저자는 친절하게 제브라 피쉬 sox10 프로모터 시약을 공유 로버트 켈쉬 감사합니다.

1. 광 변환을위한 배아 컬렉션 <ol><li> 저녁에 5 오후 6시 사이에 단풍 나무 형질 전환 제브라 피쉬 : sox10의 최소한 10 쌍을 설정합니다. </li><li> 다음 날 아침, 분배기를 끌어 정오 경 계란을 수집합니다. 페트리 접시로 전송하고 28.5 ° C의 배양기에 넣어. </li><li> 약 24 시간 게시물 수정에서 죽은 태아를 제거하여 배양 접시를 청소합니다. 라이트 필드 현미경 및 향상된 녹색 형광 단백질 (EGFP) 필터가 부착 된 형광 현미경을 이용하여 배아 (13)의 발달 단계를 확인합니다. 단풍 나무는이 필터를 통해 볼 수 있습니다. </li><li> 배아는 별도의 배양 접시에 60 HPF 전송 다섯 밝은 형광 배아에 도달하고 필요한 경우 dechorionate합니다. </li></ol> 2. 광 변환을위한 배아의 설치 <ol><li> 일반 형광 현미경을 사용하여 배아를 장착합니다. 공 초점 현미경에 배아의 설치는 매우 도전이다. </li><li> 스탠드를 사용하여ARD E3 물 또는 다음과 같은 조리법을 사용하여 신선한 E3를 준비 : 1X E3L을 확인하기 위해 다음을 추가 증류수 Dh를 2 O의 L로 다음을 추가합니다 : 0.292 g 5.0 mM의 NaCl을 0.013 g 0.17의 KCl 0.044 g 0.33mM 염화칼슘 (건조, 96 %, ACS 시약) 0.081 g · 33 mM의 망초 (anhydrated) 선택 사항 : 살균제 및 교반으로 1X E3에 0.05 % 메틸렌 블루의 200 μl를 추가합니다. 매체는 실온에서 1 주간 유지한다. </li><li> 표준 tricaine를 사용하거나 다음 조리법을 사용하여 tricaine을 준비 : 0.2 %의 5 L를 들어 다음과 같은 재료를 혼합 tricaine : 45 ㎖의 트리스로-C1 (1M pH9.5) 5 LH 2 O 10g의 tricaine </li><li> 46.​​25 ml의 E3에 4,125 ml의 0.2 % tricaine를 추가하여, &#39;영화 주스&#39;(50 ㎖ E3, 0.015 %의 tricaine 솔루션)을 준비합니다. </li><li> 50 ㎖ 유리 플라스크에 46.35 mg의 저 융점 (LMP) 아가로 오스와 46.25 ML E3 물을 혼합하여 아가로 오스 준비하고 레인지 작동에 의해 고체 입자를 용해. 그런 다음 추가아가로 오스 및 사용 때까지 50 ° C의 물을 욕조에 아가로 오스와 유리 플라스크를 넣어 3.73 ㎖의 0.2 % tricaine. </li></ol> 3. 준비 및 배아의 장착 <ol><li> 챔버 슬라이드 스텝 1.1.2 및 전송 배아에서 만든 E3/0.015 %의 tricaine 솔루션으로 배아를 마취. </li><li> 종이 조직 및 배아 상 아가 있던 모든 초과 E3를 흡수. </li><li> 위치 배아 완벽 microloader 팁과 지느러미. 전체 배아 아가로 오스 위에 떠 있지만, 몸 전체를 아래로 밀어하지 않도록하십시오. 챔버가 완전히 액체로 가득 때까지 아가 포함 된 배아에 E3/0.015 %의 tricaine 솔루션을 붓는다. </li></ol> 4. 공 초점 현미경에 소프트웨어 설정을 조정 <ol><li> 배아를 초점을 맞추기 위해, 20X 공기 내수성 목적 (구멍 크기 20 개구 825)를 사용합니다. 그런 다음 현미경 L100 라이트 버튼을 누르면 소프트웨어에서 채널을 선택합니다. 아이콘을 볼 오픈 / 수집 controls/A1settings와 카릭공 초점 설정 버튼을 K. 