이 프로젝트는 NSF (# 0905041, # 0079874), 텍사스 고급 기술 프로그램 (# ATP - 000512-0146 - 2001, # ATP - 000512-0261 - 2001), 텍사스는 (# 1R01 - NS54252) NIH / NINDS에 의해 투자되었다 A &amp; M 학술 진흥 재단, 텍사스 A &amp; M 대학, 3Scan에서 컴퓨터 과학 및 공학과. 우리는 기술 상담 KESM 장비에 대한 지원 버나드 메사 (마이크로 스타 기술)을 감사드립니다. KESM의 설계 및 구현의 주요 부분이 2007 년 사망 브루스 H. 맥코믹에 의해 수행되었다.

1. 표본 준비 : 잠돌군 Zz - 콕스 <ol><li> 이 프로토콜은 밀접하게 Mayerich 외의 프로토콜. 7을 따릅니다. </li><li> C57BL/6J 마우스 isoflurane 흡입 마취를 사용하여 깊은 anesthetized하고 다음 참수. </li><li> 두뇌는 잠돌군 Zz - 콕스 고정 솔루션 (1 % 칼륨의 크롬, 1 % 칼륨 dichromate, 그리고 탈이온수에 1퍼센트 mercuric 염화)로 제거하고 배치됩니다. </li><li> 두뇌는 10~16주 동안 상온에서, 어둠 속에서 잠돌군 Zz - 콕스 솔루션에 남아있다. </li><li> 두뇌는 어둠 속에서 밤새 탈이온수에 씻어서 있습니다. </li><li> 씻어서 두뇌는 상온에서 어둠 속에 7-10일 위해 탈이온수에 5 % 수산화 암모늄 용액에 포장되어 있습니다. 이 긴 준비 시간은 검은 침전물이 완전히 조직 형태에 대한 기회가되도록, 전체 두뇌의 침투를 보장하는 것이었다. </li><li> 두뇌는 4 시간 t에 대한 실온에서 탈이온수 다시 씻어서입니다실온에서 50 % 70% 냉장고 (4 ° C)에서 85 %, 95 % (3 변경) 및 100 % (4-5 변경) : 등급 에틸 알콜의 시리즈를 통해 탈수 암탉. 두뇌는 24 시간 동안 각각의 용액에 남아 있습니다. </li><li> 탈수 두뇌는 다음 1시 2분의 araldite - 투 - 아세톤 비율 1시 1분, 2시 1분, 마지막에있는 araldite - 아세톤 혼합하여 다음 (3-4 변경, 하루마다) 아세톤에 들어가게된다 냉장고에 100 % araldite (3 변화, 하루 때마다), (4 ° C). </li><li> 마지막으로 치료 두뇌는 100 % araldite에 포함된 및 ° C 삼일 (애보트와 Sotelo 2를 참조하라의 내장 프로토콜)에 대한 60 가열합니다. 표본은 LR 화이트에 포함된 경우 다음 표본 전에 중합에 대한 표준 절차 각 하룻밤 냉장고에 보관 LR 화이트 솔루션의 세 변화를 통해 지난 100 % 에탄올 용액에서 전송됩니다. 세 가지 변경 사항은 전체 침입을 보장하기 위해 수행됩니다. 표본은 t이다60 닫힌 용기에 polymerized는 신선한 LR 화이트로 전송 암탉 ° 적절한 중합 시간 및 온도의 필요한 금액 24 시간 C,. </li><li> 그림. 1 Araldite에 포함된 잠돌군 Zz - 콕스 묻은 머리를 보여줍니다. </li><li> 완치 표본 블록은 다음 에폭시를 사용하여 금속 표본 링에 장착하고, 블록의 측면은 (그림을 참조하십시오. 2) 필요로 손질합니다. </li></ol> 2. 