Name: real-time imaging of plant cell surface dynamics with variable-angle epifluorescence microscopy
Uploaded: 2015-12-12T14:00:00.000+00:00
Duration: 6 min 31 s
Description: Watch this Scientific Journal Video about Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy at JoVE.com

I am grateful to Dr. Masaru Fujimoto for his technical suggestions for VAEM. I am also grateful to Prof. Koh Iba and Dr. Mimi Hashimoto-Sugimoto for providing GFP-PATROL1 transgenic plants, and discussions about PATROL1. I thank Prof. Seiichiro Hasezawa for his continuing support of my work. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI grant number 25711017.

모종의 1. 준비 <ol><li> 씨앗을 소독. <ol><li> 1 μL 10 % 트리톤 X-100-500 μL 멸균 : 500 μL 차아 염소산 나트륨 (5.0 % 사용할 수 염소)를 추가하여 살균 솔루션을 준비합니다. </li><li> 장소의 약 10 형질 전환 A. 1.5 ML 튜브에 GFP - PATROL1 8을 들고 장대 씨. </li><li> 1 ml의 70 % 에탄올 용액을 추가, 5 번 반전 잘 섞는다. 1 분 동안 둡니다. </li><li> 씨앗은 튜브의 바닥에 가라 관찰한다. 깨끗한 층류 캐비닛 부드럽게 마이크로 피펫을 사용하여 70 % 에탄올을 제거하고 1 mL의 멸균 용액을 추가. 다섯 번 반전 잘 혼합하고 5 분 동안 둡니다. </li><li> 씨앗을 씻으십시오. 또 클린 벤치에 무균 상태에서 작업, 부드럽게 마이크로 피펫을 사용하여 용액을 제거하고, 1 ml의를 멸균 물을 추가합니다. 이 다섯 번 반복합니다. 살균 된 종자를 2 일 동안 4 ℃에서 멸균에 저장 될 수있다. </li></ol></li><li> 그래서0.5 % 젤란 검 화 1/2 무라 시게과 스쿡 중간 판 (PH 5.8) 9 살균 씨앗 승. 외과 테이프의 두 레이어를 사용하여 접시에 뚜껑을 테이프입니다. </li><li> 냉장 챔버, O / N과 4 ° C에서 어둠 속에서 접시를 품어. </li><li> 100 μmol m -2 초 -1 흰색 조명을 사용하여 12 시간 / 12 시간의 명암주기와 23.5 ° C로 설정 성장 챔버로 접시를 전송하고, 7 일간 배양한다. 길이 약 1mm의 자엽과 모종은 수확 할 수있다. </li></ol> 2. 스카이 드롭 떡잎 표본의 설치 주 : VAEM 관찰 용 시료 제조에 중요한 요소는 표본과 커버 유리 사이에 기포의 혼입을 피할 수있다. 거품이 크게 굴절률 차이시킴으로써 VAEM의 화질을 감소시킨다. 우리가 장착 &#39;하늘 드롭&#39;라고 한 간단한 방법은, 거품을 방지하기 위해 사용될 수있다A. 사이 장대 자엽과 커버 유리. 이 관찰 직전에 수행해야합니다. <ol><li> 76 : 기초 버퍼 [5 mM의 2- (N의 -morpholino) -ethanesulfonic의 pH 6.5에서 산 트리스 (MES 트리스), 50 mM의 KCl을, 100 μm의 염화칼슘 2] 유리 슬라이드의 중심에 (크기의 30 μl를 배치 × 26mm, 두께 : 1.0 ~ 1.2 mm). </li><li> 해부 가위를 사용하여 7 일짜리 묘목에서 자엽을 제거합니다. 관찰 측이 기초 버퍼 드롭 (그림 1, 1 단계)에 위를 향하게 자엽 플로트. </li><li> 커버 유리의 중심에 기초 버퍼의 30 μl를 놓습니다 (크기 : 18 × 18mm, 두께 : 0.12-0.17 mm) (그림 1, 2 단계). 거꾸로 부드럽게 커버 유리를 돌립니다. 표면 장력은 탈락에서 버퍼 저하를 방지 할 수 있습니다. 하나의 가장자리에 핀셋과 커버 유리를 잡고. 버퍼 강하가 약 떡잎 위에 있도록 유리 슬라이드에 반대 가장자리를 놓습니다. </li><li>이 자엽 샘플 (도 1, 단계 3) 바로 위에 있도록 슬라이드 여전히 커버 유리의 가장자리, 여전히 핀셋 반대쪽 가장자리 들고, 커버 유리 아래 버퍼 강하 위치를 조정한다. 커버 유리 가자. 기포없이 준비의 결과로 샘플에 드롭을 탑재합니다. </li><li> 보풀이없는 조직을 사용하여 과도한 버퍼를 닦아냅니다. 바로 준비를 관찰 (3 단계 참조). 참고 : 샘플을 밀봉 할 필요가 없습니다. </li></ol> 3. VAEM 관측 및 영화 취득 도립 현미경 TIRF 유닛과 1.49의 개구 수를 가지는 대물 렌즈 TIRF 장착되어 참고 : 다음과 같이, 본 연구에 사용 TIRF 현미경 시스템 (9)에 대하여 설명한다. 레이저 진입 각도의 컴퓨터 제어를 들어, 컨트롤 박스가 사용된다. 녹색 형광 단백질 (GFP)는 488 nm의 광 펌핑 반도체 레이저 및 T와 흥분그 형광은 엽록체에서자가 형광을 방지 5백10부터 5백50까지 nm의 대역 통과 필터로 검출된다. 광 출력 전력의 측정 된 최대 값은 13.0에서 13.5까지 mW였다. 검출을 위해, 전자가 사용된다 (EM-CCD) 카메라 헤드 시스템 및 C 마운트 카메라 배율 변경 단위 전하 결합 소자를 승산. <ol><li> 레이저는 제조자의 지시에 따라 중심 및 포커스 캘리브레이션. 참고 :이 단계는 정확한 VAEM 관측을 위해 중요하다. 그것은 강력 레이저 경로 조정 절차의 정기적 인 검사, 현미경에 적합한, 수행하는 것이 좋습니다. <ol><li> 현미경 실의 천장에 대물 렌즈가없는 광경으로 조명 중심 위치를 결정합니다. 