This work was supported in part by National Institutes of Health Grants HL-085887, HL-092992, S10RR027535, and MOP-93676.

1. 선박의 해부 및 이미징 <ol><li> 마틴 등 27에 설명 된대로 국내 청소년 돼지에서 수확 조직. 폴리 디메틸 실록산 (PDMS) HEPES는 생리 식염수 (PSS)를 버퍼 포함 아래 해부 접시에 수확 돼지 우심실을 놓습니다. <ol><li> 실체 현미경의 도움으로, 해부 주위 심장 조직의 층에서 혈관 세그먼트를 제거하여 집게와 봄 가위를 사용하여 주변 조직에서 관상 동맥 (~ 8mm 길이 0.5 mm 직경) 좌전 하행의 세그먼트를 제거합니다. 참고 : 혈관 벽에 구멍을하지 않도록주의하십시오. </li><li> 해부 접시에 PDMS 블록 (1 × 0.5 × 0.5 cm)를 배치하고 바닥에 핀 바늘을 사용합니다. 마이크로 핀들을 형성, 열두 ~ 0.3 cm 길이의 세그먼트로 약 40 μm의 직경의 텅스텐 와이어를 잘라 접시에 이것들을 배치합니다. 집게를 사용하여, micropi 가진 블록 용기 세그먼트의 한쪽 끝을 고정N. </li><li> 조심스럽게 혈관 내강에 작은 봄 가위를 삽입하고 완전히 엽니 용기의 한 쪽 아래로 길이 방향으로 잘라. 내피 위로 열린 용기 세그먼트의 방향을. </li><li> 선박이 평평한 사각형을 형성하도록 차단하는 열린 용기 세그먼트의 경계를 확보하기 위해 나머지 마이크로 핀들을 사용합니다. 참고 :, 마이크로 탑은 90 °로 구부려 블록 표면과 같은 높이까지 삽입해야합니다. 케어 overstretching로하지 않고 용기 준비 스트레칭주의해야한다; 최종 폭 ~ 시작 연신 폭을 1.5 배되어야한다. </li><li> 134 염화나트륨, 6의 KCl : HEPES에 플루로 닉 (0.03 %)와 DMSO에 용해 플루오 (Fluo) - 오전 4시 (10 μM)를 혼합하여 플루오 (Fluo) - 오전 4로드 솔루션의 작은 볼륨 (~ 1 ㎖)를 준비 밀리미터에 포함 PSS는 (버퍼 1 MgCl2를, 10 HEPES, 10 포도당), 그리고 어둠 속에서 실온에서 약 40 분 동안 로딩 솔루션으로 전체 블록을 삽입합니다. </li><li> 로드 한 후, 버퍼 HEPES의 블록을 씻어5 ~ 10 분 PSS. </li><li> HEPES가 PSS 버퍼가 포함 된 커버 글라스 바닥 챔버에서 50 ~ 100 μm의 두께 스페이서에 블록을 설치합니다. 