We are grateful to B. Stevenson for technical assistance and Dr Marc R. Knight for providing Aequorin transgenic line. This work was funded by the National Institutes of Health Grant R01 GM059138.

FAS 시스템을 사용하여 1 애큐 오린 기반 칼슘 이미징 <ol><li> 발광 이미징을위한 모종을 준비합니다. 0.01 % 트리톤-100를 포함하는 10 % 표백제 용액으로 애큐 오린을 표현 애기 장대 식물의 종자를 소독. 전체 강도 MS (무라 시게와 스쿡 기초 소금의 혼합물), 1 %의 자당, 1.2 % 한천을 포함하는 사각형 접시 (격자 10 × 10cm 사각형 페트리 접시)에 멸균 씨앗을 뿌리고. 수직으로 이일 4 ° C (그림 1A)에서 층화 후 성장 챔버에 넣어 판. </li><li> 필름에 묘목을 전송합니다. 접시에 성장 7-10일 된 모종의 상단에 접착 필름 (그림 1B)를 놓습니다. 조심스럽게 모종 필름 (그림 1C)을 준수하는지 확인하기 위해 손으로 필름을 밀어 넣습니다. 모종이 필름에 부착 된 상태로 유지되도록 조심스럽게 필름을 벗겨 (그림 1D 및 1E) </li><li> 보조 인자 (cofactor)로 모종을 품어. 배치포함 된 각 접시 (10 × 10cm) 상에 dhered 모종 2 μg의 15 ㎖ / ML의 H-CTZ 물 (coelenterazine). 하룻밤 (그림 1 층)에 4 시간 동안 실온에서 묘목을 품어. </li><li> 발광 이미지를 준비합니다. 두 조각을 형성, H-CTZ 솔루션에서 필름을 가지고 중간을 잘라. 모종은 두 개의 서로 다른 판에 얼굴 필름의 각 조각을 놓습니다. 5 분 동안 어둠 속에서 판을 둡니다. </li><li> 발광 이미지를 획득. 어둠 속에서 옆에 발광 이미징 시스템 (그림 1G)의 무대에서 서로 두 판을 배치합니다. 동시에 즉시 플레이트에 자극 용액 20 ㎖를 첨가시에 화상을 취득. </li><li> 발광 이미지를 분석합니다. 모든 발광 이미지에 대해 동일한 표시 범위를 선택합니다. 관심 (ROI)의 영역을 자르기 및 JPEG 파일 (그림 2A)와 같은 이미지를 생성합니다. 또한, SPE 형식 파일로 이미지를 내보내고로 가져ImageJ에 이미지 분석 소프트웨어. ROI의 평균 그레이 값의 계산을위한 설정 측정. ROI 영역의 같은 크기를 선택하고 막대 그래프 (그림 2B)로 평균 그레이와 현재의 데이터를 측정합니다. </li></ol> 2 라이브 셀 공 촛점 칼슘 이미징 <ol><li> 공 촛점 이미징을위한 모종을 준비합니다. 0.01 % 트리톤을 포함하는 10 % 표백제 용액으로 case12을 표현 애기 장대 식물의 종자를 소독. 풀 강도 MS 염, 1 % 자당, 1.2 % 한천을 포함하는 접시에 무균 씨앗을 뿌리고. 이일 4 ° C에서 층화 후 성장 챔버에서 수직으로 배치 접시. </li><li> 설치 이미징 챔버 <ol><li> 슬라이드와 커버 슬립 (상공 회의소)를 사용하여 이미징 챔버를 조립합니다. 슬라이드의 중간에 물을 적신 탈지면의 조각을 놓습니다. 그런 다음 물에 적신 탈지면의 상단에 접시에서 하나 또는 두 개의 오일 된 모종을 전송합니다. 