중앙 위치를 표시하기 위해 천장에 색칠 된 원 씰을 넣어 (그림 2, 1 단계, 2). </li><li> 대물 렌즈 (그림 2, 3 단계, 4)와 천장 조명. 나는 이동중심 위치 (그림 2, 5 단계)로 영역을 lluminated. 레이저 (그림 2, 6 단계)에 초점을 맞 춥니 다. 미세 조정 센터 (그림 2, 7 단계)에 초점을 맞춘 레이저의 위치. </li></ol></li><li> 현미경의 무대에서 표본을 설정하고 시야 조명을 사용하여 관찰 셀을 선택합니다. </li><li> 형광 단백질이 세포에서 관찰 될 수 있다는 것을 확인하고, 표면 형광 조명을 (도 3a)을 사용하여 세포 표면의 Z 위치를 설정시킴으로써 행한다. </li><li> VAEM 관측을 수행합니다. <ol><li> 제어기 상자 서서히 레이저 빔의 진입 각도 경사지게. 동시에, 신중하게 라이브 영상을 모니터링 할 수 있습니다. 초기에, 이미지는 (도 3b)을 흐리게한다. </li><li> 레이저 각도가 증가함에 따라, 화상 VAEM 결국 선명한 화상 (도 3c 및 D)의 제조, 이하 희미해질 것이다. 이 시점에서, increa을 중지레이저 각도를 노래. 형광 신호가 손실되는 경우, 더 얕은 각도로 감소한다. </li><li> 미세 조정 레이저 진입 각도는 더 나은 이미지를 얻을 수있다. 미세 조정 Z시킴으로써 행한다 위치는 이미지를 향상시킬 수 있습니다. 필요한 경우, 광학 파라미터 (레이저 파워, 파장 필터 집합)과 이미지 센서 파라미터 [화상 사이즈, 노출 시간, 게인, 디지타이저와 승산 전자 (EM)의 이득]을 조정한다. 주 : TIRF 현미경 장비의 경우 전술 한 바와 같이 다음에, 대표적인 파라미터이다. 그냥 카메라 앞에 렌즈의 배율 : 2 배; 레이저 출력 : 1.0 mW의; 이미지 크기 : 512 × 512 픽셀; 노출 시간 : 100 밀리 초; 게인 : 5 ×를; 디지타이저 : 11 MHz의; 및 EM 이득 : 100. </li></ol></li><li> 현미경 상용 소프트웨어를 사용하여 멀티 페이지 TIFF 이미지 파일로서 영화를 취득. 여기서, 영화 기공 세포의 표면 상에 점멸 GFP 표지 된 점이다. 주 : 대표 화상 취득 조건 FOL 같다최저. 획득 모드 : 스트림 RAM에; 프레임 수 : 600; 멀티 페이지 TIFF 파일 크기 : ~ 302메가바이트 </li></ol> 4. Kymograph 분석 정량 GFP 표지 점 레지던스 시간의 피지 소프트웨어를 사용하여 <ol><li> 피지를 설치 저자의 지침을 사용하여 소프트웨어 (10) ( &#39;피지 그냥 ImageJ에입니다&#39;) (http://fiji.sc/Fiji). </li><li> 피지를 실행하고 피지 메뉴 &quot;파일 - 열기&quot;를 사용하여 획득 한 멀티 페이지 TIFF 파일을 엽니 다. </li><li> 피지 도구 모음 메뉴 &quot;직선 선택 도구&quot;를 사용하여, 관심있는 사이트에 줄을 놓습니다. 선택적으로, &quot;분단 선 선택 도구&quot;또는 &quot;자유 곡선 선택 도구&quot;를 사용합니다. 여기에 제시된 결과에서 (8.0 μm의 상당) 100 픽셀의 직선 자회사 세포 (도 4a)에 위치시켰다. </li><li> 피지 메뉴 &quot;이미지 - 스택 - 동적 다시 분할을 사용하여 kymograph 이미지를 확인 </eM&gt; &quot;(http://fiji.sc/Dynamic_Reslice) (그림 4B). 선택적으로, 체크 박스 창 및 「90도 회전」 「세로 플립 &quot;도 (100 픽셀에 대응하는 (여기에 제시된 결과에서 사용되지 않음) kymograph 이미지. X 축 및 Y 축 라인의 위치를 나타내는 사용할 8.0 μm의) 및 관찰 시간은 (600 픽셀 각각) 60 초에 대응. kymograph 이미지가 만족스럽지 않으면, 위치 및 유형 (직선, 분할 및 다중 페이지 이미지 라인의 자유형)있다. 변화 될 수있다 라인 변경, kymograph 이미지가 동적으로 변경됩니다. 피지 메뉴 &quot;파일 - 다른 이름으로 저장 - 티파니&quot;를 사용하여 TIFF 파일로 kymograph 이미지를 저장합니다. </li><li> 시간축 방향에서 블롭의 길이를 측정한다. <ol><li> 노이즈 감소를 수행하려면, 피지 메뉴 &quot;프로세스 - 필터 - 가우시안 블러&quot;를 사용하여 kymograph 이미지에 가우시안 필터를 적용합니다. &quot;시그마 (라드IUS)은 &quot;조정 가능한 파라미터는 (1 화소 반경 제시된 결과에 이용된다)이다. </li><li> 세그먼트 피지 메뉴를 사용하여 임계 값에 의한 신호 영역, &quot;이미지 - 조정 - 임계 값&quot;. 주 : 선택할 수있는 여러 임계 알고리즘이 있습니다. 발표 결과에서 &quot;엔&quot;알고리즘은 (그림 4C) 선택되었다. </li><li> 메뉴를 이용하여 이동시킴으로써 행한다 블롭의 시간 길이 (y)를 측정하도록 준비 &quot;분석 설정된 측정&quot;. &quot;세트 측정&quot;창에서 &quot;경계의 구형&quot;을 확인하십시오. 이상적 영역 세트는 노이즈를 배제하는, 가능한 한 작아야한다. 여기서, 극소 영역의 화소는 50으로 설정 하였다. 시간 (Y)를 사용하여 메뉴시킴으로써 행한다 블롭의 길이를 측정 &quot;분석 - 입자 분석&quot;. 관리자에 추가 &quot;결과 값을 구하여 측정 영역의 신뢰성을 입증,&quot;결과 표시 &quot;를 확인하려면 &lt;/ EM&gt; &quot;상자. </li><li> &quot;높이&quot;열의 값 (Y)를 측정 BLOB (그림 4D)에 대한 길이를 이동시킴으로써 행한다 시간입니다. 결과 테이블 메뉴 &quot;파일 - 다른 이름으로 저장&quot;을 사용하여 값을 저장하고 계산 및 통계 패키지 및 / 또는 스프레드 시트 소프트웨어 (그림 4E)를 사용하여 BLOB 이미지 기간의 데이터 세트를 처리합니다. </li></ol></li></ol>

