽니다.</li>
		<li>제공된 스크립트 "Script2D.m"를 두 번 클릭하고 엽니다.</li>
		<li>편집기 탭으로 이동한 다음 실행을 클릭합니다. 폴더 선택 창이 나타납니다.</li>
		<li>3.8단계에서 만든 디렉토리를 선택한 다음 열기 를 클릭하여 진행합니다. 분할 매개 변수의 입력을 묻는 대화 상자가 자동으로 나타납니다. 참고: 테스트 목적으로 제공된 데이터 집합("Sample_2D")을 선택합니다. 샘플 데이터 집합은 압축 된 파일로 제공되며 사전에 추출해야합니다.</li>
		<li>분할 매개 변수 입력: 이미지 비나화에 대한 이미지 Binarization (0-1) = 0.45, 셀 영역(픽셀) = 200, 셀 영역(픽셀) = 10, 검출 기 수 = 32에 대한 낮은 임계값. 확인 을 클릭하여 진행합니다. 참고: 이러한 매개 변수는 이미지 품질에 따라 조정이 필요할 수 있습니다.</li>
		<li>CRM 이미지를 표시하는 새 이미지 창이 나타나야 합니다. 백그라운드 영역(예: 셀이 없는 영역)을 선택하여 백그라운드 빼기에 사용합니다. 마우스 드래그하여 CRM 이미지 내에서 사각형을 그립니다. 선택한 영역 내에서 두 번 클릭하여 선택을 확인합니다.</li>
		<li>4.2.4 단계에서 선택한 동일한 디렉터리에서 서명 이라는 새 디렉터리를 찾습니다. 참고: 제공된 코드는 자동으로 이 디렉터리를 만듭니다. "서명" 디렉터리는 일반적인 접두사 "서명"후에 일련 번호가 매겨진 .png 파일로 각 미생물 셀의 타고난 형광 서명을 저장합니다.</li>
	</ol>
</li>
</ol>5.3D 이미지 분석
<ol><li>워크스테이션에 이미지 분석 소프트웨어(재료 표)를 장착합니다.</li>
	<li>단세포 선천적 형광 서명의 세포 세분화 및 재구성을 수행합니다.<ol>
<li>이미지 분석 소프트웨어를 엽니다.</li>
		<li>제공된 스크립트 "Script3D.m"를 두 번 클릭하고 엽니다.</li>
		<li>편집기 탭으로 이동한 다음 실행을 클릭합니다. 폴더 선택 창이 나타납니다.</li>
		<li>3.8단계에서 만든 디렉토리를 선택한 다음 열기 를 클릭하여 진행합니다. 분할 매개 변수의 입력을 묻는 대화 상자가 자동으로 나타납니다. 참고: 테스트 목적으로 제공된 데이터 집합("Sample_3D")을 선택합니다. 샘플 데이터 집합은 압축 된 파일로 제공되며 사전에 추출해야합니다.</li>
		<li>분할 매개 변수 입력: 이미지 비나화에 대한 이미지 비나화 임계값 (0-1) = 0.01, 셀 볼륨(픽셀) = 1,000, 셀 영역(픽셀) = 20, X 픽셀 크기 [μm/픽셀] = 0.26, Y 픽셀 크기 [μm/픽셀] = 0.26, Z 픽셀 크기 [μm/픽셀] = 0.42= 0.42, 및 검출기 의 수 = 32. 확인 을 클릭하여 진행합니다. 여기 파장의 수에 대한 입력을 묻는 대화 상자가 나타납니다. 참고: 이러한 매개 변수는 이미지 품질에 따라 조정이 필요할 수 있습니다.</li>
		<li>이미지 수집에 사용되는 파장의 수를 입력합니다(예: 405, 488, 561, 630이 사용되는 경우 대화 상자에 4 를 입력). 확인을 클릭합니다.</li>
		<li>가장 짧은(즉, 상자 이름: 여기 1번)에서 대화 상자에 가장 긴 파장에 이르는 순서로 여기 파장을 입력합니다. 확인 을 클릭하여 진행합니다. CRM 이미지를 제공하는 새 이미지 창이 팝업되어야 합니다.</li>
		<li>백그라운드 빼기에는 임의백 영역(즉, 셀이 없는 영역)을 선택합니다. 마우스 드래그하여 CRM 이미지 내에서 사각형을 그립니다. 선택한 영역 내에서 두 번 클릭하여 선택을 확인합니다.</li>
		<li>5.2.4 단계에서 선택한 디렉터리에서 서명 이라는 디렉토리를 찾습니다. 참고: 제공된 코드는 자동으로 이 디렉터리를 만듭니다. "서명" 디렉터리는 일반적인 접두사 "서명"을 한 후 일련 번호가 매겨진 .png 파일로 각 단일 미생물 셀의 타고난 형광 서명을 저장합니다.</li>
	</ol>
</li>
</ol>6. 통계 분석
참고: 치수 감소 기술(예: 주 성분 분석[PCA])을 수행하여 세포 집단의 하이퍼스펙트럼 분포를 시각화합니다. 제공된 스크립트(PCA.py)는 두 개의 셀 모집단(즉, 두 클래스)에 대해 PCA를 실행합니다.
<ol><li>워크스테이션에 프로그래밍 언어와 함께 제공되는 라이브러리 및 모듈(재료 표)을 장착합니다.</li>
	<li>C 드라이브(또는 이와 동등한)에 빈 디렉터리를 만들고 디렉토리 "Parent_directory"(예: C:/)의 이름을 지정합니다. Parent_ 디렉토리).</li>
	<li>두 셀 모집단의 형광 서명(예: 4.2.7단계에서 생성된 .png 파일)을 두 개의 개별 디렉터리로 저장합니다. 참고: 두 디렉터리 모두 "Parent_directory"에 있어야 합니다. C:/Parent_directory/ <img alt="image 12" class="xfigimg" src="/files/ftp_upload/61120/61120img1.jpg">푸티다KT2440/ <img alt="image 13" class="xfigimg" src="/files/ftp_upload/61120/61120img2.jpg"> <img alt="image 9" class="xfigimg" src="/files/ftp_upload/61120/61120img3.jpg">시그니처01.png <img alt="image 14" class="xfigimg" src="/files/ftp_upload/61120/61120img2.jpg"> <img alt="image 10" class="xfigimg" src="/files/ftp_upload/61120/61120img3.jpg">시그니처02.png <img alt="image 4" class="xfigimg" src="/files/ftp_upload/61120/61120img2.jpg"> <img alt="image 11" class="xfigimg" src="/files/ftp_upload/61120/61120img3.jpg">: <img alt="image 5" class="xfigimg" src="/files/ftp_upload/61120/61120img1.jpg">푸티다KT2442/ 
	 <img alt="image 6" class="xfigimg" src="/files/ftp_upload/61120/61120img3.jpg">시그니처01.png 
	 <img alt="image 7" class="xfigimg" src="/files/ftp_upload/61120/61120img3.jpg">시그니처02.png 
	 <img alt="image 8" class="xfigimg" src="/files/ftp_upload/61120/61120img3.jpg">:</li>
	<li>"Parent_directory"에 PCA.py 다운로드합니다.</li>
	<li>워크스테이션의 명령줄 인터페이스를 엽니다.</li>
	<li>명령줄 인터페이스에 "파이썬 C:/Parent_directory/PCA.py"를 입력합니다.</li>
	<li>'대상 디렉토리 선택'이라는 메시지가 표시된 후 'Parent_directory'을 선택합니다.</li>
	<li>"C:/Parent_directory"에서 결과 PCA 플롯이 포함된 "PCA.png"를 찾습니다.</li>
</ol>

