Name: visualizing stromule frequency with fluorescence microscopy
Uploaded: 2016-11-23T14:00:00.000+00:00
Duration: 8 min 27 s
Description: Watch this Scientific Journal Video about Visualizing Stromule Frequency with Fluorescence Microscopy at JoVE.com

J.O.B. and A.M.R. were supported by predoctoral fellowships from the National Science Foundation.

참고 :이 프로토콜을 위해, 우리는 N의 표피에 stromule 주파수을 분석에 초점을 맞추고있다 benthamiana 나뭇잎. FNRtp : EGFP 13 NRIP1 6 세룰 리안 여러 안정한 형질 전환 라인 35S PRO 포함하여 이러한 목적을 위해 사용될 수있는 생성되었다. 이 라인은 모두 광범위한 조건 하에서 성장 잎의 엽록체 기질의 형광의 강력한 표현을 보여줍니다. 대안 적으로, 엽록체 표적 형광체는 일시적 N. 표현 될 수있다 benthamiana하여 아그로 13 변형. 아그로 박테 리움이 침입 N. 일부 기초 방어 반응을 유도하기 때문에 이것은 트랜스 제닉 라인보다 적합 아그로 박테 리움과 benthamiana과 상호 작용 가능성이 결과의 해석을 복잡하게 잎 14 stromule 주파수를 변경할 수 있습니다. 마지막으로, 시험 관내에서 stromule 형성을 시각화하기 위해,아래의 섹션 5에 기술 된 바와 같이 엽록체는 유전자 코드 형광체 또는 형광 염료를 사용하여, 임의의 종의 식물에서 추출 할 수있다. 9,15 참고 :. 식물 재배에 대한 자세한 방법은 이전에 기술되어있다 (16) 간단히, N. 성장 좋은 배수를 제공하는 전문적인 토양 혼합 가득 4 &quot;냄비에 benthamiana 식물입니다.. 발아 습한 환경을 제공하는 최초의 10-14일에 대한 명확한 플라스틱 돔 모종을 커버 14-에 제조업체의 지침에 따라 표준 비료 혼합 추가 일 된 식물. ~ 100 μmol 광자 m -2 초 -1 빛의 강도를 사용하여, 백색광에서 식물을 성장. 물 식물을 정기적으로. 시각화 1. 준비 잎 샘플 참고 : 조직 준비가 stromule를 시각화하기 직전에 수행되어야하므로 Stromule 역학은 8 부상에 의해 영향을받는공 초점 형광 현미경으로의. 이상적으로, 샘플은 공장에서 제거 후 15 분으로 시각화한다. <ol><li> 매우 날카로운 면도날을 사용하여 잎의 작은 부분을 잘라. 항상 일관성 잎의 같은 영역을 사용하고 모든 실험에서 (향축 또는 배축) 같은 표면을 시각화 확신 할 수. </li><li> 즉시 잎 부분을 절단 한 후, 물을 가득 5 ㎖ 무 바늘 주사기의 잎 부 잠수함 주사기로부터 공기를 제거하고, 손가락으로 주사기 구멍을 덮는 플런저 당기는 진공을 적용한다. <ol><li> 잎 부분이 손상되지 않도록 부드럽게 플런저를 놓습니다. 이 두 개 또는 세 번 반복, 또는 공기의 대부분이 제거 될 때까지 리프 깊은 녹색으로 보인다. 참고 :이 단계는 stromules의 정확한 시각화를 방해 할 수있는 잎에 공기를 제거합니다. </li></ol></li><li> 슬라이드에 물 한 방울을 추가하고이 드롭의 잎 부분을 배치합니다. 그런 다음, 다른 닥터를 추가잎의 상단에 물 연산, 그리고 잎 위에 커버 슬립을 배치합니다. 기포가있는 경우가 제거 될 때까지 천천히 커버 슬립을 탭. 슬라이드는 이제 현미경에 대한 준비가되어, 즉시 가시화되어야한다. </li></ol> 공 초점 형광 현미경 2. 떠올리 Stromules <ol><li> 투과광으로 (멀리 면도날에 의해 손상된 세포) 판 단면의 중심 부근의 시야에 초점을 맞춘다. 많은 엽록체 동시에 시각화 할 수 있도록하는 20X 목표를 사용합니다. 필요한 경우 세포 유형은 나중에 구별 될 수 있도록 전송 된 광 화상을 저장한다. </li><li> 형광 물질이 사용되기에 적합한 여기 및 방출 필터를 레이저로 조명 원 스위치. 예를 들어, 488 nm의 레이저로 GFP를 여기 500 nm 내지 525 nm의 사이에 배출을 모은다. <ol><li> 현미경의 소프트웨어를 사용하여 상기 GFP 채널을 선택하고, 핀홀 개구 아이 1을 조정 클릭스피 장치. 레이저 주사 선택 샘플 시각화 시작하고 레이저 강도 여전히 명확 stromules 시각화하면서 레이저 파워를 최소화하기 위해 상기 검출기 이득을 조정하는 GFP 채널을 클릭. 이러한 설정이 결정되고 나면, 그 모든 실험을 통해 가능한 한 일관성을 유지한다. </li></ol></li><li> 시야의 잎 표피를 통해 일련의 이미지를 수집하는 Z -stack 실험을 준비한다. <ol><li> 의 Z -stack를 들어, (잎 표면 근처 엽록체 테스트 조건에서 더 stromules이있는 경우, 예를 들어) 편견을 피하기 위해 시야에있는 모든 표피 엽록체를 포함한다. </li><li> A에서 Z의 -stack에 필요한 시간을 모든 stromules을 포함하지만 최소화 할 수있는 최대 간격 수의 이미지를 가져 가라. 