참고 : 금속 핀이 스페이서로 사용하고 혈관 세그먼트가 아래로 향하게하고 스페이서를 만지고되지 않도록 할 수 있습니다. </li><li> 공 촛점 이미징 장비 거꾸로 현미경의 무대에 실을 배치하고 내피 세포의 층에 초점을 맞 춥니 다. </li><li> 3 ~ 20 배 배율 분, 공 초점 이미지 시퀀스 aquisition 소프트웨어를 사용하여 초 당 ~ 8 프레임의 프레임 속도를위한 기초 상대 형광 캡처 시간 경과 이미지 시퀀스. </li><li> 기저 형광을​​ 기록 ~ 3 분 후에는 HEPES한다 추가 3 분간 동일 HEPES에서 PSS 버퍼링 용해 물질 P (100 PM)의 부피 (~ 1 ㎖), 및 레코드와 PSS 용액을 버퍼링 대체. </li></ol></li></ol> 2. 자동 분석 <ol><li> 8 비트, G 등의 공 촛점 수집 소프트웨어에서 형광 칼슘 활동의 이미지 시퀀스 (들)을 렌더링없는 규모의 정보 rayscale &#39;. TIF&#39;형식의 파일. </li><li> 오픈 ImageJ에는 파일 메뉴에서 &#39;열기&#39;를 클릭하고, ImageJ에있는 이미지 시퀀스 (들)을 볼 수있는 탐색기 창에서 해당 이미지 시퀀스를 선택합니다. </li><li> 이미지 시퀀스 (들) 내의 활동 공간 확산의 상부 및 하부 경계를 추정하는 직사각형 ROI 도구를 사용하여 적절한 ROI 직경을 결정한다. 주 : 평균 직사각형의 직경은 투자 수익 (ROI)의 직경을하는 경우 적합한 선택입니다. </li><li> 컴퓨터 하드 드라이브에 새 폴더를 만들고 폴더 디렉토리에 이미지 시퀀스 (들)을 추가합니다. </li><li> ImageJ에에서 &#39;플러그인&#39;창을 클릭 한 다음 분석을 시작하는 &#39;LC_Pro&#39;을 클릭합니다. </li><li> 투자 수익 (ROI)의 지름 값을 입력하고 필터 임계 값 (0.05 또는 0.01 중 하나)를 선택합니다. </li><li> &#39;약물 치료&#39;확인란을 클릭하고 바로 전후에 약이 추가 된 시간 (초) 포인트 값을 입력합니다. </li><li> &#39;확인&#39;을 클릭하고파일 탐색기 창에 이미지 시퀀스 디렉토리를 입력합니다. </li></ol> 3. 그래픽 출력 <ol><li> 에서 R 버전 3.0.2을 다운로드 <a href="http://www.r-project.org/">http://www.r-project.org/</a> . </li><li> R.의 32 비트 버전을 엽니 다 </li><li> &quot;파일&quot;다음 &#39;스크립트 열기&#39;를 클릭하고 그래픽 실험 보고서를 생성하는 &#39;traceplot.R&#39;R 스크립트를 선택합니다. </li><li> &#39;패키지&#39;를 선택하여 보정하고, gplots 패키지를 설치, &#39;패키지를 설치&#39;, 그리고 appropirate 패키지를 선택. </li><li> &#39;디렉토리 변경&#39;명령 다음, &#39;파일&#39;을 클릭하고 LC_Pro 분석 출력에 해당하는 디렉토리를 선택 탐색기 창을 사용합니다. </li><li> 스크립트 창을 클릭 한 후 &#39;실행하는 모든&#39;스크립트를 실행하는 명령. </li></ol>