표본 준비 : Nissl <ol><li> 마우스 케타민을 및 xylazine (1.7 MG 케타민을 / 20g의 체중 당 0.26 MG의 xylazine) 주입 intraperitoneally를 사용하여 깊은 anaesthetized하고 다음 transcardially 실온 50 ML을 사용 perfused 인산 버퍼 객실 250 ML 다음 염분을 (산도 7.4) 온도 중립 10 % (산도 7.4) 포르말린을 버퍼. 그리고 마지막으로 undiluted 인도 잉크의 3.0 CC와 함께. </li><li> 염분과 정착액과 함께 온몸 재관류는 심장 혈관 시스템에서 혈액을 취소하고 TI를 해결하는 데 필요한<html> ssues. 인도 잉크와 재관류 완전히 심장 혈관 시스템의 vasculature을 채우기 위해 필요합니다. </li><li> 그 결과 뇌는 등급 에틸 알콜 (25 % -100 %)의 시리즈를 통해 다음 탈수하고 다음 위의 1.8-1.9에서 다음과 같은 프로토콜 araldite 플라스틱에 포함된. LR 화이트 포함 매체있다면 위의 1.10의 프로토콜은 다음입니다. </li></ol> 4. 표본 준비 : 일반 종, 일반 기관 <ol><li> 일반적인 경우를, 고정가 중 10% 중성 포르말린 버퍼 또는 4 % paraformaldehyde로 할 수 있습니다. </li><li> 작은 조직 볼륨 대신 재관류의 보급은 고정 및 얼룩을 권장합니다. 작은 생물이나 기관의 경우도 얼룩은 탈수 과정에서 할 수 있습니다. </li><li> 솔루션 침투를위한 시간이 기관 또는 전체 마우스 뇌보다 훨씬 작은 생물에 대한 줄일 수 있지만 내장 프로토콜은 상기와 동일합니다. </li></ol>jove_title &quot;&gt; 5. KESM 설정 및 이미징 <ol><li> 펌프를 끄고, 무대 설정 카메라 설정, 조명기 켭니다. </li><li> 나이프와 목적을 올립니다. </li><li> 표본 반지를 설치합니다. </li><li> 측정 표본의 크기와 KESM 스테이지 Controller2 응용 프로그램 구성 파일을 만듭니다. </li><li> KESM 단계 Controller2를 시작하고 무대를 초기화합니다. </li><li> 낮은 칼 / 목표, 펌프를 켜십시오. </li><li> 광학 기차에 초점 노브를 돌려 칼날에 상대적인보기의 카메라의 필드를 조정하여, 객관적이고 카메라를 중점을두고 있습니다. </li><li> 이미징을 시작 [이동]을 누르십시오. </li><li> 참조은 무화과. 3-8. 기술적인 세부 9, 7을 참조하십시오. </li></ol> 6. 영상 처리 및 데이터 준비 <ol><li> 설정 파일 폴더 (00000, 00001 등)에서 데이터 폴더로 KESM 스택 프로세서 실행 파일을 복사합니다. </li><li> 조명 유물을 제거하고 모든 배경 강도를 정상화하기 위해 KESM 스택 프로세서를 실행이미지를 표시합니다. 모든 데이터 하위 폴더에 대한 반복합니다. </li><li> 데이터 multiscale 타일을 준비 설정하고 업로드하고 KESM 두뇌 아틀라스 웹 서버에있는 파일을 설정합니다. </li><li> 그림을 참조하십시오. 9. </li></ol> 7. 데이터 시각화 및 분석 <ol><li> KESM 뇌 아틀라스를 사용하여 시각화. 