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Methods. 9 (7), 676-682 (2012).</li><li>Chebli, Y., Kaneda, M., Zerzour, R., Geitmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+wall+of+the+Arabidopsis+pollen+tube-spatial+distribution,+recycling,+and+network+formation+of+polysaccharides.">The cell wall of the Arabidopsis pollen tube-spatial distribution, recycling, and network formation of polysaccharides.</a> Plant Physiol. 160 (4), 1940-1955 (2012).</li><li>Fujimoto, M., Suda, Y., Vernhettes, S., Nakano, A., Ueda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol+3-kinase+and+4-kinase+have+distinct+roles+in+intracellular+trafficking+of+cellulose+synthase+complexes+in+Arabidopsis+thaliana.">Phosphatidylinositol 3-kinase and 4-kinase have distinct roles in intracellular trafficking of cellulose synthase complexes in Arabidopsis thaliana.</a> Plant Cell Physiol. 56 (2), 287-298 (2015).</li><li>Li, X., Wang, X., Yang, Y., Li, R., He, Q., Fang, X., Luu, D. T., Maurel, C., Lin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+analysis+of+PIP2;+1+dynamics+and+partitioning+reveals+multiple+modes+of+Arabidopsis+plasma+membrane+aquaporin+regulation.">Single-molecule analysis of PIP2; 1 dynamics and partitioning reveals multiple modes of Arabidopsis plasma membrane aquaporin regulation.</a> Plant Cell. 23 (10), 3780-3797 (2011).</li><li>Li, R., Liu, P., Wan, Y., Chen, T., Wang, Q., Mettbach, U., Baluska, F., Samaj, J., Fang, X., Lucas, W. 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저자는 공개 아무것도 없습니다.