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+non-invasive+cell+characterisation+and+discrimination+based+on+multispectral+autofluorescence+features.">Quantitative non-invasive cell characterisation and discrimination based on multispectral autofluorescence features.</a> Scientific Reports. 6, 23453 (2016).</li><li>Yawata, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-+and+interspecies+variability+of+single-cell+innate+fluorescence+signature+of+microbial+cell.">Intra- and interspecies variability of single-cell innate fluorescence signature of microbial cell.</a> Applied and Environmental Microbiology. 85, 00608-00619 (2019).</li><li>Nelson, K. 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Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas.</a> Gene. 16 (1-3), 237-247 (1981).</li><li>Luo, Y., Vijaychander, S., Stile, J., Zhu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+analysis+of+DNA-binding+proteins+by+yeast+one-hybrid+and+one-two-hybrid+systems.">Cloning and analysis of DNA-binding proteins by yeast one-hybrid and one-two-hybrid systems.</a> Biotechniques. 20 (4), 564-568 (1996).</li><li>Yawata, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+biofilm+development+in+a+microfluidic+device+using+modified+confocal+reflection+microscopy.">Monitoring biofilm development in a microfluidic device using modified confocal reflection microscopy.</a> Journal of Bioscience and Bioengineering. 110 (3), 377-380 (2010).</li><li>Lakowicz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Fluorescence Spectroscopy. Third edition. , (2006).</li><li>Blacker, T. S., Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+mitochondrial+redox+state+using+NADH+and+NADPH+autofluorescence.">Investigating mitochondrial redox state using NADH and NADPH autofluorescence.</a> Free Radical Biology &amp; Medicine. 100, 53-65 (2016).</li><li>Croce, A. 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저자는 공개 할 것이 없습니다.