이동 <a href="http://kesm.cs.tamu.edu">http://kesm.cs.tamu.edu</a> 하고 해당 아틀라스를 선택합니다. <ol><li> Google지도에서와 같이 이동합니다. </li><li> Google Maps에로와 축소. </li><li> [-] Z 깊이를 증가 또는 감소에 다른 깊이로 이동하려면, 중 [+] 또는 누른 다음 풀다운 메뉴에서 각각의 단계에서 이동하는 방법에 깊이를 선택합니다. </li><li> 풀다운 메뉴를 사용하여 오버레이 번호를 조정합니다. </li><li> 풀다운 메뉴를 사용하여 오버레이 간격을 조정합니다. </li><li> 그림을 참조하십시오. 11. </li></ol></li><li> MeVisLab를 사용하여 시각화. <ol><li> MeVisLab를 시작합니다. </li><li> 새 프로젝트를 만듭니다. </li><li> 다음에, 새로운 모듈을 타이로 만들 수 있습니다명령 상자에서 모듈 이름으로 Ping을 실행합니다. </li><li> 생성 모듈 [Compose3Dfrom2DFiles], 그리고 원하는 폴더로 이동합니다. 파일 문자열을 입력하고 [GetFileList]을 클릭합니다. 선택된 이미지가 메모리에 들어갈 수 있는지 확인하십시오. 클릭하여 [Create3D]는 볼륨을 만들 수 있습니다. </li><li> 생성 모듈 [SetWorldMatrix] 아래에 &quot;크기 조정&quot;을 설정 해당 voxel 크기 (0.6, 0.7, 10X 0.45NA 목적을 위해 1.0)로 X, Y, Z &quot;를 초등학교가 변환&quot;. 링크 [Compose3Dfrom2DFiles]을 [SetWorldMatrix]. </li><li> 매트릭스를 설정하고 &quot;앞으로&quot;, &quot;무시&quot;, &quot;무시&quot;를 &quot;매트릭스 구성&quot;아래 변환. </li><li> 생성 모듈 [Arithmetic1]와 &quot;(맥스 IMG) 반전&quot;에 &quot;기능&quot;을 설정합니다. 링크 [SetWorldMatrix]을 [Arithmetic1]. </li><li> 생성 모듈 [View3D] 및 [Arithmetic1]를 연결합니다. </li><li> View3D에서 마우스 오른쪽 버튼을 누를 동안 임계값을 조정 주위 마우스를 이동합니다. 당신은 지역 변경 방향 (왼쪽 마우스 버튼을 누를있는 동안 마우스 이동), 변경 조명을 (볼륨 renderin 작물 수 있습니다G 또는 MIP) 등에 / 배경 해제 </li><li> 그림을 참조하십시오. 10. </li></ol></li></ol> 8. 대표 결과 : 여기, 우리는 전체 - 뇌 데이터와 정보를 제시한다. 무화과. 12-15보기 전체 - 뇌 인도 잉크, 잠돌군 Zz, 그리고 Nissl 데이터는 1, 4, 3을 설정합니다. <img alt="그림 1" src="/files/ftp_upload/3248/3248fig1.jpg" /> 그림 1., 고정 스테인드 및 임베디드 마우스 뇌 <img alt="그림 2" src="/files/ftp_upload/3248/3248fig2.jpg" /> 그림 2. 임베디드 마우스 뇌 표본은 표본 링에 장착. <img alt="그림 3" src="/files/ftp_upload/3248/3248fig3.jpg" /> 그림 3. 나이프 에지 스캐닝 현미경 (KESM). KESM의 사진은 자사의 주요 구성 요소가 표시와 함께 표시됩니다 : (1) 고속 라인 스캔 카메라 (2) 현미경의 목적, (3) 다이아몬드 나이프 어셈블리 및 조명 콜리 메 이터를 (4) specimen 탱크 (물 침지 영상에 대한), (5) 세 축 정밀 에어 베어링 스테이지 (6) 흰색 가벼운 현미경 조명기, sectioned 조직의 제거 (7) 펌프 (뒤쪽), (8) 무대 제어 및 이미지 수집, (9) 화강암베이스, 그리고 (10) 화강암 다리를 PC 서버. <img alt="그림 4" src="/files/ftp_upload/3248/3248fig4.jpg" /> 그림 4. KESM의 이미징의 원칙. KESM 운영의 주요한는 그림입니다. 