예를 들어, 1 에어리 단위마다 2 ㎛의 가능성 이상적인 이미지를 촬영하고, 2 ㎛의 깊이 인 - 포커스 촛점 부에 상당한다. 참고 : 잎 epidermiS는 요철면이므로 시료에 따라 달라질 -stack는 z의 총 깊이; 대부분의 Z -stacks 10-20 이미지가 필요하지만 더 포함 할 수있다. </li><li> (가능하면, 일반적으로 5 ~ 10 분 이내)의 Z -stack 신속하게 수집되어 있는지 확인하기 위해 스캔 속도와 필요한 이미지 해상도를 조정합니다. 나중에 분석을위한 Z의 -stack을 저장합니다. </li></ol></li></ol> 3. 이미지 처리 <ol><li> 모든 이미지 분석 소프트웨어를 사용 stromule 주파수를 결정한다. 이 프로토콜을 위해, 우리는 국립 보건원 (http://imagej.nih.gov/ij/), 또는 ImageJ에, 피지의 인기 업그레이드에서 공개적으로 사용할 수 ImageJ에, (http://fiji.sc를 사용하는 것이 좋습니다 / 피지). </li><li> 소프트웨어의 최대 강도 투사를 사용하여 하나의 이미지로 Z의 -stack을 병합합니다. </li><li> 확인하고 수동으로 이미지의 모든 엽록체를 계산합니다. </li><li> 각 엽록체를 들어, 시각적으로인지하는 하나 이상의 stromules를 결정병합 된 Z -stack 이미지의 엽록체에서 연장 볼 수 있습니다. 예를 들어, 그림 1을 참조하십시오. 참고 : stromules의 생물학적 기능이 알려져 있지 않기 때문에, 엽록체에서 어떤 얇은 기질 가득한, 관 확장 길이에 관계없이,에 &quot;stromule&quot;으로 간주 될 수있다. 그것은 약 1 ㎛ 길이 7의 대부분을 따라 직경보다 작 으면 일반적으로 엽록체에서 기질 가득 확장은 stromule이다. 한 사람이 엽록체가 stromule을 가지고 있는지의 여부를 결정하는 주관적인 차이를 제어하는 ​​모든 이미지를 분석 있는지 확인합니다. 두 번째 연구는 독립적으로 더 주관적인 편견을 줄이기 위해 stromule 주파수를 확인해야합니다. </li></ol> 4. 실험 설계 및 표본 추출 참고 : Stromule 주파수는 잎 사이에서 매우 다양하지만, 몇 가지 보고서는 stromule 주파수에 약간의 변화가 INDIVI 내에서이 있음을 시사이중 잎 9,17. <ol><li> 한 잎의보기의 단일 필드에서 잎 stromule 주파수를 계산합니다. 잎을 폐기하고,이 독립적 인 샘플을 고려합니다. 독립적 인 샘플 하나 잎에서보기 별도의 필드를 고려하지 마십시오. 참고 : stromules의 기능을보다 명확하게 정의 stromule 활동 때까지 방법에 가장 측정 변경, 연구자 stromule 역학을 정량화 창조적 계속한다에 강한 공감대가 없습니다. 문헌에 걸쳐, 비교를 위해, 그러나,보고의 공통 표준은 창의적인 방법에 추가로 사용한다. 최소한, 항상 하나 이상의 stromules이 엽록체의 주파수를보고한다. </li><li> 보기의 필드에 stromules이 엽록체의 주파수를 결정합니다. 시야 모두 엽록체 ImageJ에 식별을 사용한다. 그리고, 각 엽록체 들어 엽록체 적어도 하나 stromule 형성 여부를 결정한다. percenta으로 보고서 stromule 주파수stromule와 엽록체의 GE. 주 : 일부 연구자 stromule 주파수를보고하기 전에, 아크 사인 변환하여, 예를 들어, 백분율 변환; 이 통계적 분석에 대한 옵션 인 반면, stromule 주파수 정현 직관적 값이되지 않고, 연구의 본문 또는 도면에보고 할 필요가 없다. <ol><li> 다른 방법으로, stromules의 총 수를 계산하고, 엽록체의 총 개수로 나눈다. 주 : 대부분의 경우,이 4.2에 접근하는 유사한 결과를 얻을 것입니다; 단일 엽록체 많은 stromules을 형성 한 경우는, 그러나, stromule 주파수 과대 평가된다. 대표적인 결과를 아래에 도시 된 예에서, 예를 들어, 여러 엽록체 (87분의 33의 주파수 대 stromule) 87분의 40에 엽록체 당 stromules 수를 높여 둘 이상의 stromules있다. </li><li> 또한, 대신 stromule 주파수의 stromule 길이를 결정합니다. 길이를 측정 할 수있다프리 핸드 선 도구를 사용하여 stromule을 추적하여 ImageJ에있다. 참고 stromules의 생물학적 역할은 알려져 있지 때문에,이 stromule 길이가 그 기능에 영향을 미치는 것을 명확하지 않다. 그들이 관찰하는 경우 stromule 길이가 유의 한 차이를보고하지만 (4.2에 설명 된대로)도 stromule 주파수를보고합니다. 참고 : Stromule 주파수는 동일한 성장 조건에서 다른 나뭇잎 사이에서 매우 다양 할 수있다. 