이 비디오 기사에서는 프로토콜 모니터링 및 애기 장대의 기공 단지에 GFP - PATROL1 점의 동적 거동을 측정하기 위해 제공됩니다. 여기에 도시 된 바와 같이, VAEM 관찰 식물 세포 표면의 라이브 영상을위한 강력한 도구입니다. 고감도 EM-CCD는 VAEM 광학계에 비교적 약한 여기 레이저의 사용을 허용하기 때문에 GFP-PATROL1 모니터링 여기 사용 된 실험 조건 하에서, 비디오 캡쳐의 1 분에 사용 시료 거의 형광 광표백가 있었다. 레이저 센터링 및 각 실험의 시작하기 전에, 포커스, 성공적인 VAEM 관찰을 위해 중요하다. 사용자는 VAEM를 사용하기 전에 선택한 현미경 회사에서 전문 직원에 의해 훈련해야한다. VAEM는 GFP - PATROL1는 세포막에 깜박이는 움직이지 점 같은 구획 동적 현지화를 가지고 있음을 알 수있다. 형광 표지 된 세포막 프로톤 펌프 AHA1 오 -1-, 공변 세포와 보조 세포 patrol1 돌연변이에 ocalizes 이전에 8,9를보고. 따라서, 시각 VAEM-GFP-PATROL1 도트 역학 원형질막으로 프로톤 펌프의 전달을 나타낼 수있다. GFP-PATROL1 이러한 동적 동작은 식물 세포 표면 상에 실시간 단백질 역학 잠복 VAEM에서의 유용성을 나타내는 종래의 검류계 미러 형의 공 초점 레이저 주사 현미경을 사용하여 시각화하는 것은 곤란하다. GFP - PATROL1 관찰 식물 세포 생물학 VAEM 응용 프로그램의 한 예입니다. VAEM는 (식물 세포에서의 세포질의 얇은 외피 층의 다수의 이벤트를 감시에 적용 할 것 등, 효소 세포벽 합성 분비 또는 수정 11,12, 세포막 단백질 역학 7,13,14, 피질 미소 관 역학 4 액틴 미세 섬유 4,15의 재 배열 및 세포 소기관 운동 6). 식물 세포 과학자의 다양한 베네한다VAEM 기술에서 맞습니다. 생물학적 화상 데이터의 정량적 평가 최근 현미경 장비 및 컴퓨팅에서의 발전에 힘 입어 중요 해지고있다. 600 (t) × 512 화소 (Y) × 512 (X)를 함유 VAEM 동영상 데이터를 쉽게 몇 분 밖에 얻을 수있다. 이미지 분석은 형광 표지 된 단백질의 수명이나 움직임을 측정하는 데 도움이뿐만 아니라 대형 다차원 이미지 데이터 세트로부터 생체 정보를 채굴한다. kymograph는 세포막 단백질의 2 상으로 순차적 채용에 의해 도시 된 바와 같이, VAEM 무비 데이터로부터 목적 단백질의 이동을 결정하는 단계에 기초하지만, 가치있는 방법이다. 도 4에 도시 된 바와 같이 kymograph는 GFP-PATROL1 도트 &#39;체류 시간의 시각화 및 세포막에 낮은 운동을 허용. 반대로 kymograph는 세포질 스트리밍과 같은 더 복잡한 움직임을 평가하는 효과를 제한하고있다. 더 compli 들어광학 플로우 (16)가 도움이 될 것으로 cated 운동, 다른 이미지 분석은 접근한다.