커버 슬립,의 각 모서리에 점토의 작은 조각 스틱커버 슬립 및 슬라이드 사이의 간격 (챔버) (그림 3A, 왼쪽 패널)를 만들 수있는 모종의 상단에 D 장소 coverslip에. 1 ML의 주사기에 폴리에틸렌 관 (0.58 mm 직경)의 한쪽 끝을 연결하고 챔버에 바로 인접 튜브의 다른 쪽 끝을 배치합니다. 테이프 (그림 3A, 오른쪽 패널)와 장소에 튜브를 잡습니다. </li><li> 플렉시 글라스 챔버 (챔버 B)를 사용하여 촬상 챔버를 조립한다. <ol><li> 플렉시 글라스 챔버의 양쪽에있는 채널로 코팅 된 튜브를 두 폴리에틸렌 관 (0.58 mm 직경)의 주위에 실리콘 그리스를 얇게 확산 누릅니다 (그림 3b는 왼쪽 패널). 튜브 홈을 포함하는 챔버의 표면 상에 실리콘 그리스를 얇게 펴고 챔버 개구 (잘라)의 한쪽을 밀봉하는 그리스로 커버 슬립을 누른다. </li><li> 챔버로 판에서 오일 된 묘목을 전송하고 상단 오에 물에 흠뻑 탈지면의 조각을 배치묘목 바. 챔버의 다른 표면에 실리콘 그리스를 확산하고, 인감 위에 다른 커버 슬립을 누릅니다. 1 ML의 주사기에 튜브 중 하나의 끝을 연결합니다. 다른 관 오픈 (그림 3B 오른쪽 패널)의 끝을 둡니다. </li></ol></li><li> 챔버 커버 유리 (챔버 C)를 사용하여 촬상 챔버를 준비한다. 70 % 에탄올로 챔버 커버 유리를 소독하고 건조까지 후드에 두십시오. 0.6 ML 또는 각각이 잘 챔버의 각 웰 또는 8 잘 챔버로 (그림 3C 오른쪽 패널)를 1 % 자당 및 0.5 % phytagel를 포함하는 전체 강도 MS 매체의 0.2 ML을 추가합니다. 종자를 소독하고 명확한 젤의 얇은 층을 포함하는 챔버 커버 유리에 직접 씨앗을 뿌리고 그들을 오일 (그림 3C)에 대해 수직으로 성장 할 수 있습니다. </li></ol></li><li> 공 초점 현미경을 사용하여 이미지를 획득. 얼음-COL 천천히 150 mM의 NaCl을 약 200 μl를 주입, 염분, 감기 나 과산화수소와 같은 스트레스 자극을 적용하려면직전에 각각의 이미지를 획득하거나 부드럽게 2 또는 8 잘 챔버의 웰에 자극 용액 500 ㎕를 100 μl를 추가로 실 (챔버 A 또는 B)에 D 물 또는 1 ㎜ H 2 O이 솔루션 . 이미지 캡처 즉시 20X 물 침지 렌즈 (개구 수 0.75) 반전 니콘 A1R 공 초점 레이저 주사 현미경을 사용하여 자극 솔루션을 적용한 후. 각각 550 나노 미터, 512 X 512 픽셀의 해상도에서 - 488 nm 내지 500의 여기 및 발광 파장의 4 초 간격으로 화상의 시계열을 모은다. </li><li> 이미지 분석 니콘 요소를 사용하여, ROI를 원하는 각 셀 (또는 영역)의 주위에 그려졌다. 각 투자 수익 (ROI) 내에서 전체 강도는 시간 측정 대화 상자 (ImageJ에 대신 사용될 수있다)를 사용하여 시간이 지남에 따라 측정 하였다. 전체 강도 측정은 수출 및 DataGraph에서 처리되었다. 칼슘 스파이크 진폭 번째로 피크 강도 마이너스 휴식 강도, 지속 기간으로 정의 하였다이 스파이크의 스파이크 인접한 피크 사이의 시간 간격 등의 개시 및 종료 스파이크 및 기간 간의 전자 시간. -test는 수단과 비교하는 데 사용 된 T. </li></ol>