따라서, 시간 소모적 일 수 stromule 주파수를 결정하지만, 실험은 신중하게 큰 샘플 크기를 포함하도록 설계되어야한다. 우리 실험실에서 데이터 세트를 사용하여, 우리는 기준 (각 처리에 대해 적어도 16 개의 식물의 샘플 크기는 두 조건 사이 stromule 주파수의 적어도 1.5 배의 변화가 통계적으로 유의 한 차이가 존재 하는지를 판정하기 위하여, 일반적으로 충분히 강력하다고 판단 α = 0.05 β는 = 0.80). </li></ol></li><li> 결과의 통계 분석. <ol><li> 중동을 비교하려면이 치료의 stromule 주파수는, (도 동일하지 않은 분산을 가정 heteroscedastic t 테스트 또는 t 테스트라고도 함) 웰치 t 테스트를 사용합니다. 주 :이 테스트는 종래 스튜던트 t 테스트처럼 강력하지 않다하지만 stromule 주파수 분포의 변화는 치료와 유사하다고 가정하지 않으므로 웰치 t 테스트가 바람직하다. 참고 : stromule 주파수는 &quot;비율&quot;이기 때문에, 그 샘플링 분포는 이론적으로 스큐 통계 분석을 표본 크기가 매우 낮은 경우 또는 stromule 주파수 (근접 0 %까지) 또는 매우 매우 낮은 경우 수있는, 이항보다는 정상적인 것으로 예상된다 높은 (100 %에 가까운). 몇몇 보고서는 정규 분포 이항 분포를 변환함으로써 학생의 t- 시험 또는 ANOVA 표준 요건을 충족하기위한 통계적 분석 전에 아크 사인 변환을 사용했다. 실제로, 그러나rcsine 변환은 통계 분석에 거의 또는 전혀 영향을 미치지 때문에 사용하지 않는 것이 좋습니다. 그 대신에, 통계적 전력을 증가 및 데이터의 해석의 오류를 줄일 샘플 크기를 증가시키는 실험을 반복한다. </li><li> 사용 통계 어떤 접근 방법,주의 깊게 각 실험에 대한 표본 추출 방법, 표본의 크기, 통계 테스트 및 p 값을보고해야합니다. 주 : 생물학의 다른 분야와 같이, p 값 0.05는 &quot;통계적으로 유의 한&quot;고려 될 수있다, 연구자들은 확실히 얻은 정확한 p 값을보고해야하지만. p는 두 개 또는 세 개의 실험 시료의 크기를 증가 약간 0.05 이상이면 관찰 된 차이가 통계적으로 유의하고 재현 가능인지의 여부를 명확히하고있다. </li></ol></li></ol> 5. Stromule 역학을 시각화하기 위해 본래 엽록체 추출 참고 : 여러 가지 방법관내 15 stromule 형성에 대한 최근의 연구에서 약간 다른 프로토콜을 포함, 잎에서 엽록체를 분리하기 위해 사용되어왔다. 아래에 설명 된 프로토콜은 생화학 엽록체 순수한 샘플을 생성하지 않는 비교적 간단한 방법을 사용하는 대신 그대로 건강 엽록체 9,18 다량 격리 않는다. <ol><li> 냉 추출 버퍼 준비는 50mM 헤 페스의 NaOH를 330 mM의 소르비톨, 2 mM의 EDTA, 1.0 밀리미터의 MgCl 2와 1.0 mM의 MnCl 2. 사용하기 전에 수산화 나트륨과 염산, 냉장 6.9로 pH를 조정합니다. 참고 : 비록 필요하지, 감기 버퍼를 사용하여 그대로 엽록체의 높은 비율을 얻을 것입니다 가능하면 얼음에 샘플을 유지. </li><li> 분리 준비 완충액 : 50 mM의 헤 페스의 NaOH를 330 mM의 소르비톨, 2 mM의 EDTA, 1.0 mM의 MnCl 2, 1.0 밀리미터의 MgCl 2, 10mM의 KCl을, 1.0 mM의 염화나트륨. 사용하기 전에 수산화 나트륨과 염산 냉장와 7.6 pH를 조정합니다. </li><li> 잎이없는 경우디메틸 설폭 사이드에서 50 mM의 CFDA 주식 솔루션 (DMSO) (1.0 ml의 DMSO 당 2.3 mg을 CFDA) : 유전자 인코딩 된 기질의 형광을 발현, 카르복시 디 아세테이트 (CFDA) 솔루션을 준비합니다. 참고 : 정확한 농도는 중요하지 않다. 항상 어둠 속에서 CFDA의 재고를 유지합니다. -20 ° C에서 50 mM의 CFDA의 작은 분취 량을 저장합니다. </li><li> 엽록체를 분리하기 위해 여러 식물에서 ~ 5~10g 잎을 제거하고 간단하게 찬물에 헹군다. 즉시 50 ㎖ 차가운 추출 버퍼에 전송합니다. 여러 짧은 펄스로 믹서기를 사용하여 분쇄 잎. 이 50 ㎖ 원심 분리기 튜브로 추출 된 엽록체를 분할, 잎 파편을 제거하기 위해 투박한 무명의 두 개 또는 세 개의 레이어를 통해 필터, 750 X g에서 1 분간 원심 분리기. </li><li> 뜨는을 취소하고 10 ml의 분리 버퍼에 녹색 엽록체를 재현 탁. 원심 분리기는 다시 750 XG에 1 분 동안, 상층 액을 버리고, 및 절연 버퍼에 resuspend의 엽록체 5 ml의 최종 부피를 가지고. </li><li> 전송 (20)1, 슬라이드에 엽록체의 리터는 커버 슬립으로 커버하고, 현미경을 시작합니다. 참고 : 플라 타겟 형광 단백질을 발현하는 형질 전환 식물에서 엽록체 이제 공 초점 형광 현미경으로 시각화를위한 준비가되어 있습니다. </li><li> stromules을 시각화 플라 타겟 형광 단백질을 발현하지 않는 식물에서 엽록체를 얼룩. <ol><li> 분리 버퍼에 엽록체가 탁 5 ㎖의 50 mM의 CFDA 주식의 5 μl를 추가합니다. 