The plant cell surface, including the plasma membrane and its immediately adjacent cytoplasm, is the main region of a plant cell&#8217;s perception and integration of biotic and abiotic cues from the extracellular environment. In response to these cues, cell surface components including plasma membrane proteins and the cortical cytoskeleton undergo dynamic changes, on a time scale of seconds to minutes1-4. Thus, real-time and high-resolution imaging of fluorescent proteins on the cell surface can illuminate a plant&#8217;s responses to environmental cues at the molecular level.
Confocal laser scanning microscopy is a powerful tool for determination of fluorescently-tagged protein localization3, however, it is often difficult to monitor the real-time protein dynamics because of its relatively long capturing times. An emerging technique for real-time monitoring of proteins in the plant cell is variable angle epifluorescence microscopy (VAEM), which is an adaptation of equipment usually used for total internal reflection fluorescence (TIRF) microscopy. In TIRF microscopy, the fluorescence-excitation light source is an evanescent light field that is generated when the entry angle of the laser is shallow enough to totally internally reflect light at the glass&#8211;water interface. The penetration depth of the evanescent light field is around 100 nm. TIRF microscopy is an outstanding tool for single molecule imaging, such as the detection of exocytosis in animal cells5. However, evanescent light cannot reach plasma membranes or the cortical cytoplasm in plant cells, because they have thick cell walls. Recently, TIRF microscopy equipment has been adapted by plant cell biologists, observing that a laser, if angled slightly more deeply than when being used to induce total internal reflection phenomena, could excite the surface of plant cell samples, resulting in high-quality plant cell imaging6,7. The excitation illumination depth is varied by adjusting the entry angle of the laser; therefore, this technique is described as VAEM. This optical system is also called variable angle TIRF microscopy (VA-TIRFM) because there is a possibility that total reflection may take place at the cell wall-periplasm interface7, however, the term VAEM is used in this article, as per the first report in plants6.
The goal of this protocol is to demonstrate practical procedures for using VAEM to visualize fluorescently-tagged protein dynamics on plant cell surfaces. Additionally, an image analysis protocol to quantify the residence time (duration of presence) of molecules is described for VAEM movie analysis. GFP-PATROL1 dot blinking on stomatal complex cells in Arabidopsis thaliana cotyledons is used as an example. PATROL1 was identified by forward genetic approaches as a causal gene of a stomatal response defect mutant in A. thaliana8. PATROL1 is a plant homolog of MUNC-13, which is a priming factor in synapse vesicle exocytosis8. In response to environmental cues, such as light or humidity, it is thought that PATROL1 reversibly regulates the delivery of a proton pump to plasma membranes in the stomatal complex. Stomatal complexes each comprise a pair of guard cells8 and subsidiary cells9, and they require a proton pump for stomatal movement. In these cells, GFP-tagged PATROL1 localizes to dot-like structures that remain on the plasma membrane for less than 1 min9.

