이 작품은 Hukriede, NCI / NIH (P01 CA78039) 포그트하고, NHLBI / NIH (1R01HL088016) Tsang로하는 NICHD / NIH (1R01HD053287)에 의해 재정 지원을합니다.

1) zebrafish matings 및 BCI 처리를 설정합니다. <ol><li> 개별적으로 짝짓기 탱크 하나에 pt6 zebrafish 및 장소 : 이틀 전에 치료, homozygous 남성 TG를 (d2eGFP dusp6) 식별합니다. 구분 및 야생 타입 AB * 여성의 물고기를 추가하고 야간 짝짓기 새장에 둡니다. </li><li> 다음날 아침은 구분 기호를 제거하고 1 시간 뒤에 배아를 수집합니다. E3 (5 MM NaCl, 0.17 MM KCl, 0.33 MM CaCl2, 0.33 MM MgSO4), 6 시간 동안 28 ° C에서 솔루션과 부화와 100mm 요리 배아를 전송합니다. 죽은 제거하고 배아를 unfertilized, 신선한 E3 솔루션을 추가하고 밤새 품어. </li><li> 유사한 단계 (24hpf)과 균일한 GFP 발현을위한 유전자 변형 배아를 정렬하십시오. 96 - 웰 플레이트의 각 우물에 개별적으로 배열 배아. </li><li> micropipette과 초과 E3 솔루션을 제거합니다. 각 우물에 멀티 채널 pipettor와 E3 솔루션의 200μl를 추가합니다. </li><li> 로 표시된 다음과 같은 화합물을 추가합니다 : <table border="1"><tbody><tr><td></td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td><td> 8 </td><td> 9 </td><td> 10 </td><td> 11 </td><td> 12 </td></tr><tr ><td> </td><td> 2μl DMSO </td><td> 2μl BCL (0.1mM) </td><td> 2μl BCL (0.5mM) </td><td> 2μl BCL (1mM) </td><td> 2μl BCL (2mM) </td><td> 2μl BCL (2.5mM) </td><td> 2μl BCL (0.1mM) </td><td> 2μl BCL (0.5mM) </td><td> 2μl BCL (1mM) </td><td> 2μl BCL (2mM) </td><td> 2μl BCL (2.5mM) </td><td> 2μl DMSO </td></tr><tr ><td> B </td><td> 2μl DMSO </td><td> 2μl BCL (0.1mM) </td><td> 2μl BCL (0.5mM) </td><td> 2μl BCL (1mM) </td><td> 2μl BCL (2mM) </td><td> 2μl BCL (2.5mM) </td><td> 2μl BCL (0.1mM) </td><td> 2μl BCL (0.5mM) </td><td> 2μl BCL (1mM) </td><td> 2μl BCL (2mM) </td><td> 2μl BCL (2.5mM) </td><td> 2μl DMSO </td></tr><tr ><td> C </td><td> 2μl DMSO </td><td> 2μl BCL (0.1mM) </td><td> 2μl BCL (0.5mM) </td><td> 2μl BCL (1mM) </td><td> 2μl BCL (2mM) </td><td> 2μl BCL (2.5mM) </td><td> 2μl BCL (0.1mM) </td><td> 2μl BCL (0.5mM) </td><td> 2μl BCL (1mM) </td><td> 2μl BCL (2mM) </td><td> 2μl BCL (2.5mM) </td><td> 2μl DMSO </td></tr><tr ><td> D </td><td> 2μl DMSO </td><td> 2μl BCL (0.1mM) </td><td> 2μl BCL (0.5mM) </td><td> 