재현 가능한 결과를 얻기 위해 밀접하게 따라야 하는 두 가지 중요한 점이 있습니다: 1) 심내 목표하에서 레이저 전력 출력을 관찰하고, 2) 정확한 세포 세분화를 수행한다.
첫 번째 점은 다른 실험 중 타고난 형광 서명을 비교할 때 특히 중요합니다. 최대 전력 출력이 레이저 라인 간의 크기 순서까지 다를 수 있기 때문에 단순히 동일한 "백분율 출력" 설정을 여기 파장(예: 405, 4...

셀1 내의 다양한 생체 분자는 자가 형광 신호를 방출하고, 세포의 타고난 형광 서명은 이러한 신호의 조립으로 구성된다. 이 시그니처 형광은 다양한 세포 특성과 생리적 상태의 차이를 반영합니다. 선천적 형광의 분석은 최소 침습적이며 온화한 신진 대사 수정에서 완전한 세포 파괴에 이르기까지 다양한 흔적을 남기는 전통적인 침습적 미생물 프로브를 보완 할 수 있습니다. DNA 또는 세포 함량 추출2,3, 시투 혼성화4의 형광등, 그리고 게놈에 형광 기자 유전자의 도입과 같은 전통적인 기술은 세포 유형 또는 생리적 상태를 결정하는 데 효과적이지만, 일반적으로 세포의 조작또는 침습적 태깅이 필요합니다.
벌크 미생물 배양 현탁액5,6, 활성 슬러지7, 포유류 조직8,9 및 포유류 세포를 포함한 다양한 살아있는 및 그대로 미생물 식민지의 타고난 형광에 대한 연구는 선천성 형광 분석이 세포 유형 및 생리 상태의 태그 없는 분석을 용이하게 하는 것으로 나타났습니다. 선천적 형광 서명은 전통적으로 단일 세포 수준이 아닌 인구 수준에서 분석되어 클론 배양을 필요로합니다. 대조적으로, confocal refction microscopy-보조 단세포 innate fluorescence 분석 (CRIF) 기술11 여기에 설명된 각 개별 살아있는 미생물 세포의 타고난 세포 형광 서명을 재구성하고 카탈로그합니다. 더욱이, CRIF는 3차원(3D) 공간에 분포되는 집단 내에서 단일 미생물 세포의 선천적 형광 서명을 체계적으로 대조할 수 있다.