<table><tbody><tr><td>Inverted microscope</td><td>Olympus</td><td>IX-73</td><td></td></tr><tr><td>TIRF unit</td><td>Olympus</td><td>IX3-RFAEVAW</td><td></td></tr><tr><td>TIRF objective lens&amp;nbsp;</td><td>Olympus</td><td>UAPON 100 &amp;times; OTIRF&amp;nbsp;</td><td>NA = 1.49</td></tr><tr><td>Laser angle control box</td><td>Chuo Seiki</td><td>QT-AK</td><td></td></tr><tr><td>Optically pumped semiconductor laser</td><td>Coherent</td><td>Sapphire&lt;sup&gt;TM&lt;/sup&gt; LP USB 488-20 CDRH Laser</td><td></td></tr><tr><td>510&amp;ndash;550 nm band-pass filter</td><td>Olympus</td><td>U-FBNA</td><td></td></tr><tr><td>EM CCD camera</td><td>Hamamatsu Photonics</td><td>ImagEM C9100-13</td><td></td></tr><tr><td>C-mount camera magnification change unit&amp;nbsp;</td><td>Olympus</td><td>U-TVCAC</td><td></td></tr><tr><td>MetaMorph software</td><td>Molecular Devices</td><td>MetaMorph version 7.7.11.0</td><td></td></tr><tr><td>TIRF microscopy manual</td><td>Olympus</td><td>AX7385</td><td>Instructions: Total Internal Reflection Illumination System (Printed in Japan on August 24, 2012)</td></tr><tr><td>Immersion oil</td><td>Olympus</td><td>Immersion Oil Typr-F</td><td>ne = 1.518 (23 degrees)</td></tr></tbody></table>

real-time imaging of plant cell surface dynamics with variable-angle epifluorescence microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