2μl BCL (1mM) </td><td> 2μl BCL (2mM) </td><td> 2μl BCL (2.5mM) </td><td> 2μl BCL (0.1mM) </td><td> 2μl BCL (0.5mM) </td><td> 2μl BCL (1mM) </td><td> 2μl BCL (2mM) </td><td> 2μl BCL (2.5mM) </td><td> 2μl DMSO </td></tr><tr ><td> E </td><td> 2μl DMSO </td><td> 2μl BCL (0.1mM) </td><td> 2μl BCL (0.5mM) </td><td> 2μl BCL (1mM) </td><td> 2μl BCL (2mM) </td><td> 2μl BCL (2.5mM) </td><td> 2μl BCL (0.1mM) </td><td> 2μl BCL (0.5mM) </td><td> 2μl BCL (1mM) </td><td> 2μl BCL (2mM) </td><td> 2μl BCL (2.5mM) </td><td> 2μl DMSO </td></tr><tr ><td> F </td><td> 2μl DMSO </td><td> 2μl BCL (0.1mM) </td><td> 2μl BCL (0.5mM) </td><td> 2μl BCL (1mM) </td><td> 2μl BCL (2mM) </td><td> 2μl BCL (2.5mM) </td><td> 2μl BCL (0.1mM) </td><td> 2μl BCL (0.5mM) </td><td> 2μl BCL (1mM) </td><td> 2μl BCL (2mM) </td><td> 2μl BCL (2.5mM) </td><td> 2μl DMSO </td></tr><tr ><td> G </td><td> 2μl DMSO </td><td> 2μl BCL (0.1mM) </td><td> 2μl BCL (0.5mM) </td><td> 2μl BCL (1mM) </td><td> 2μl BCL (2mM) </td><td> 2μl BCL (2.5mM) </td><td> 2μl BCL (0.1mM) </td><td> 2μl BCL (0.5mM) </td><td> 2μl BCL (1mM) </td><td> 2μl BCL (2mM) </td><td> 2μl BCL (2.5mM) </td><td> 2μl DMSO </td></tr><tr ><td> H </td><td> 2μl DMSO </td><td> 2μl BCL (0.1mM) </td><td> 2μl BCL (0.5mM) </td><td> 2μl BCL (1mM) </td><td> 2μl BCL (2mM) </td><td> 2μl BCL (2.5mM) </td><td> 2μl BCL (0.1mM) </td><td> 2μl BCL (0.5mM) </td><td> 2μl BCL (1mM) </td><td> 2μl BCL (2mM) </td><td> 2μl BCL (2.5mM) </td><td> 2μl DMSO </td></tr></tbody></table> 최종 농도는 1 %의 DMSO에 5μM, 10μM, 20μM, 25μM BCI 1μM 있습니다. 알루미늄 호일의 플레이트 커버 28 6 시간 동안 품어 ° C.를 </li></ol> 96 - 웰 플레이트 및 분석 2) 자동 이미징. <ol><li> 30hpf 배아를 마취 각 잘하는 tricaine (0.61mM)의 10μl를 추가합니다. </li><li> 분자 장치 Imagexpress 울트라에 플레이트를로드합니다. </li><li> 오픈 Metaxpress 소프트웨어. 4X 객관적이고 최대 핀홀 직경과 구성을 선택, 레이저 오토포커스는 접시 바닥을 감지하도록 구성하고, Z - 비행기의 관심 영역을 감지하는 최적의 결정합니다. 525분의 488 NM (GFP)의 여기 / 발광 파장의 형상을 습득. 노 비닝과 2000x2000 픽셀 (4 X 4mm)의 단일 사이트로 영역을 스캔 설정합니다. 