<table><tbody><tr><td>Agarose</td><td>Wako Chemicals</td><td>312-01193</td><td></td></tr><tr><td>Beam splitters</td><td>Carl Zeiss, Nikon</td><td></td><td>MBSInVis405, MBS458, MBS488, MBS458/514, MBS488/543, or MBS 488/543/633 beam splitters (Carl Zeiss)</td></tr><tr><td>Confocal microscope</td><td>Carl Zeiss, Nikon</td><td></td><td>Model LSM 880 (Carl Zeiss), Model A1R (Nikon)</td></tr><tr><td>Cover slips</td><td>Matsunami Glass</td><td>C024601</td><td></td></tr><tr><td>Glass slides</td><td>Matsunami Glass</td><td>S011120</td><td></td></tr><tr><td>Half-reflection mirror</td><td>Carl Zeiss, Nikon</td><td></td><td>NT80/20</td></tr><tr><td>Laser power meter</td><td>Thorlabs</td><td></td><td>PM400 (power meter console) and S175C (sensor)</td></tr><tr><td>LB Broth</td><td>Nacalai tesque</td><td>20066-95</td><td>For bacteria culture</td></tr><tr><td>Image analysis software</td><td>The MathWorks</td><td></td><td>MATLAB version 2019a or later, Image Processing Toolbox is needed</td></tr><tr><td>Microscope objective</td><td>Carl Zeiss, Nikon</td><td>440762-9904</td><td>e.g. 63x plan Apochomat NA = 1.4 (Carl Zeiss)</td></tr><tr><td>Microscope software</td><td>Carl Zeiss, Nikon</td><td></td><td>ZEN (Carl Zeiss),NIS-elements (Nikon)</td></tr><tr><td>PBS(-)</td><td>Wako Chemicals</td><td>166-23555</td><td></td></tr><tr><td>Programming language</td><td></td><td></td><td>Python and libraries, modules (numpy, scikit-learn, scikit-image, os, glob, matplotlib, tkinter) are rquired to run the supplied PCA script.</td></tr><tr><td>Silicone gasket</td><td>ThermoFisher Scientific</td><td>P24744</td><td></td></tr><tr><td>Workstation</td><td></td><td></td><td>A high-performance workstation with discrete GPUs is recommended.</td></tr><tr><td>Yeast extract-peptone-dextrose (YPD) agar medium</td><td>Sigma-Aldrich</td><td>Y1500-250G</td><td>For yeast culture</td></tr><tr><td>YPD medium</td><td>Sigma-Aldrich</td><td>Y1375-250G</td><td></td></tr></tbody></table>

reconstruction of single-cell innate fluorescence signatures by confocal microscopy

여기서 설명된 공초점 반사 현미경 보조 단세포 인천성광 분석(CRIF),3차원(3D) 공간에 분포된 인구에서 각 개별 라이브 셀로부터 선천적인 세포 형광 시그니처를 재구성하는 최소 침습적 방법이다. 세포의 타고난 형광 시그니처는 세포 내의 다양한 생체 분자에 의해 방출되는 형광 신호의 모음입니다. 이전 연구는 타고난 형광 서명이 다양한 세포 특성과 생리적 상태의 차이를 반영하고 세포 특성화 및 식별을위한 풍부한 정보 소스임을 확립했습니다. 선천적 형광 서명은 전통적으로 인구 수준에서 분석되어 복제 배양을 필요로하지만 단일 세포 수준에서는 그렇지 않습니다. CRIF는 개별 세포에서 형광 신호의 3D 해상도 및/또는 선택적 추출을 요구하는 연구 결과에 특히 적합합니다. 형광 서명은 세포의 타고난 특성이기 때문에 CRIF는 그대로 및 단일 세포의 유형 및 / 또는 생리 적 상태의 태그없는 예측에도 적합합니다. 이 방법은 이종 집단에서 각 단일 세포의 표현형이 세포 태그없이 현미경의 밑에 그것의 자동 형광 서명에 의해 직접 평가될 수 있는 능률적인 세포 분석을 위한 강력한 공구일 지도 모릅니다.