다음 &#39;자동&#39;을 클릭하고 다시 창을 닫기 전에 다음 채널을 선택 : CH1 : DAPI 채널 403.4 NM (350-410 나노 미터 사이의 파장이 사용될 수있다) 채널 2 : 488.0 nm의 3 장 : 561.9 nm의 </li></ol> 5. 광 변환 <ol><li> 채널 1과 3의 전원을 끄고 제거 연동 버튼 및 스캔을 클릭하기 전에 채널 2를 켭니다. </li><li> 광 변환에 대한 관심의 세포에 집중하는 것입니다 1frame/sec 고전압 (HV) 200로 시작하는 다음 프레임 / 초에 예정하고 그에 따라 약 1 / 32 프레임 / 초와 HV에 도달 할 때까지 HV를 낮추는 방법으로 수행하는 것이 가장 100 ~ 120, 배경 잡음에 따라. </li><li> 배아에 초점이 맞으면, 화면의 오른쪽 가장자리에 보이는 녹색 테두리를 잡고는 광 변환하고 마우스 오른쪽 버튼을 클릭에 대한 관심의 영역을 맞도록 더 작게 만든다. 광 변환은 되돌릴 수 있기 때문에 작은 좋습니다. </li><li> (200)에 1 HV에 프레임 / 초를 켜고 충돌스캔 할 수 있습니다. 광 변환의 영역에서 확대 된 이미지는 이제 볼 수 있습니다. </li><li> 채널 1 (403.4 nm의)에 돌려 Photoconvert 세포. GFP 채널을 통해 본 녹색 단풍 나무가 거의 없을 때까지이 지역의 크기에 따라, 어디 5-60초 photoconverting 레이저에 노출. </li><li> 녹색 단풍 나무의 신호가 거의없는 경우, 채널 1을 끄십시오. 채널 2와 3을 켭니다. 그런 다음 A1 스캔 영역 필드에 창에 마우스 오른쪽 버튼을 클릭하고 원하는 세포 photoconverted했다 있는지 확인하는 &#39;원래 크기로 돌아 가기&#39;를 누르십시오. </li></ol> 6. Z-스택 만들기 <ol><li> 메뉴 표시 줄의 아이콘 &#39;캡처 Z-시리즈&#39;를 찾아서 시작하기 전에 Z-스택의 상한과 하한을 설정합니다. LUT를 설정합니다. </li></ol> 7. 소프트웨어 설정 시간 경과 <ol><li> A1Simple GUI 상자로 이동 한 다음 설정을 선택해서 1 / 32 프레임 / 초 크기 1024 보통 4 또는 8X 한 루프에서 촬영되어야하는 Z-스택의 수에 따라 </li><li> :로 이동보기 / 인수 컨트롤 / ND 순서 수집 및 설정 기간 (8 - 30 분), 연속 클릭하고 Z-스택을 클릭합니다. 테스트 이전의 경계를 설정하고 영화를 시작합니다. </li><li> 하루 동안 E3/0.015 %의 tricaine 솔루션 챔버 슬라이드를 계속 추가. 밤에는 완전히 실을 입력합니다. 그 다음날 아침까지 지속됩니다. </li><li> 다음 날 아침, E3/0.015 %의 tricaine 솔루션을 보충하고 태아가 아직 살아 있는지 확인. 형광의 손실이 죽음을 나타냅니다. 하루 동안 보충 및 검사를 계속합니다. </li><li> 구조가 형성 완료, 또는 태아가 사망 한 때, 영화를 완성합니다. </li></ol> 8. 포스트 프로덕션 처리 <ol><li> 도구 모음에 &#39;쇼 최대 강도 투사&#39;를 클릭 한 다음 이미지를 마우스 오른쪽 버튼으로 클릭하고 새 문서를 만듭니다. 동영상은 현재 최고의 품질을 볼 수 있습니다. 필요없는 프레임을 삭제하고의 LUT를 조정에 대한 이동합니다. 마지막으로. AVI로 저장합니다. 이 형식은 Windows 용 잘 될 것입니다. 맥 소프트웨어 다운로드 T에 대한오. MOV 또는. wmv로 변환합니다. </li></ol>