50 μM에 최종 농도를 가져옵니다. </li><li> 엽록체는 5 분 동안 배양 한 다음 슬라이드에 작은 나누어지는 (~ 50 μL)를 전송하는 커버 슬립으로 커버하고, 현미경을 시작 할 수 있습니다. </li><li> FITC 또는 GFP 필터 세트를 사용합니다. 하나의 고립 된 엽록체를 시각화하는 동시에 많은 엽록체를 시각화하는 20X 목표, 또는 더 높은 목표를 사용합니다. 참고 : CFDA 그대로 엽록체의 기질 내부에 형광 것입니다. </li><li> 강한 배경 형광이있는 경우, chlo 씻어다시 CFDA, 750 XG에 1 분 동안 원심 분리기로 5 분 부화 후 10 ml의 분리 버퍼에 roplasts은 5 ml의 분리 버퍼에 뜨는, 그리고에 resuspend 폐기합니다. </li></ol></li></ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium-derived+cytokinin+influences+plastid+morphology+and+starch+accumulation+in+Nicotiana+benthamiana+during+transient+assays.">Agrobacterium-derived cytokinin influences plastid morphology and starch accumulation in Nicotiana benthamiana during transient assays.</a> BMC Plant Biol. 14 (1), 127 (2014).</li><li>Ho, J., Theg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formation+of+stromules+in+vitro+from+chloroplasts+isolated+from+Nicotiana+benthamiana.">The formation of stromules in vitro from chloroplasts isolated from Nicotiana benthamiana.</a> PLoS One. 11 (2), e0146489 (2016).</li><li>Brunkard, J. O., Burch-Smith, T. M., Runkel, A. M., Zambryski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+plasmodesmata+genetics+with+virus-induced+gene+silencing+and+an+Agrobacterium-mediated+GFP+movement+assay.">Investigating plasmodesmata genetics with virus-induced gene silencing and an Agrobacterium-mediated GFP movement assay.</a> Method. Mol. Biol. , 185-198 (2015).</li><li>Schattat, M. H., Kl&#246;sgen, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+stromule+formation+by+extracellular+sucrose+and+glucose+in+epidermal+leaf+tissue+of+Arabidopsis+thaliana.">Induction of stromule formation by extracellular sucrose and glucose in epidermal leaf tissue of Arabidopsis thaliana.</a> BMC Plant Biol. 11, 115 (2011).</li><li>Joly, D., Carpentier, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+intact+chloroplasts+from+spinach+leaves.">Rapid isolation of intact chloroplasts from spinach leaves.</a> Method. Mol. Biol. 684, 321-325 (2011).</li><li>Waters, M. T., Fray, R. G., Pyke, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromule+formation+is+dependent+upon+plastid+size,+plastid+differentiation+status+and+the+density+of+plastids+within+the+cell.">Stromule formation is dependent upon plastid size, plastid differentiation status and the density of plastids within the cell.</a> Plant J. 39 (4), 655-667 (2004).</li></ol>