도 1A는 전통적인 스펙트럼 플롯(top)과 히트맵(middle)으로 제시된 세균 세포의 전형적인 단세포 형광 서명을 나타낸다. 도 1B는 토양 박테리아집단의 원래 CRM 이미지 위에 중첩된 정확한 2D 세포 세분화의 결과를 나타낸다(슈도모나스 푸티다 KT2440)12. 인구에 대한 그 결과 타고난 형광 서명은 도 1D의 히트맵으...

Watch this Scientific Journal Video about Reconstruction of Single-Cell Innate Fluorescence Signatures by Confocal Microscopy at JoVE.com

Reconstruction of Single-Cell Innate Fluorescence Signatures by Confocal Microscopy

여기서, 3차원 공간에 분포된 모든 개별 라이브 셀로부터 광적으로 추출 및 분류하는 프로토콜(즉, 세포 자동 형광)을 제시한다. 이 방법은 박테리아, 곰팡이, 효모, 식물 및 동물의 세포를 포함하여 단일 세포 해상도에서 다양한 생물학적 시스템의 타고난 형광 서명을 연구하는 데 적합합니다.

공초점 현미경 검사법에 의한 단세포 인천성형광서명의 재구성

Described here is confocal reflection microscopy-assisted single-cell innate fluorescence analysis (CRIF), a minimally invasive method for reconstructing ...

reconstruction-single-cell-innate-fluorescence-signatures-confocal

Research

JoVE Journal

Biology

2.7K 조회수.  University of Tsukuba. 여기서, 3차원 공간에 분포된 모든 개별 라이브 셀로부터 광적으로 추출 및 분류하는 프로토콜(즉, 세포 자동 형광)을 제시한다. 이 방법은 박테리아, 곰팡이, 효모, 식물 및 동물의 세포를 포함하여 단일 세포 해상도에서 다양한 생물학적 시스템의 타고난 형광 서명을 연구하는 데 적합합니다.

비디오: Reconstruction of Single-Cell Innate Fluorescence Signatures by Confocal Microscopy

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors1. This process leads to bacterial invasion of endothelial cells2. More recently, we observed an interaction of Opc with a 100kDa protein found in whole cell lysates of human cells3. We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as &alpha;-actinin. As no surface expressed &alpha;-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and &alpha;-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with &alpha;-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &#945;-actinin by Confocal Imaging

Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human &alpha;-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular &alpha;-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).

의 세포 상호 작용 Visualisation 및 부량 Neisseria meningitidis</em공촛점 이미징에 의해 α - actinin>과 인간

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

연구

면역학 및 감염병학

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

단일 분자 형광 현미경에 의한 수용체 역학의 고해상도 시공간 분석

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

생체공학

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

실시 한 형광 현미경으로 여러 영상 모드

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

단일 세포 형광 현미경 검사 법에 의해 Efferocytosis의 정량화

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

정확한 세포 표현과 정확한 단백질 국소화를 위한 세균 세포의 3차원 이미징

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

세균 본부 기계의 슈퍼 해상도 이미징

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

생물학

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

공 초점 및 다중 광자 현미경을 사용하여 다섯 형광 단백질에 의해 표시됨 조혈 줄기 및 전구 세포의 생체 내 클론 추적에서

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

일렉트로을 통해 생활 미생물의 국제화 및 형광 생체 분자의 관측

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

Confocal 현미경 검사 법 밝혀 세포 표면 수용 체 이미지 상관 분광학을 통해 집계

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...