레이저 전원을 구성하고 포화하지 긍정적인 컨트롤 이미지 같은 것을 밝은 지역을 습득합니다. 몇 가지 긍정적이고 부정적인 제어 우물에 적절한 이미지 수집을 확인합니다. </li><li> 판 검사를 시작합니다. 악기는 자동으로 모든 우물 및 아카이브들을 SQL 서버 데이터베이스에서에서 이미지를 획득합니다. </li><li> Cellenger 응용 프로그램 모듈과 분자 장치 Imagexpress 울트라 플랫폼에 의해 생성된 이미지 포맷 및 파일 구조를 인식 가져오기 필터를 사용하여 Definiens 개발자에 이미지를 업로드합니다. </li><li> 사용자 정의 설계 인식 네트워크 기술 알고리즘 (또한 ruleset 칭했다)과 프로세스 이미지. 우리가 정의가 최근에 출판된 ruleset 2를 기반으로 개발된 알고리즘은, 자동으로 배아, 난황, 그리고 머리를 식별하고, GFP - 표현 헤드 구조의 밝기와 영역을 계량하기 위해 설계되었습니다. 더 배아가 감지되지되는거나 머리가 unambiguously (예를 들어, 초점 이미지의 높은 화합물 독성 또는 밖으로)를 할당할 수 없습니다 어디 웰스는 분석에서 제외됩니다. 몇 가지 부정적인 통제 이미지 (차량 취급)와 긍정적인 이미지 제어 (BCI이 대접) 머리와 노른자가 올바르게 모두에서 확인할 수 있습니다 그러한 사용하여 알고리즘을 구성합니다. 밝은 머리 구조의 검색이 시각적 관찰과 일치하는 때까지 의미 노른자 강도의 배수으로 &quot;머리 구조&quot;검출 임계값을 설정합니다. 수출에 매개 변수를 정의합니다. 이 예제에서, 가장 유익한 매개 변수 이하 &quot;전체 헤드 구조의 밝기&quot;(그림 1) 칭했다 머리 이내 GFP 양성 지역의 전체 통합 밝기되었습니다. <img src="/files/ftp_upload/1900/1900fig2.jpg" alt="그림 1" /> 그림 1 그래프는 유전자 변형 배아에서 GFP 표현에 BCI의 복용량에 의존 효과를 depicitng. </li><li> 통합 작업 스케줄러를 사용하여 모든 플레이트 이미지 알고리즘을 실행합니다. Definiens 소프트웨어는 여러 개의 이미지를 동시에 분석함으로써 여러 프로세서 ( &quot;엔진&quot;)에서 분산 컴퓨팅을 사용합니다. 알고리즘 적용 분석 처리 이미지의 사본과 함께 데이터베이스에 저장되는 이미지 분석,의 결과에 따라 수치 조치를 계산합니다. </li></ol> 대표 결과 : 모든 이미지는 분석 후 데이터 Cellenger 이내 시각하거나 추가 분석 및 그래픽 표현에 대한 타사 소프트웨어 (엑셀, GraphPad 프리즘)에 수출하고 있습니다. 그림 2 보여줍 대우 자동차에서 스캔 이미지를 보관하고 적용과 CNT 분석없이 잘 BCI - 대우. 그림 1 문서 TG에서 GFP 리포터 유전자 발현 (dusp6 : d2EGFP)에 BCI의 복용량에 의존 효과 pt6 배아. 반 최대한 응답 (EC50)을 이끌어내는 데 필요한 농도는 약 12​​ μm의되었습니다. <img src="/files/ftp_upload/1900/1900fig1.jpg" alt="그림 2" /> 그림 2 유전자 변형 zebrafish의 배아의 자동 이미지 캡처 및 분석. 30hpf에서 pt6 배아 (A) DMSO 차량은 TG를 (d2eGFP dusp6) 치료. (B) 20μM BCI 쇼 취급 트렌스 제닉 배아는 GFP 발현 증가. 배아의 (C) 이미지에서 (A) 머리에 GFP 발현을 찾을 수 ruleset Definiens를 실행한 후. 오렌지 명소 소프트웨어가 GFP 강도를 측정 어디 상징. (D) 배아의 이미지에서 (B) 머리에 GFP 발현을 찾을 수 ruleset Definiens를 실행한 후. 오렌지 명소 소프트웨어가 GFP 강도를 측정 어디 상징. ruleset은 치료 배아에서 GFP의 표현의 확장 영역을 찾을 수 있다고합니다.