The authors have nothing to disclose.

stromules을 조사 할 때, 세 가지 중요한 요인 전반에 걸쳐 고려되어야한다 : 절대 최소로 유지해야 식물 조직의 (ⅰ) 조작, (ⅱ) 실험 시스템은 일관성을 유지해야하며, (ⅲ) 샘플링 전략은 신중을 계획해야합니다 강력한 보장, 재생 가능한 데이터 분석된다. Stromules는 매우 동적 : 그들은 확장하고 현미경 관찰자의 눈 앞에 빠르게 철회 할 수 있습니다. 또한 stromule 주파수 (예 : 잎...

Chloroplasts are dynamic organelles in plant cells responsible for photosynthesis and a host of other metabolic processes. Signaling pathways from the chloroplast also exert significant influence on plant physiology and development, coordinating plant responses to environmental stress, pathogens, and even leaf shape1-6. Recently, biologists have gained interest in a poorly understood aspect of chloroplast structure: stromules, very thin stroma-filled tubules that extend from the surface of the chloroplast7.
The biological functions of stromules remain unknown, although stromule frequency is known to vary in response to environmental stimuli7-9, and stromules may be capable of transmitting signaling molecules between organelles6. All types of plastids (not only the green, photosynthetic chloroplasts, but also clear leucoplasts, starch-filled amyloplasts, and pigmented chromoplasts, to name a few types of plastids) make stromules, and stromules are found in all land plant species that have been examined to date. Stromules can extend and retract dynamically, appearing or disappearing within seconds, or they can remain relatively stationary for long times. One of the major hurdles facing stromule biologists is that stromules are often studied using dramatically different methods, tissues, and species, making comparisons across the stromule biology literature difficult. Going forward, standard practices and thorough descriptions of the experimental systems used to study stromules will be critical to discovering the function of these ubiquitous features of chloroplast morphology.
Here we describe methods for visualizing stromule formation in the epidermal chloroplasts of Nicotiana benthamiana leaves. In the mesophyll, chloroplasts are densely packed into large, three-dimensional cells, which makes it difficult to accurately and rapidly visualize stromules by confocal microscopy. By contrast, epidermal cells are relatively flat, contain fewer chloroplasts, and are at the surface of the leaf, allowing for easy and rapid visualization of stromules. N. benthamiana is an ideal model system for these experiments because, unlike many plant species, all cells in the epidermis of N. benthamiana make chloroplasts10. In the epidermis of most plants, including Arabidopsis thaliana, only the stomatal guard cells have chloroplasts, while other epidermal cells have "leucoplasts", plastids that are clear, relatively amorphous, and nonphotosynthetic9,11,12. Thus, whereas a single field of view of an A. thaliana epidermis might show only a handful of chloroplasts in a pair of guard cells, a field of view of an N. benthamiana epidermis will include dozens or even hundreds of chloroplasts. All of the methods described here, however, can be modified to investigate other questions in stromule biology; for example, we have used the same approach to study leucoplast stromules of A. thaliana9.