이 비디오 기사, A에 GFP - PATROL1의 VAEM 관찰을위한 프로토콜에서 장대 떡잎 기공 복잡한 세포가 제공됩니다. 하늘 강하 실장은 A. VAEM 제제의 기포의 발생을 감소시킬 수있는 간단한 제조 방법 장대 자엽 (그림 1). 엔트리 레이저 및 / 또는 VAEM에 대한 표본의 Z-위치의 과도하게 기울 것은 분명 이미지를 제공합니다. 그렇게되면, 그것은 표면 형광 조명에 의해 판단으로 즉시 샘플 위의 위치부터 다시 시작하는 것이 좋습니다. VAEM 관찰 며칠 경험 한 결과, 이러한 점멸 도트 GFP-PATROL1의 여기에 도시 한 바와 같이, 성공적으로 일상적 VAEM 동영상을 취득 할 수 있어야한다. 도 4, A. 12 독립 법인 세포에서 GFP-185 PATROL1 도트의 체류 시간에서 제시 kymograph 분석을 이용하여 장대 자엽을 측정 하였다. 장착 밀도기능은 대부분의 GFP - PATROL1 점은 빠른 세포 외 유출 / 양성자의 엔도 사이토 시스는 자회사 세포 표면에 펌프 제안, 약 3.5 초 (그림 4E)의 피크와, 2 내지 10 초 동안 자회사 셀에 거주하는 것으로 확인된다. <img alt="그림 1" src="/files/ftp_upload/53437/53437fig1.jpg" width="700" /> . 하늘의 그림 1. 도식은 방법을 장착 드롭 1 단계 : 표본 애기 장대를 장대 자엽이 유리 슬라이드에 기초 버퍼의 드롭에 떠있다. 단계 2 : 기저 완충액 강하 강하는 커버 유리에 배치된다. 3 단계 : 드롭 커버 유리는 거꾸로되어 드롭 유리 슬라이드에 표본 위에 배치된다. 단계 4 : 커버 유리 풀어 놓기 시험편에 빠진다. (TOP) 새 눈보기. (아래) 측면과 확대도. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53437/53437fig1large.jpg" target="_blank">PLE</a>ASE이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오. <img alt="그림 2" src="/files/ftp_upload/53437/53437fig2.jpg" width="700" /> 도 2 레이저 센터링의 보정 및 초점 1 단계 :. 현미경 리볼버의 빈 위치는 현미경 상기 천장에 레이저 광을 전송하도록 선택된다. 2 단계 : 중앙 위치가 천장에 색칠 된 원 씰을 사용하여 표시됩니다. 단계 3 : 대물이 선택된다. 4 단계 : 대물 렌즈를 통해 레이저 광에 의해 조명의 위치를​​ 확인합니다. 5 단계 : 조명이 표시된 중앙 위치로 조정됩니다. 단계 6 : 레이저 광이 집중된다. 7 단계 :. 초점을 맞춘 레이저 빛이 표시된 중앙 위치로 이동 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53437/53437fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 표면 형광 현미경 및 가변 각도 표면 형광 현미경 (VAEM)도 3의 비교. (A) 표면 형광 현미경 레이저 광로의 개략도. (B) 애기 장대의 7 일 된 모종의 떡잎 표면에 GFP - PATROL1의 대표적인 표면 형광 이미지. 스케일 바는 5 μm의를 나타냅니다. VAEM되는 레이저의 광 경로 (C) 회로도. 레이저의 항목이 기울어 져 있습니다, 그것은 변수 깊이와 시료의 표면을 조명한다. (B)에 도시 된 바와 같이 동일한 영역 (D) VAEM 이미지. 스케일 바는 5 μm의를 나타냅니다. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53437/53437fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 그림 4. Kymograph 분석은 GFP - PATROL1 점의 체류 시간을 정량화한다. (A) VAEM 이미지를 kymograph를 만들기 위해 배치 직선으로. 스케일 바는 5 μm의를 나타냅니다. (B) (A)에 도시 된 라인을 따라 kymograph 이미지. (B)의 (C) 치 화상 피지 생성 엔 소프트웨어의 알고리즘에 기초. 노란색 선은 GFP - PATROL1 신호의 자동 감지 영역을 보여줍니다. (D) 스크린 샷은 피지의 결과 테이블의 기능을 &quot;입자를 분석&quot;. (E) 자회사 세포에서 GFP - PATROL1 점의 체류 시간의 히스토그램. 빨간색 선은 밀도 함수를 보여줍니다. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53437/53437fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy at JoVE.com