<ol>
	<li>Molina, G. A., Watkins, S. C., Tsang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+FGF+reporter+transgenic+zebrafish+and+their+utility+in+chemical+screens.">Generation of FGF reporter transgenic zebrafish and their utility in chemical screens.</a> BMC Dev Biol. 7, 62-62 (2007).</li><li>Vogt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+image-based+phenotypic+analysis+in+zebrafish+embryos.">Automated image-based phenotypic analysis in zebrafish embryos.</a> Dev Dyn. 238, 656-663 (2009).</li><li>Molina, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+chemical+screening+reveals+an+inhibitor+of+Dusp6+that+expands+cardiac+cell+lineages.">Zebrafish chemical screening reveals an inhibitor of Dusp6 that expands cardiac cell lineages.</a> Nat Chem Biol. 5, 680-687 (2009).</li></ol>

zebrafish 화학 화면의 처리량을 제한하는 주요 요인은 체계적으로 처리 이미징 및 분석을위한 96 - 웰 플레이트에 방법론의 부족이다. 다세포 생물의 이미지가 복잡하고, 낮은 콘트라스트, 그리고 자연의 이기종 때문에, 기존의 영상 분석 알고리즘은 유기체 내의 특정 구조를 감지하고 수치 실패합니다. 대부분의 출판 zebrafish 화학 화면 따라서 절차 전반에 걸쳐 전담 요원에 의해 개인 우물의 영상 검사를 포함하고 있습니다. 이것은 일반적으로 수치 설치 했어요의 생성 및 화면 이미지와 데이터의 보관 완전히 방지합니다. 또한, 수동 평가 즉 이미지 기반의 심사, 화면 성능의 회고 분석을 수행하기 위해 심사 캠페인 (예 : 독성 또는 발달 결함)의 주요 초점이되지 않은 phenotypic 변경 사항을 검토하고 교차하는 기능의 몇 가지 주요 장점을 제거 같은 화합물 라이브러리를 사용하여 과거 또는 미래 스크린 - 참조 검사 데이터입니다. 이 보고서에서 우리는 사용자의 개입없이 multiwell 플레이트에 zebrafish 배아의 자동 이미징 및 분석을위한 방법론을 강조. 96 잘 접시에 pt6 배아 및 Definiens &#39;인식 네트워크 기술되는 계량 GFP에게 기반으로 이미지 분석 알고리즘을 개발 : 이미지 TG (d2EGFP dusp6)에 공촛점 리더를 (분자 장치, 서니 베일, CA) 스캔 ImageXpress 울트라 레이저에서 우리는 자동 이미지 캡처 유전자 변형 배아의 머리에 표현. 방법은 등급 반응을 전달하고 작은 분자 FGF 신호의 활성의 생체내 활동에 계량. 비슷한 결과는 그것이 상용 판 독자의 다양한 2 zebrafish 배아의 자동 이미지 캡처를 구현 가능하다 문서화 epifluorescence 기반 ArrayScan II (Cellomics 주식 회사, 피츠버그 PA)로 획득했다. 자동으로 다세포 생물 이미지를 분석하는 방법론의 개발은 인간의 관찰자에 의해 시각 점수에 대한 필요성을 제거하고 미래의 스크린 회고 분석 및 비교를위한 수치 데이터 및 이미지의 생성 및 보관을 활성화.

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<td>Tricaine (MS222)</td>
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development of automated imaging and analysis for zebrafish chemical screens.

우리는 zebrafish의 배아에서 FGF / RAS / MAPK 경로를 조절 수있는 작은 분자를 식별하는 이미지 기반의 높은 콘텐츠 심사 (HCS) 방법론의 응용 프로그램을 보여줍니다. zebrafish 배아는 생체내 높은 컨텐츠 화학 화면에서 이상적인 시스템입니다. 1 일 이전 난자는 직경이 약 1mm이고 쉽게 96 - 웰 플레이트, 높은 처리량 검사를위한 표준 형식으로 못하였 느니라 수 있습니다. 개발의 첫날 동안 배아 따라서 embryogenesis 동안 조직 형성의 시각화를 가능하게 현재 주요 기관의 대부분 투명하고 있습니다. zebrafish 화학 화면의 완벽한 자동화는 특히 자동화된 이미지 수집 및 분석의 개발에, 그러나, 여전히 도전입니다. 우리는 이전에 FGF 활동의 통제하에 녹색 형광 단백질 (GFP)을 표현하는 유전자 변형 기자 라인을 생성하고 화학 화면에서 유틸리티를 보여주<sup&gt; 1</sup&gt;. 높은 처리량 전체 유기체 화면에 대한 방법론을 확립하기 위해, 우리는 96 - 웰 플레이트에 24-48시간 포스트 수정 (HPF)에서 zebrafish 배아의 자동 이미징 및 분석을위한 시스템을 개발<sup&gt; 2</sup&gt;. 이 비디오에서 우리는 24hpf에서 multiwell 플레이트에 유전자 변형 배아를 arraying위한 절차를 강조 표시하고 FGF 신호를 hyperactivates 작은 분자 (BCI)의 추가<sup&gt; 3</sup&gt;. 접시는 분자 장치 ImageXpress 울트라 레이저 스캐닝 공촛점 HCS 리더에서 사전에 자동으로 이미지에 애벌레를 마취하는 tricaine의 또한 다음에 6 시간 동안 incubated 수 있습니다. 이미지는 우리가 유전자 변형 배아의 머리에 GFP의 표현을 감지하고 수치 개발한 인식 네트워크 기술 알고리즘을 사용하여 Definiens 개발자 소프트웨어에 의해 처리됩니다. 이 예제에서 우리는 치료 배아에서 GFP 리포터 유전자 표현에 대한 BCI의 복용 종속적인 효과를 측정하는 알고리즘의 능력을 강조.