객관적이고 나이프는 (폭 5mm 표본 20 NM 및 1-5의 여행 속도의 해상도에서 위치 단계 이동 (실선과 화살표)에 부착된 동안 장소에서 개최되며, 다이아몬드 나이프에 대한 스크랩한 도착 10X 목적을 위해), 칼 (실선과 화살표)를 통해 흐르는 얇은 섹션을 생성. 라인 스캔 영상은 칼로의 매우 끝 가까이에 이루어집니다. <img alt="그림 5" src="/files/ftp_upload/3248/3248fig5.jpg" /> 그림 5. KESM 카메라와 목표를 중심으로 제어합니다. <img alt="그림 6" src="/files/ftp_upload/3248/3248fig6.jpg" /> 그림 6. KESM 나이프 어셈블리 및 제어합니다. <img alt="그림 7" src="/files/ftp_upload/3248/3248fig7.jpg" /> 그림 7. 관측 포트를 통해 초점을 초기. <img alt="그림 8" src="/files/ftp_upload/3248/3248fig8.jpg" /> 그림 8. KESM 스테이지 컨트롤러 2 응용 프로그램 (스크린샷). <img alt="그림 9" src="/files/ftp_upload/3248/3248fig9.jpg" /> 그림 9. KESM 스택 프로세서 응용 프로그램 (스크린샷). <img alt="그림 10" src="/files/ftp_upload/3248/3248fig10.jpg" /> 그림 10. MeVisLab 응용 프로그램 (스크린샷). <img alt="그림 11" src="/files/ftp_upload/3248/3248fig11.jpg" /><br/&gt; 그림 11 KESM 뇌 아틀라스 :. 웹 인터페이스 (스크린샷). <img alt="그림 12" src="/files/ftp_upload/3248/3248fig12.jpg" /> 그림 12. KESM 전체 - 뇌 인도 잉크 데이터입니다. KESM 데이터 스택의 볼륨 시각화는 혈관 데이터 집합에 대한 표시됩니다. (A) 클로즈업 혈관 데이터. 폭 ~ 100 음. 전체 마우스 뇌 vasculature의 (BD) 3 표준보기 (고해상도 데이터 subsampled). 너비는 10mm ~. <img alt="그림 13" src="/files/ftp_upload/3248/3248fig13.jpg" /> 그림 13. KESM 전체 - 뇌 마우스 잠돌군 Zz 데이터입니다. <img alt="그림 14" src="/files/ftp_upload/3248/3248fig14.jpg" /> 그림 14. KESM 잠돌군 Zz 데이터 세부 사항. <img alt="그림 15" src="/files/ftp_upload/3248/3248fig15.jpg" /> 그림 15. KESM 전체 - 뇌 Nissl 데이터입니다. (A) 클로즈업 N의issl 데이터가 없습니다. ~ 300 음 3. 전체 마우스 뇌 Nissl 데이터 (BD) 3 표준보기 (고해상도 데이터 subsampled). 크로스 섹션은 내부 구조의 명확한 보려면 표시됩니다.