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

이 프로토콜의 목적은 기공 복합체의 세포 피질에, GFP - 태그 PATROL1, 막 인신 매매 단백질의 점을 점멸 표시 가변 각도 표면 형광 현미경으로 식물 세포 표면에 형광 표지 된 단백질의 역학을 모니터링하는 방법을 설명하는 것입니다 애기 장대.

가변 각도 표면 형광 현미경으로 식물 세포 표면 역학의 실시간 영상

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

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JoVE Journal

Biology

8.9K 조회수.  The University of Tokyo.  이 프로토콜의 목적은 기공 복합체의 세포 피질에, GFP - 태그 PATROL1, 막 인신 매매 단백질의 점을 점멸 표시 가변 각도 표면 형광 현미경으로 식물 세포 표면에 형광 표지 된 단백질의 역학을 모니터링하는 방법을 설명하는 것입니다 애기 장대. 

비디오: Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

미생물에 접속 조직 및 역학 시각화, 총 내부 반사의 형광 현미경을 사용하여

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

연구

생체공학

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

애기 장대 꽃 개발의 공 촛점 이미징 라이브

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

발생학

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

뿌리와 배설물 개발의 장기 공 촛점 이미징을위한 단순한 챔버

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and ...

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 ...

생물학

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens

Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. In general, for observations over a long period, the technique requires transformation of a fluorescent protein; however, for most systems, genetic transformation is either time-consuming or technically unavailable. This manuscript presents a protocol for 3-D time-lapse imaging of cell wall dynamics over a 3 day period using calcofluor dye (which stains cellulose in the plant cell wall), developed in the moss Physcomitrium patens. The calcofluor dye signal from the cell wall is stable and can last for 1 week without obvious decay. Using this method, it has been shown that the detachment of cells in ggb mutants (in which the protein geranylgeranyltransferase-I beta subunit is knocked out) is caused by unregulated cell expansion and cell wall integrity defects. Moreover, the patterns of calcofluor staining change over time; less intensely stained regions correlate with the future cell expansion/branching sites in the wild type. This method can be applied to many other systems that contain cell walls and that can be stained by calcofluor.

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens

This manuscript presents a detailed protocol to image the 3-D cell wall dynamics of living moss tissue, allowing the visualization of the detachment of cell walls in ggb mutants and thickening cell wall patterns in the wild type during development over a long period.

이끼 피스코미트리움 파텐스에서 칼코플루오르를 사용한 세포벽 역학의 3D 타임랩스 이미징

Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. In ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

이미징을위한 간단한 방법 Arabidopsis는 Infiltrative 영상 매체로 Perfluorodecalin를 사용하여 잎

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

의 장기, 고해상도 공 촛점 시간 경과 영상<em&gt; Arabidopsis 떡잎</em발아시&gt; 표피

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

겔의 표면에 성장 식물 뿌리의 가벼운 시트 형광 현미경

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

Intravital 시간 경과 영상에 대 한 애기 를 장착 하는 다양 한 방법을 나뭇잎합니다

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

컨포칼 이미징을 통한 ERL1 동역학의 시공간 조사

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

저자 스포트라이트: 기공 계통 세포에서 막 이동 이벤트를 연구하기 위한 이미지 기반 방법  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

기공 계통 세포에서 막 트래피킹 사건을 연구하기 위한 이미지 기반 방법

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...