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The authors have nothing to disclose.

Decoding complex Ca2+ signals at the cellular and multicellular level will require rigorous experimental and analytical approaches. Here, an approach is described in which time resolved confocal image sequences of Ca2+ dependent fluorescence are subjected to an automated analysis that identifies and quantifies statistically relevant Ca2+ signals within intact cellular fields In the specific case presented, an artery segment was isolated from pig heart, pinned opened to expose the endothel...

칼슘 2 +는 분자 및 세포 내 칼슘 2 + 수준을 높게 조절되는 신호 유비쿼터스 두 번째 메신저입니다. 세포 내 칼슘 2 + 신호가 복잡하고 고립 과도, 진동, 파도 1 전파 포함 - 4. 칼슘 2 +의 공간과 시간 제어 생체 신호 특이성을 기초로 생각, 따라서 칼슘 2 + 신호 패턴의 분석은 여러 필드 5의 조사에 상당한 관심입니다. 예컨대 플루오 (Fluo) -4의 Fura-2와 같은 칼슘 2 + 인디케이터 염료는 일반적으로 형광 현미경 5-12로 내 Ca 2 + 신호를 측정하기 위해 사용된다. 16 - 일반적으로, 시간적 칼슘 2 + 신호는 시간에 따른 사용자 정의 지역 내의 평균 형광의 변화, 또는 관심 영역 (ROI) 5,6,13로 평가됩니다. 현재, 수동 ROI 분석은 시간과 노동력에19 -가 많은 로아를 확인하고 반복적 인 연산 17을 수행하는 사용자가 필요로하기 때문에 긴장의. 이 기술은 인공 신호 모드와 낮은 진폭 또는 확산 신호 (18, 20)의 배제의 도입을 포함, 많은 사용자의 오차가있을 수 있습니다. , 자동 ROI 검출 알고리즘은 이전에 최적의 투자 수익 (ROI)의 위치를 결정하기 위해 통계적 다양한 접근 방법을 사용하여 구현되었지만, 일반적으로 라인 스캔 또는 의사 선 시간이 17에서 하나의 공간 차원으로 분석을 제한 스캔 이미지의 분석에 국한되어 19-22. 또한, 기존의 많은 알고리즘 캘리포니아주기, 지역화 과도에서 전파 파도 (23, 24)에 이르기까지 다양 2 + 릴리스 이벤트의 다양성을 포괄하기에 충분하지 않습니다. 생리 칼슘 2 + 신호의 종합 평가는 종종 더 큰 이미지의 인형도 만들고의 존재에 의해 복잡그 많은 실험 시스템의 잡음 차별 신호를 혼동 역할을합니다. 이전에, 캘리포니아에 자동 ROI 검출 알고리즘 솔루션 2 +는 (건강, 베데스다, MD의 국립 연구소)가 개발하고 (25), (26)을 검증 된 NIH ImageJ에 소프트웨어를위한 플러그인으로 구현, 과도 감지 신호. LC_Pro 불리는이 알고리즘은 두 가지 차원의 시간 경과 이미지 시퀀스에서 칼슘 2 + 과도 신호를 포괄의 ROI를 확인하고 분석 할 수 있도록 설계되었습니다. 여기서 실제 실험 프로토콜 및 돼지 관상 동맥 내피의 알고리즘의 적용이 가능한 대표적인 데모 그래픽 출력을 생성하기 위해 오픈 소스 통계 처리 소프트웨어 R을 후 처리하여 추가로 제공된다.

<table border="0" cellpadding="0" cellspacing="0" width="573">
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			<td height="21" style="height:21px;width:216px;">dissection dish</td>
			<td style="width:71px;">Fisher Sci</td>
			<td style="width:119px;">#08-772-70</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:216px;">polydimethylsiloxane (PDMS)</td>
			<td style="width:71px;">Fisher Sci</td>
			<td style="width:119px;">#NC9644388</td>
			<td>elastomer kit, must be molded into dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES-buffered PSS</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">#H3375-250G</td>
			<td>HEPES acid</td>
		</tr>
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			<td height="42" style="height:42px;width:216px;">stereomicroscope</td>
			<td style="width:71px;">Nikon Inst.</td>
			<td style="width:119px;">#MNA42000</td>
			<td></td>
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			<td height="63" style="height:63px;width:216px;">forceps</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">#11223-20</td>
			<td></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">spring scissors</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">15003-08</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">tungsten wire</td>
			<td style="width:71px;">Scientific Inst Svcs</td>
			<td style="width:119px;">#406</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Fluo-4 AM</td>
			<td style="width:71px;">Life Tech.</td>
			<td style="width:119px;">#F-14201</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">pluronic F-127</td>
			<td style="width:71px;">Life Tech.</td>
			<td style="width:119px;">#P3000MP</td>
			<td></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">metal pins</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">#26002-10</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">cover-glass bottom chamber</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Custom designed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">spinning disc confocal microscope</td>
			<td style="width:71px;">Perkin Elmer</td>
			<td style="width:119px;">RS-3</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">imageJ software</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="http://rsbweb.nih.gov/ij/download.html">download at: http://rsbweb.nih.gov/ij/download.html</a></td>
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			<td height="21" style="height:21px;width:216px;">LC_Pro plugin for imageJ</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="http://rsbweb.nih.gov/ij/plugins/lc-pro/index.html">download at: http://rsbweb.nih.gov/ij/plugins/lc-pro/index.html</a></td>
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			<td height="21" style="height:21px;width:216px;">R software</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="http://www.r-project.org/">download at: http://www.r-project.org/</a></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">R traceplot script</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="https://docs.google.com/file/d/0B-PSp1D9e2fjV3NIcGppUzkxdEk/edit?usp=sharing">download at: https://docs.google.com/file/d/0B-PSp1D9e2fjV3NIcGppUzkxdEk/edit?usp=sharing</a></td>
		</tr>
	</tbody>
</table>