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저자는 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

다음은 제브라 피쉬 모델에서 두개 안면 개발의 시각화를위한 새로운 방법이 표시됩니다. sox10 : 단풍 나무 형질 전환 제브라 피쉬 라인이 성공적으로 자세히 CNCC의 이주 패턴을 설명하는 데 사용 된이 모델 생물 5로 사용됩니다. 이전 연구는 세포를 대상으로 같은 눈으로 총 랜드 마크를 사용하고 단풍 나무 mRNA의 주입, 광 변환 분석 또는 광 변환 10, 11, 14, 15 ?...

구강 안면 쪼개진 조각이 1/700-1로, 가장 널리 두개 안면 기형을 나타내고, 000의 배송은 1에 영향을 미쳤다. 초기 배아 두개 안면 개발의 중단은 갈라진 입술과 구개 (CL / P)의 형성으로 이어질 수 있습니다. syndromic 분열에 대한 원인은 크게 표시되어 있지만, 구강 안면 clefting의 nonsyndromic 형태의 유전과 후생 유전 학적 기초는 여전히 2-4를 발견해야합니다. 이러한 기형의 병인 및 발병 기전을 이해하기 위해, 셀룰러 기초 두개 안면 구조의 발달을 규명 할 필요가있다. 모든 척추 동물 종에서 두개골 신경 능선 세포 (CNCC)는 구강 안면 구조의 형성에 기여 인두 아치를 채우는 등의 신경 튜브에서 마이그레이션. 초기 배아 신경 능선 개발 중단 CL / P 5-7를 포함한 두개 안면 기형의 형성으로 이어질 수 있습니다. 광고에제브라 피쉬와 포유류의 두개 안면 개발 (CNCCs는 상동 지역에 거주) 사이의 구조적 유사성에 dition은 유전자 조절 네트워크는 매우 절약됩니다. 또한 CNCCs은 제브라 피쉬에게 CL / P.의 발달과 유전 적 기초 연구를위한 강력한 유기체를 만드는 amniote 종과 제브라 피쉬 8과 같은 방식으로 개발하는 것으로 나타났습니다 그것은 작은 크기, 신속하고 전 자궁 내 배아 발달, 높은 번식 속도 등 많은 장점을 가지고 있습니다. 또한, 배아는 현미경 9에서 복잡한 발달 사건의 관찰에이 의무 만들기, 광학적으로 투명합니다. 그것은 마이그레이션 및 두개골 신경 능선 세포의 분화 연구를위한 이상적인 동물 모델입니다. 단풍 나무 형질 전환 모델 5 : 이전에 출판 된 작품 8, 10, 11, CNCC의 이주 패턴에 확장하는 sox10를 사용하여 상세하게 설명했다. 단풍 나무는 광 변환 단백질입니다 TU사진을 활성화 한 후 녹색에서 빨간색으로 RNS와는 가능한 정확하게 CNCCs를 추적 할 수 있습니다. 이 변환 동안 펩타이드 골격은 변환이 세포가 자신의 최종 목적지 (12)에 추적 할 수있는 의미, 안정 것을 제안, 절단됩니다. sox10의 전사 제어에 단풍 나무로 표지 유전자 변형 라인은 amniote 구개 및 제브라 피쉬의 사골 판은 Y 모양의 융합 솔기 사이 유사하다 frontonasal 돌기 ​​(FNP)와 그 양측 상악 홍염 (MXP)의 융합에 의해 상동 형성되는 것으로 나타났다 종. 다른 응용 중에서도 sox10 : 단풍 나무 형질 전환 지브라 피쉬 모델은 정상 및 비정상 두개 안면 구조의 형성을 보여주기 위해 다양한 개발 단계에 제브라 피쉬 배아의 영상을 생성하는 데 사용 하였다. 세포의 특정 그룹의 광 변환은 가능한 그들의 발달을 추적 할 수있다. craniofac 개발의 라이브 영상을 만들려면이 방법 접근 방식제브라 피쉬의 원점이라 구조는 쉽게 시각적으로 복잡한 발달 과정을 설명하기 위해 제작, 도입된다. 예를 들어 형질 전환 지브라 피쉬 단풍 나무 :이 프로토콜은 sox10있는 사골 플레이트의 정상적인 발전을 이용하여 동영상을 생성하는 이들의 경험을 공유 할 목적으로한다. 이 프로토콜은 더 제브라 피쉬의 뇌 신경 능선 세포에서 파생 된 구조의 시간 경과 비디오를 만들기에 적용 할 수 있습니다.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>sox10: kaede transgenic zebrafish line</td>
			<td>MGH</td>
			<td></td>
			<td>Available via the Liao lab</td>
		</tr>
		<tr>
			<td>Petri dishes 100x15 mm</td>
			<td>BD Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 35 mmx10 mm</td>
			<td>BD Falcon</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure Low melting point (LMP) Agarose</td>
			<td>Invitrogen</td>
			<td>15517022</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Tek 2 Chamber SlideSystem</td>
			<td>LabTek</td>
			<td>154453</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloaders 200/pk</td>
			<td>Fisher</td>
			<td>E5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R Si Confocal Ti series</td>
			<td>Nikon</td>
			<td>No Catalog number</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements Software AR3.2 64-bit</td>
			<td>Nikon</td>
			<td>No Catalog number</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of craniofacial development in the sox10: kaede transgenic zebrafish line using time-lapse confocal microscopy

척추 palatogenesis는 두개골 신경 능선 (CNC) 세포 융합과 얼굴 홍염의 확장 및 두개 안면 골격 성숙의 마이그레이션을 포함하는 고도의 안무와 복잡한 발달 과정이다. 단풍 나무 형질 전환 제브라 피쉬 라인이 생성되었습니다 제브라 피쉬 미각의 특정 지역 sox10에 두개골 신경 능선의 기여를 공부합니다. Sox10함으로써 기존의 염료 또는 기자 mRNA의 주입보다 더 정확한 과정을 라벨 세포를 만드는 신경 능선에 단풍 나무 기자 단백질의 계보 제한을 제공합니다. 단풍 나무는 사진을 활성화 한 후 녹색에서 적색으로 점등하고 가능한 정확하게 세포를 수행 할 수있게 광 변환 단백질이다. sox10 : 단풍 나무 유전자 변형 라인은 하악 요소에 비해 상악에 상승을 제공하고 양막 류 (amniotes)에 얼굴 홍염의 상 동성을 설명 CNC의 세포 집단을 묘사하는 혈통 분석을 수행하는 데 사용되었다. 이 프로토콜은 공정을 설명단풍 나무 제브라 피쉬 배아 : sox10의 라이브 시간 경과 비디오를 생성하는의. 사골 판의 개발은 실용적인 예제가 될 것입니다. 이 프로토콜은 형질 전환 제브라 피쉬의 모든 단풍 나무 또는 유사한 photoconvertible 기자 단백질의 시간 경과 공 촛점 촬영하기에 적용 할 수 있습니다. 또한, 제브라 피쉬 돌연변이있는 두개 안면 구조의 정상 만 아니라 비정상적인없는 발전을 캡처하는 데 사용될 수있다.

sox10의 : 단풍 나무 형질 전환 라인, 이주 및 사후 이주 CNCCs은 형광 녹색 표시되어 있습니다. 녹색 형광 단풍 나무로 표지 CNCC 세포는 내생 sox10의 mRNA 발현 5 요점을 되풀이. 다른 애플리케이션 사이에서,이 동물 모델은 더 CNCC 종속 두개 안면 구조의 발달을 시각화하는데 사용되었다. 특정 구조 또한 두개 안면 기형의 병리학 적 개발, 특히 갈라진 입술과 구개의 정상적...

Watch this Scientific Journal Video about Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy at JoVE.com

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

실험 데이터의 시각화는 과학계에 결과를 제시하는 핵심 요소가되고있다. 성장하는 배아의 살아있는 시간 경과 기록의 생성은 복잡한 발달 과정의 더 나은 표현과 이해에 기여한다. 이 프로토콜은 제브라 피쉬의 단풍 나무 단백질의 광 변환을 통해 셀 라벨에 대한 단계별 가이드입니다.

sox10의 두개 안면 개발의 시각화 : 단풍 나무 형질 전환 제브라 피쉬의 선은 시간 경과 공 초점 현미경을 사용하여

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

visualization-craniofacial-development-sox10-kaede-transgenic

Research

JoVE Journal

Biology

12.7K 조회수.  Massachusetts General Hospital.  실험 데이터의 시각화는 과학계에 결과를 제시하는 핵심 요소가되고있다. 성장하는 배아의 살아있는 시간 경과 기록의 생성은 복잡한 발달 과정의 더 나은 표현과 이해에 기여한다. 이 프로토콜은 제브라 피쉬의 단풍 나무 단백질의 광 변환을 통해 셀 라벨에 대한 단계별 가이드입니다. 

비디오: Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Monitoring Actin Disassembly with Time-lapse Microscopy

Okay, so what I'd like to do today is to show you how to construct a flow chamber, a fusion chamber for imaging on an upright microscope. What then I then do is I create two spaces using film. So what I do is I take the para film, I cut it into small strips.Next I'm going to, this will basically form the channels for the, the walls, and I dry them with a tilted air. I put the cover slip on top of the para, And here's the finished. Then I heat it on a heat block to melt the param and basically create a seal.So what this is, is the, the cover glass here, and then on top of the cover glass is two layers of phim as you see right here. And then on top of this, again, there's a, it's a 22 by two two mil meter cover slip. This one right here, which I pressed against the param to fall the seal.Okay, so the liquid goes in in one direction here and I can suck it out with a filter paper here in this direction. And I'll show you how to do that in a bit. So what I like to do is I like to put it in a humidified chamber, which is very simple.Take two kind of elevated strips and have a filter of paper underneath. And to keep it noisy as I, I put the cover, cover slip on the flow chamber on. Now the way you introduce a solution to the chamber is that you just put the pipet tip at the entrance of the chamber and you just split.Now what I do is I incubate the chamber with the lid closed for 10 minutes. What I usually do to suck the liquid out from the other side is to use it, get a filter paper and cut it into thin strips like this. And I take one of these thin strips and I simultaneously apply the solution into the entrance while putting the filter paper at the exit to suck the exchange, the solution.So you'll see the liquid flowing through the solution. And then I wait for five minutes for the mid close. Now what I'm ready to do is to actually put that the actin it and the actin, I actually have to polymerize, I polymerize inside the flow chamber and it's just the same procedures before.So because like this, and then now that the action is polymerized, I wash out all the un excess un polymerized action using a large volume of just buffer. So here I have about a hundred microliters and it's a few reaction chamber volumes worth of buffer. Just, you know, when the filter paper becomes saturated, I have to change the direction.Okay?And so I'm ready to do imaging with this. So perfusion chamber with actin attached to it. Now I take the slide, mount it under the microscope, and since I'm imaging with an oil objective, I'm gonna put some immersion oil on the top surface of the cover, grass touch, such so assess solution or protein or anything on acting inside.So what I do is I can actually exchange the solution side while I'm doing the time lapse imaging. As such.

시간 경과 현미경과 분해 굴지 모니터링

연구

생물학

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

라이브 zebrafish의 배아에서 두 광자의 axotomy하고 시간이 경과 공촛점 영상

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

공촛점 현미경으로 Zebrafish 배아 뇌 영상 라이브

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNA Transfection 후의 유사 분열의 시간 저속 영상

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

트렌스 제닉 Zebrafish의 여러 가지 빛깔의 시간 저속 영상 : 타겟의 연결을 세포 절제에 의해 활성화 떠올리 망막 줄기 세포

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

Craniofacial 개발 과정 Ectodermal 상피 조직의 신호 특성을 평가

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

상순 Embryogenesis의 시간 경과 현미경 Caenorhabditis 엘레간스

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

Craniofacial Mesenchyme의 Electroporation

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

시간 경과 비디오 현미경을 이용한 세포의 무작위 마이 그 레이션의 정량 분석

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

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 ...