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The authors have nothing to disclose.

우리는 애기 장대 모종의 공간 - 시간 칼슘 응답을 기록하는 FAS 시스템을 증명하고있다. 이 FAS 칼슘 기록 시스템은 칼슘 여기되게됩니다 비 생물 적 스트레스 자극뿐만 아니라 다양한 자극에 의해 발생 2 + 역 동성을 측정하기 위해 적용 할 수있는, 간단한 민감하고 강력한 접근 방식을 제공합니다. 이 시스템을 사용하여, 우리는 쉽게 전체 플랜트 레벨에서 조직 - 또는 자?...

식물 세포는 세포의 행동을 통해 시그널링 좌표 환경에 응답한다. 환경 자극에 대한 응답으로 이벤트를 신호 초기 셀은 일시적인 칼슘 증가이다. 자유 칼슘 농도의 패턴, 또는 일시적인 증가의 서명은 그 진폭, 주파수 및 시간에 의해 특징된다. 고유 시공간 칼슘 서명은 다른 세포 활동 한을 조절한다. 열, 냉기, 소금, 가뭄, 빛, 또는 식물 호르몬과 같은 특정 자극, 미세 조정할 수 막 화 된 칼슘 채널 및 / 또는 수송의 시공간적 활동을, 특정 칼슘 서명 결과. 칼슘 수송이 잘 특징으로되어 있지만, 작은 식물 하나의 칼슘 채널의 분자 정체성과 기능에 대한 알려져있다. 자극을 강조하는 변경 칼슘 반응과 돌연변이에 대한 유전 스크린은 효과적인 APPR 수 있습니다칼슘 서명을 구성하는 구성 요소를 식별하기위한 oach. 최근 몇 애큐 오린 기반 칼슘 탐지 시스템은 병원균의 공격과 비 생물 적 스트레스 2-4에 대한 응답으로 구성 요소를 신호 칼슘 2에 대한 유전 화면을 용이하게 개발되었다. 애큐 오린 처음 1990 년대 초에 5 칼슘보기 식물체에서 2 + 신호를 검출하는데 사용되었다. 그 이후로, 애큐 오린은 같은 칼슘이 특정 세포를 모니터링 할 수있는 루트에서 8 tonoplast 세포질 5, 핵 6, 엽록체 7, 미토콘드리아 9, 간질 열뿐만 아니라, 서로 다른 종류의 세포 등 다양한 세포 구획을 타겟으로하고있다 + 신호 11. 애큐 오린 기반 칼슘 측정은 자극을 강조하는 세포 집단의 공간과 시간 칼슘 반응을 알 수있다. 그러나 대부분의 경우, 하나의 셀의 칼슘 반응 unsynchroni 아르응답 조직 사에 데이빗. 따라서, 애큐 오린 칼슘 기록은 반드시 각각의 세포에서 칼슘 신호를보고하지 않습니다. 최근 몇 년 동안, 유전자 인코딩 형광 단백질 (FP)는 높은 해상도로 세포 내 신호 칼슘 2를 연구하는 데 사용 된 칼슘에게 같은 노란색 카멜레온 (YCS) 12 CASEs12 (13) 등 2 + 지표를 기반. YCS는 형광 공명 에너지 전달 (FRET)는 CFP를 포함, 칼슘에게 2 + 지표를 기반 및 YFP이 단백질 칼 모듈 린과 칼 모듈 린 결합 펩티드 M13을 -binding 2 + 칼슘에 의해 연결된 변종입니다. 이 칼슘 결합으로 칼 모듈 린함으로써 증가 된 에너지 전달 (FRET 강화)의 결과로, 더 가깝게 CFP와 YFP을 제공, 구조적 변화를 겪는다. CFP 신호 강도에 YFP의 비율로 대략적으로 계산 된 시간에 따른 FRET 레벨은, 내 Ca 2 +보기 역학을 반영한다. 여러 YC 버전은 식물에서 사용되어왔다. YC3.6은 t이었다세포질 14, 15, 16 핵, 미토콘드리아 (17) 및 세포막 (18), 그리고 YC4.6 및 D4ER에 argeted는 ER 15,19를 대상으로하고, D3cpv는 퍼 옥시 솜 (20)을 대상으로 하였다. YCS를 발현하는 형질 전환 식물은 서로 다른 세포 유형의 서로 다른 세포 구획 내 칼슘 역학의 라이브 세포 이미징을 할 수 있습니다. 케이스 (아마도 칼슘 lcium 자체 nsor)는 칼 모듈 린과 칼 모듈 린 결합 펩티드 M13을 품고 하나의 원 순열 형광 단백질 (cpFPs)입니다. 칼슘 이온에 결합되면, 경우는 형광 강도의 증가로 이어지는 구조 변화를 겪는다. CASE의 형광 반응과 칼슘 이온 농도의 상관 관계는 세포 내 칼슘 역학을 정량적으로 측정 할 수 있습니다. Case12 변형이 12 배는 칼슘 포화 지방 형태로 형광을 증가했다. N. benthiminana 식물 일시적으로 EXPR싱 Case12 또는 안정적으로 케이스 (12)를 표현하는 애기 장대 식물은 방어와 비 생물 적 스트레스 4,21에서 칼슘 신호 전달을 연구하는 데 사용되었다. 또는 탈수 스트레스에 병원균의 공격에 대응하는 세포에서 비동기 공간과 시간 칼슘 진동은 Case12 기반의 칼슘 이미징 공개되었다. 여기서 우리는 애기 모종 2 + 역학 칼슘 특정하고, 세포질의 공 초점 이미징 및 핵 CA의 조직 -와 자극의 애큐 오린 기반 발광 이미징에 대한 자세한 지침을 제시 FAS의 케이스 (12) 발광 영상을 표현 애기 루트 세포 2 + 역학 그대로 식물 또는 여기에 설명되지 않은 조직에서 스트레스에 의한 칼슘 역학을 분석하기 위해, 또는 2 + 신호 칼슘에 의한 변형 된 스트레스와 돌연변이에 대한 돌연변이 애기 장대 식물 집단을 선별 적용 할 수있다. 라이브 세포 칼슘 영상 설정은 칼슘을 분석하기에 적합 할 수다른 칼슘 지표를 사용하여 다른 subcelluar 구획 내에서 또는 서로 다른 세포 유형에서 2 + 역학.