automated analysis of dynamic ca2+ signals in image sequences

세포 내 칼슘 2 + 신호는 일반적으로 형광 칼슘 2 + 지표 염료 및 현미경 기술을 연구한다. 그러나, 칼슘 2 + 영상 데이터의 정량 분석 시간과 바이어스에 따라 달라질 수 있습니다. 또한 (ROI)의 검출 영역에 기초하여 자동화 된 신호 분석 알고리즘은 일차원 라인 스캔 측정에 대해 구현되어 있지만 이차원 이미지 시퀀스에서 최적화 된 신분증과의 ROI 분석을 통합 전류 알고리즘은 없다. 여기서 이미지 시퀀스에서 수집 및 급속의 ROI 분석을위한 알고리즘을 설명한다. 이 타원이 최적의 투자 수익 (ROI)의 위치를 결정하기 위해 노이즈 필터링 된 신호에 맞게 활용하고, 진폭, 시간 및 공간 확산의 칼슘 2 + 신호 파라미터를 계산한다. 이 알고리즘은 ImageJ에 (NIH) 소프트웨어를 자유롭게 사용할 수 플러그인으로 구현되었습니다. 함께 오픈 소스 통계 처리 소프트웨어 R 용으로 작성된 분석 스크립트와,이러한 접근법은 실험 출력 퀵 통계 분석을 수행하기위한 고용량 파이프 라인을 제공한다. 저자는이 분석 프로토콜의 사용은 생리적 칼슘 2 + 신호의보다 완전하고 공정한 특성으로 이어질 것입니다 것이 좋습니다.

A custom algorithm, LC_Pro, was developed and implemented in order to perform automated analysis of Ca2+ dynamics on confocal image sequences. As depicted in Figure 1, the algorithm utilizes sequential processing modules that A) detect and track sites of dynamic Ca2+ change above statistical (p &#60; 0.01) noise, B) define regions of interest (ROI) automatically at active site centers, and C) calculate average fluorescence intensities at ROIs to determine specific event parameters. ...

Watch this Scientific Journal Video about Automated Analysis of Dynamic Ca2+ Signals in Image Sequences at JoVE.com

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

동적 칼슘의 자동 분석<sup&gt; 2 +</sup이미지 시퀀스에서&gt; 신호

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

automated-analysis-of-dynamic-ca2-signals-in-image-sequences

Research

JoVE Journal

Biology

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

17.0K 조회수.  University of South Alabama. Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

비디오: Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

만성 실험에 장착 전극 또는 Microdialysis 프로브를위한 라이트 - 치료 치과 접착제 수지의 응용

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

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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 ...

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non coding sequences. One prominent function carried out by the non coding fraction of the genome is to regulate gene transcription; however, there are no effective methods to broadly predict cis-regulatory elements from primary DNA sequence. We have developed an efficient protocol to functionally evaluate potential cis-regulatory elements through zebrafish transgenesis. Our approach offers significant advantages over cell-culture based techniques for developmentally important genes, since it provides information on spatial and temporal gene regulation. Conversely, it is faster and less expensive than similar experiments in transgenic mice, and we routinely apply it to sequences isolated from the human genome. Here we demonstrate our approach to selecting elements for testing based on sequence conservation and our protocol for cloning sequences and microinjecting them into zebrafish embryos.

We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.

향상제 시퀀스를 평가하기위한 모자이크 Zebrafish의 Transgenesis

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

로보틱스 및 식물 조직에서 유전자 발현의 연구 동적 이미지 분석

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Development of automated imaging and analysis for zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

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 ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

이미징 CA의 기술 2 + 인간 정자의 시그널링

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent 
proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest1, recent 
developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association 2, 3. One small 
tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH 
(green fluorescent), with high specificity even in live cells 2. The TC/biarsenical dye system offers far less steric constraints to the host protein than 
fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions 4-7. We recently developed 
a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington 
disease 7. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only 
binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer 
content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) 
with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).

The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.

ReAsH / 플래시 라벨링 및 전지 Tetracysteine​​ 센서 단백질의 이미지 분석

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells.  While fluorescent 
proteins ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

측정 공간적 칼슘<sup&gt; 2 +</sup애기 장대 식물에서&gt; 신호

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

이미징 로컬 CA<sup&gt; 2 +</sup&gt; 배양 된 포유 동물 세포에서 신호

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

고해상도 시간 경과 이미징 및 생활 인간 제대 정맥 내피 세포의 미세 소관 역학의 자동 분석

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...