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Development of automated imaging and analysis for zebrafish chemical screens.

우리는 multiwell 플레이트에 zebrafish 배아 유전자 변형의 자동 이미징 및 분석을위한 시스템의 개발을보고합니다. 이것은 Fibroblast 성장 팩터 리포터 유전자의 표현에 작은 분자, BCI의 복용 부양 효과를 측정하고 높은 처리량 zebrafish 화학 화면 설립을위한 기술을 제공하는 능력을 보여줍니다.

zebrafish 화학 화면을위한 자동화 이미징 및 분석의 개발.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

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JoVE Journal

Biology

11.3K 조회수.  University of Pittsburgh Drug Discovery Institute. 우리는 multiwell 플레이트에 zebrafish 배아 유전자 변형의 자동 이미징 및 분석을위한 시스템의 개발을보고합니다. 이것은 Fibroblast 성장 팩터 리포터 유전자의 표현에 작은 분자, BCI의 복용 부양 효과를 측정하고 높은 처리량 zebrafish 화학 화면 설립을위한 기술을 제공하는 능력을 보여줍니다.

비디오: Development of automated imaging and analysis for zebrafish chemical screens.

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Neurotoxicity 높은 컨텐츠 분석의 연결 및 Astrocyte 공동 문화 분석

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

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생물학

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

작은 하트의 세미 - 자동 광학 도면 분석

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

의 이식 GFP - 표현 키메라 Zebrafish의 태아의 망막과 두뇌 개발의 라이브 영상에 대한 Blastomeres을

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

신진 대사 라벨링 및 Bioorthogonal 클릭 화학에 의해 Zebrafish 태아의 영상 Glycans

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

의 정량 및 자동화된 고속 처리량 게놈 전체의 RNAi 화면 C. elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Zebrafish 개발하는 동안 Photoconvertible 단백질을 사용하여 셀 추적

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Zebrafish의 태아의 조직 특이 형광의 반 자동화 이미징

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

자동 형광 수명 이미징 현미경을 활용하여 오픈 소스 높은 콘텐츠 분석

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Zebrafish 독도에서 단일 셀 해상도 칼슘 이미징 사용 하 여 베타 세포 기능 분석

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Automated Detection and Analysis of Exocytosis

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic events in cell culture. To perform an unbiased, efficient identification and analysis of such events, a computer-vision based approach was developed and implemented in MATLAB. The analysis pipeline consists of a cell segmentation and exocytic-event identification algorithm. The computer-vision approach includes tools for investigating multiple parameters of single events, including the half-life of fluorescence decay and peak &#916;F/F, as well as whole-cell analysis of the frequency of exocytosis. These and other parameters of fusion are used in a classification approach to distinguish distinct fusion modes. Here a newly built GUI performs the analysis pipeline from start to finish. Further adaptation of Ripley&#39;s K function in R Studio is used to distinguish between clustered, dispersed, or random occurrence of fusion events in both space and time.

We developed automated computer vision software to detect exocytic events marked by pH-sensitive fluorescent probes. Here, we demonstrate the use of a graphical user interface and RStudio to detect fusion events, analyze and display spatiotemporal parameters of fusion, and classify events into distinct fusion modes.

엑소시토시스의 자동 검출 및 분석

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic ...