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. J., 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=A+membrane+microdomain-associated+protein,+Arabidopsis+Flot1,+is+involved+in+a+clathrin-independent+endocytic+pathway+and+is+required+for+seedling+development.">A membrane microdomain-associated protein, Arabidopsis Flot1, is involved in a clathrin-independent endocytic pathway and is required for seedling development.</a> Plant Cell. 24 (5), 2105-2122 (2012).</li><li>Staiger, C. J., Sheahan, M. B., Khurana, P., Wang, X., McCurdy, D. W., Blanchoin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+filament+dynamics+are+dominated+by+rapid+growth+and+severing+activity+in+the+Arabidopsis+cortical+array.">Actin filament dynamics are dominated by rapid growth and severing activity in the Arabidopsis cortical array.</a> J. Cell Biol. 184 (2), 269-280 (2009).</li><li>Ueda, H., 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=Myosin-dependent+endoplasmic+reticulum+motility+and+F-actin+organization+in+plant+cells.">Myosin-dependent endoplasmic reticulum motility and F-actin organization in plant cells.</a> Proc. Natl. Acad. Sci. USA. 107 (15), 6894-6899 (2010).</li></ol>

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 거...

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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted microscope</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">IX-73</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TIRF unit</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">IX3-RFAEVAW</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TIRF objective lens&#160;</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">UAPON 100 &#215; OTIRF&#160;</td>
			<td>NA = 1.49</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laser angle control box</td>
			<td style="width:71px;">Chuo Seiki</td>
			<td style="width:119px;">QT-AK</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Optically pumped semiconductor laser</td>
			<td style="width:71px;">Coherent</td>
			<td style="width:119px;">SapphireTM LP USB 488-20 CDRH Laser</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">510&#8211;550 nm band-pass filter</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">U-FBNA</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">EM CCD camera</td>
			<td style="width:71px;">Hamamatsu Photonics</td>
			<td style="width:119px;">ImagEM C9100-13</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">C-mount camera magnification change unit&#160;</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">U-TVCAC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MetaMorph software</td>
			<td style="width:71px;">Molecular Devices</td>
			<td style="width:119px;">MetaMorph version 7.7.11.0</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">TIRF microscopy manual</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">AX7385</td>
			<td style="width:167px;">Instructions: Total Internal Reflection Illumination System (Printed in Japan on August 24, 2012)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Immersion oil</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">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 판...

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 ...

real-time-imaging-plant-cell-surface-dynamics-with-variable-angle

Research

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

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

형광등을 통해 F - 굴지 역학 라이브 셀 이미징은 현미경을 스페클 (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

연구

생물학

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

조립, 로딩 및 분석 초원 심 분리기 샘플 셀 정렬

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

낮은 친화력 세포 수용체 - 리간드 상호 작용의 확장 가능한 감지를위한 욕망 기반의 세포 상호 작용 검사 (AVEXIS)

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

탠덤 고압 냉동 및 투과 전자 현미경을위한 식물 조직의 빠른 동결 교체

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

막 단백질의 실시간 측정 : 수용체 상호 작용은 표면 플라즈몬 공명 (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

대사 프로파일의 실시간 분석<em&gt; 전의 VIVO</em&gt; 마우스 장내 크립트 Organoid 문화

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

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.

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 ...

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 ...

Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, &#34;flash-and-freeze,&#34; that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution.

We developed a novel technique in electron microscopy, &#34;flash-and-freeze,&#34; that enables the visualization of membrane dynamics with ms temporal resolution. This technique combines the optogenetic stimulation of neurons with high-pressure freezing. Here, we demonstrate the procedures and describe the protocols in detail.

플래시 및 동결 : 전자 현미경으로 막 역학을 캡처하는 소설 기법

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

Confocal 현미경 검사 법 밝혀 세포 표면 수용 체 이미지 상관 분광학을 통해 집계

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...