<ol>
	<li>Abbott, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+imaging+of+whole+small+animal+brains+with+the+knife-edge+scanning+microscope.">High-throughput imaging of whole small animal brains with the knife-edge scanning microscope.</a> Neuroscience Meeting Planner. Program No. 504.2, (2008).</li><li>Abbott, L. C., Sotelo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+catecholaminergic+innervation+in+weaver+and+normal+mouse+cerebellar+cortices.">Ultrastructural analysis of catecholaminergic innervation in weaver and normal mouse cerebellar cortices.</a> Journal of Comparative Neurology. 426, 316-329 (2000).</li><li>Choe, Y., Abbott, L. C., Miller, D. E., Han, D., Yang, H. -. F., Chung, J. R., Sung, C., Mayerich, D., Kwon, J., Micheva, K., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+imaging,+analysis,+and+integration+of+mouse+brain+networks.">Multiscale imaging, analysis, and integration of mouse brain networks.</a> Neuroscience Meeting Planner. Program No. 516.3, (2010).</li><li>Choe, Y., Han, D., Huang, P. -. S., Keyser, J., Kwon, J., Mayerich, D., Abbott, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+submicrometer+scans+of+mouse+brain+microstructure:+Neurons+and+vasculatures.">Complete submicrometer scans of mouse brain microstructure: Neurons and vasculatures.</a> Neuroscience Meeting Planner. Program No. 389.10, (2009).</li><li>Choe, Y., Abbott, L. C., Han, D., Huang, P. S., Keyser, J., Kwon, J., Mayerich, D., Melek, Z., McCormick, B. H., Ravi Rao, A., Cecchi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knife-edge+scanning+microscopy:+High-throughput+imaging+and+analysis+of+massive+volumes+of+biological+microstructures.">Knife-edge scanning microscopy: High-throughput imaging and analysis of massive volumes of biological microstructures.</a> High-Throughput Image Reconstruction and Analysis: Intelligent Microscopy Applications. , 11-37 (2008).</li><li>Choe, Y., Abbott, L. C., Ponte, G., Keyser, J., Kwon, J., Mayerich, D., Miller, D., Han, D., Grimaldi, A. M., Fiorito, F., Edelman, D. B., McKinstry, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charting+out+the+octopus+connectome+at+submicron+resolution+using+the+knife-edge+scanning+microscope.">Charting out the octopus connectome at submicron resolution using the knife-edge scanning microscope.</a> BMC Neuroscience. 11, 136-136 (2010).</li><li>Mayerich, D., Abbott, L. C., McCormick, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knife-edge+scanning+microscopy+for+imaging+and+reconstruction+of+three-dimensional+anatomical+structures+of+the+mouse+brain.">Knife-edge scanning microscopy for imaging and reconstruction of three-dimensional anatomical structures of the mouse brain.</a> Journal of Microscopy. 231, 134-143 (2008).</li><li>Mayerich, D., Kwon, J., Sung, C., Abbott, L. C., Keyser, J., Choe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+macro-scale+Transmission+Imaging+of+Microvascular+Networks+Using+KESM.">Fast macro-scale Transmission Imaging of Microvascular Networks Using KESM.</a> Biomedical Optics Express. 2, 2888-2896 (2008).</li><li>McCormick, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+Brain+Tissue+Scanner.">Development of the Brain Tissue Scanner.</a> Technical Report. , (2002).</li><li>Sporns, O., Tononi, G., K&#246;tter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+connectome:+A+structural+description+of+the+human+brain.">The human connectome: A structural description of the human brain.</a> PLoS Computational Biology. 1, 42-42 (2005).</li></ol>