<table border="0" cellpadding="0" cellspacing="0" width="887">
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			<td height="21" style="height:21px;width:356px;">Name of Material/ Equipment</td>
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			<td height="21" style="height:21px;">10X10 cm square petri dish&#160;</td>
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			<td height="21" style="height:21px;">luminescence imaging system&#160;</td>
			<td>Princeton Instruments</td>
			<td style="width:175px;">N/A</td>
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			<td height="21" style="height:21px;">inverted confocal laser-scanning microscope&#160;</td>
			<td>Nikon Instruments Inc.</td>
			<td style="width:175px;">N/A</td>
			<td style="width:184px;">Nikon A1R&#160;</td>
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			<td height="21" style="height:21px;width:356px;">Imaging software</td>
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			<td>Nikon Elements&#160;</td>
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			<td height="21" style="height:21px;width:356px;">ImageJ</td>
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			<td>Public domain, Java-based image processing program</td>
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			<td>Visual Data Tools Inc</td>
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			<td>DataGraph 3.1.1 is the newest version</td>
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			<td style="width:172px;">Phytotechnology Labs</td>
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			<td height="21" style="height:21px;width:356px;">sucrose</td>
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			<td height="21" style="height:21px;width:356px;">Agar</td>
			<td style="width:172px;">Sigma</td>
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			<td height="21" style="height:21px;width:356px;">Phytagel</td>
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</table>

measuring spatial and temporal ca2+ signals in arabidopsis plants

발육 및 환경 단서 칼슘보기 식물 세포에서 2+ 변동을 유도한다. 자극 특정 시공간 칼슘 패턴이 칼슘 신호 폭포를 시작 세포의 칼슘 결합 단백질에 의해 감지된다. 그러나, 우리는 여전히 특정 칼슘 신호가 생성되는 자극 방법에 대한 약간 알고있다. 칼슘 신호의 특이성은 주어진 자극에 응답하여 칼슘 이온 채널들 및 / 또는 전송기의 활동 정교한 조절에 기인 할 수있다. 이들 세포 성분을 식별하고 그들의 기능을 이해하기 위해서는 조직 및 세포 수준에서 모두 칼슘 신호를 구분하고 강력한 기록을 허용 시스템을 사용하는 것이 중요하다. 서로 다른 세포 구획을 대상으로 유전자 인코딩 칼슘 지표 세포 칼슘 신호의 라이브 세포 공 촛점 이미징을위한 플랫폼을 제공하고 있습니다. 여기에서 우리는 명령을 설명이 칼슘 감지 시스템의 용도들 : FAS (필름 점착 모종), 발광 칼슘 이미징 기반 case12 생균 공 초점 형광 칼슘 이미징 기반의 애큐 오린. Case12를 이용한 생균 공 촛점 이미지는 높은 해상도 세포질과 핵 칼슘 신호의 동시 검출을 제공하면서 FAS 시스템을 사용하여 발광 이미징은 조직 수준에서 시공간적 칼슘 신호의 단순 견고하고 민감한 검출을 제공한다.