<table><tbody><tr><td>Hepes</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>NaOH</td><td>Fischer-Scientific</td><td>S320-1</td><td></td></tr><tr><td>Sorbitol</td><td>Sigma-Aldrich</td><td>S1876</td><td></td></tr><tr><td>EDTA</td><td>Fischer-Biotech</td><td>BP121</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>221279</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M0250</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9625</td><td></td></tr><tr><td>Laser Scanning Confocal Microscope&amp;nbsp;</td><td>Carl Zeiss Inc</td><td>Model: LSM710</td><td></td></tr><tr><td>Carboxyfluorescein diacetate (CFDA)</td><td>Sigma-Aldrich</td><td>21879&amp;nbsp;</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>EMD</td><td>MX1458-6</td><td></td></tr><tr><td>Waring blender</td><td>Waring&amp;nbsp;</td><td>Model: 31BL92</td><td></td></tr><tr><td>Fiji</td><td>fiji.sc</td><td></td><td>Open-source software for analyzing biological images</td></tr></tbody></table>

visualizing stromule frequency with fluorescence microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

이 프로토콜은 젊은 N.의 자엽에서 하루 밤에 stromule 주파수를 시각화하는 데 사용 된 benthamiana 모종. Z- 스택에서 슬라이스는 하나의 이미지 (그림 1A)에 병합되었다. 시각적 인 목적을 위해, 그 이미지는 흐릿한과 기질 블랙 (그림 1B)를 나타나도록 반전했다. 엽록체는 하나 더 stromules (녹색 별표)를 갖지 않는 또는 적어도 하나 st...

Watch this Scientific Journal Video about Visualizing Stromule Frequency with Fluorescence Microscopy at JoVE.com

Visualizing Stromule Frequency with Fluorescence Microscopy

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

형광 현미경으로 Stromule 주파수를 시각화

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

visualizing-stromule-frequency-with-fluorescence-microscopy

Research

JoVE Journal

Biology

8.0K 조회수.  USDA. Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

비디오: Visualizing Stromule Frequency with Fluorescence Microscopy

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins.
Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

표면 식민지 미생물 집단에 공간 분리를 모니터링

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

연구

유전학

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

생활 세포의 분자 Rotors의 형광 수명 이미징

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

생체공학

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

라이브 셀 이미징 바실러스 subtilis과 연쇄상 구균 pneumoniae 자동 시간 저속 현미경을 사용하여

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

면역학 및 감염병학

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 &#181;m x 500 &#181;m region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

포토닉 칩을 사용한 고처리량 총 내부 반사 형광 및 직접 적층 광학 재건 현미경

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

생화학

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

단일 분자 단지를 떠올리 VIVO에서 고급 형광 현미경을 사용하여

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

생물학

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. 
Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series.

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Streptomyces are characterized by a complex life cycle that has been experimentally challenging to study by cell biological means. Here we present a protocol to perform fluorescence time-lapse microscopy of the complete life cycle by growing Streptomyces venezuelae in a microfluidic device.

전체의 형광 시간 경과 영상<em&gt; S. venezuelae</em미세 유체 장치를 사용&gt; 수명주기

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of ...

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

세포 수준에서 곰팡이 - 미생물 상호 작용을 조사하기위한 미세 유체 도구

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

단일 효모 세포에서 MAPK 유도 표현의 시간적 정량화

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

단일 분자 형광 제자리에서 교 잡 실험에 대 한 개별 대장균 세포에 성적표를 라벨에 대 한 방법

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

형광 현미경으로 Stromule 주파수를 시각화

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