토드 호프만은 3Scan, KESM 기술 (미국 특허 # 6,744,572)를 제조하고 시장에 상업적인 회사입니다.

KESM은 생물 학적 표본 (~ 1cm 3) 대량의 submicrometer 수준의 조사를 허용합니다. 볼륨의이 종류가 같은 뇌, 폐, 심장, 신장 등 모든 작은 동물 장기를 가질 정도이며, 등 이러한 기관의 구조 조직에 대한 전례없는 양적 정보를 제공하고, 다양한 기능의 전산 모델링을 활성화할 수 같은 기관에서 이미지를 검색 회로 및 네트워크 역학, 전기적 특성, 유체 및 공기 흐름 역학, 그리고 근육의 역학을 포함하여 이들 기관의 측면. 이 문서에서 자세한 표본 준비 프로토콜 및 사후 분석 프로토콜 KESM에 쉽게 액세스하여 결과 데이터를 가지고 외부 연구 그룹을 도울 것으로 예상된다. KESM 작업 프로토콜이 독특한 이미징 양상의 장점과 제한 사항을 감사하기 위해 이러한 외부 연구자 도움이 될 것입니다.

A correction was made to <a href="http://www.jove.com/video/3248">Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy</a>. Important modifications were made to the Nissl staining protocol.

A correction was made to <a href="http://http://www.jove.com/video/3248">Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy</a>. Important modifications were made to the Nissl staining protocol.

Erratum: Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

specimen preparation, imaging, and analysis protocols for knife-edge scanning microscopy

높은 처리량, 높은 해상도, 3D 현미경 기술의 주요 발전 submicrometer 해상도 neuroanatomical 큰 데이터 볼륨의 인수를 사용할 수있다. 전체 - 뇌 규모의 데이터를 생산 최초의 악기 중 하나는 나이프 - 에지 스캐닝 현미경 (KESM) 7, 5, 9, 개발 및 저자의 실험실에서 호스팅됩니다. KESM는 복잡한의 연결 네트워크의 세부 사항 (잠돌군 Zz) 1, 4, 8, 혈관 네트워크 (인도 잉크) 1, 4, 및 세포 본문 배포 (Nissl) 3 공개, 섹션 및 submicrometer 해상도에서 이미지 전체를 마우스 뇌에 사용되고 있습니다 . KESM의 사용은 마우스이나 두뇌에 제한되지 않습니다. 우리는 성공적으로 문어의 두뇌 6, 마우스 폐, 및 쥐 두뇌를 몇 군데있다. 현재 전체 제브라 다 니오의 배아에 노력하고 있습니다. microcirculation 및 hemodynamic 연구,, 이런 데이터는 크게 connectomics 연구 10 기여할 수 있으며, provi에 의해 stereology 연구에땡 지상 진실을 정확하게. 이 문서에서는, 우리는 표본 준비 (고정, 얼룩 및 포함)를 포함 파이프라인, KESM 구성 및 설정, sectioning 및 이미징 KESM와 함께, 이미지 처리, 데이터 준비, 데이터 시각화 및 분석에 대해 설명합니다. 강조 표본 준비와 얻은 KESM 데이터의 시각화 / 분석됩니다. 우리는이 문서에서 제시한 자세한 프로토콜 KESM하고 활용도를 높이기 위해 접근을 확대 데 도움을 기대합니다.

Watch this Scientific Journal Video about Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy at JoVE.com

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

뇌 표본 준비에서 데이터 시각화 및 분석에, 나이프 - 에지 스캐닝 현미경을 사용하여 시리얼 sectioning 이미징의 전체 과정을 설명합니다. 이 기술은 현재 마우스 뇌 데이터를 취득하는 데 사용하지만, 다른 기관, 다른 수종에 적용됩니다.

나이프 - 에지 스캐닝 현미경에 대한 표본 준비, 이미징 및 분석 프로토콜

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

specimen-preparation-imaging-analysis-protocols-for-knife-edge

Research

JoVE Journal

Bioengineering

17.6K 조회수.  Texas A&M University. 뇌 표본 준비에서 데이터 시각화 및 분석에, 나이프 - 에지 스캐닝 현미경을 사용하여 시리얼 sectioning 이미징의 전체 과정을 설명합니다. 이 기술은 현재 마우스 뇌 데이터를 취득하는 데 사용하지만, 다른 기관, 다른 수종에 적용됩니다.

비디오: Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

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

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

원자 힘 현미경에 의해 영상에 대한 세균 고정

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

비디오 - 속도 검색 공촛점 현미경과 Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

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

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

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

형광 현미경과 영상 셀 마이그레이션을위한 접착제 및 가용성식이 그라디언트를 만들기

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

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

동적 상호 작용의 3 차원 구조 분석을위한 상호 현미경

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

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., ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

운모의 준비 고해상도 광학 현미경 이미징 지질 이중층 지원

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Hybrid &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Hybrid  &#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

하이브리드 μCT-FMT 및 촬상 화상 해석

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

원자 힘 현미경 이미징 및 지원 지질 이중층의 힘 분광학

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

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

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

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

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

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

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