만니톨, 염화나트륨 및 H 2 O 2는 각각, 탈수, 소금 및 산화 스트레스 자극에 대한 프록시로서 사용 하였다. 중금속 이온의 Cu 2 +이 세 스트레스 자극 중 어느 synergizes 여부를 확인하기 위해, 우리는 2 +의 Cu의 존재 또는 부재의 각 자극에 칼슘 응답을 비교 하였다. 그림 2에 나타낸 바와 같이, FAS 발광 이미징 Arabidoposis 모종 탈수, 소금, 산화 스트레...

Watch this Scientific Journal Video about Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants at JoVE.com

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

칼슘 신호는 식물에서 다양한 생물학적 과정을 조절한다. 여기에 우리가 공간과 시간 칼슘에 의한 비 생물 적 스트레스를 모니터링하기위한 방법을 제시 애기 세포와 유전자 인코딩 칼슘 지표 애큐 오린 또는 Case12를 사용하여 조직에 2 + 신호.

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+ ...

measuring-spatial-and-temporal-ca2-signals-in-arabidopsis-plants

Research

JoVE Journal

Biology

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

12.0K 조회수.  Purdue University.  칼슘 신호는 식물에서 다양한 생물학적 과정을 조절한다. 여기에 우리가 공간과 시간 칼슘에 의한 비 생물 적 스트레스를 모니터링하기위한 방법을 제시 애기 세포와 유전자 인코딩 칼슘 지표 애큐 오린 또는 Case12를 사용하여 조직에 2 + 신호. 

비디오: Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

회로 기판과 생물 세포막을 모델링하고 Axons에 전기 신호를 측정 : 학생 실험실 연습을

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

연구

생물학

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

얼음 총액 : 성장을위한 방법 Arabidopsis높은 처리량 Genotyping을위한 96 - 웰 플레이트에와 토마토 식물

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

세포 내 칼슘 측정<sup&gt; 2 +</sup개의 기술하여 인간 정자에서&gt; 변경 : 기존 Fluorometry은 흐름 Fluorometry, 유동 세포 계측법 및 단일 세포 이미징을 중지

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

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+ ...

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, ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

실시간 Vivo에서 녹음 애기 칼슘 신호의 곤충 먹이 형광 바이오 센서를 사용 하 여 중

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

세포내 캘리포니아2 + 제자리에서 신호 계량 Spatio 시간적 매핑 및 입자 분석 기법에의 응용

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

레이저 캡처 미세 절 RNA-시퀀싱 용 공간 및 측두 조직 -특정 유전자 발현 분석

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. Upon wounding of a leaf, an increase in cytosolic calcium ion concentration (Ca2+ signal) occurs at the wound site. This signal is rapidly transmitted to undamaged leaves, where defense responses are activated. Our recent research revealed that glutamate leaking from the wounded cells of the leaf into the apoplast around them serves as a wound signal. This glutamate activates glutamate receptor-like Ca2+ permeable channels, which then leads to long-distance Ca2+ signal propagation throughout the plant. The spatial and temporal characteristics of these events can be captured with real-time imaging of living plants expressing genetically encoded fluorescent biosensors. Here we introduce a plant-wide, real-time imaging method to monitor the dynamics of both the Ca2+ signals and changes in apoplastic glutamate that occur in response to wounding. This approach uses a wide-field fluorescence microscope and transgenic Arabidopsis plants expressing Green Fluorescent Protein (GFP)-based Ca2+ and glutamate biosensors. In addition, we present methodology to easily elicit wound-induced, glutamate-triggered rapid and long-distance Ca2+ signal propagation. This protocol can also be applied to studies on other plant stresses to help investigate how plant systemic signaling might be involved in their signaling and response networks.

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

아라비도시스에서 지역 및 전신 상처 신호의 넓은 필드, 실시간 이미징

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. ...

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

애기장대 및 기타 식물의 흰가루병 감염 표현형을 정확하게 평가하기 위한 쉽고 유연한 접종 방법

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...