우리는 극지 :: 극지-eGFP 건설에 대한 시간 경과 프로토콜과 린 Pillitteri 개발에 도움을 아만다 Rychel 감사드립니다. 우리는 또한 PM-RB 구조로 제공 ABRC에 감사하고 있습니다. 이 프로토콜은 일본 과학 기술 청에서 프레스토 수상의 지원을 통해 개발되었습니다. 극지에 관한 연구는 워싱턴 왕실 연구 기금 (RRF-4098)와 국립 과학 재단 (National Science Foundation) (MCB-0855659)의 대학에 의해 지원되었다. 키 관리 프로토콜은 NSF 대학원 연구원 (DGE-0718124)이며, KUT는 HHMI - GBMF 수사관입니다.

1. 종자 살균 <ol><li> 33 % 가정용 표백제, 0.1 % 트리톤 X-100 : 종자 살균 솔루션을 준비합니다. </li><li> 원하는 형광 기자 구조 (들)과 유전자형 (들) 1.7 ML 튜브 및 살균 솔루션 1 ML을 적용 들고 장소 종자. 15 분에 nutator에 품다. </li><li> 멸균 후드에 뒤에 종자를두고, 튜브에서 살균 솔루션을 제거 할 pipettor를 사용합니다. 한 ML 멸균 물을 씻어. 네 번 반복합니다. </li><li> 이일 이상 4 ° C에서 알을 품다. </li></ol> 2. 상공 회의소 슬라이드 미디어 준비 <ol><li> 용해 될 때까지 Bacto의 200 ML 플라스크의 물에 한천 (순수하지 아가로 오스), 그리고 전자 레인지의 0.5 % 솔루션의 20 ML을 섞는다. 솔루션을 끓고 할 때는 신중을 기해야합니다. </li><li> 약 60 ° C.에 솔루션을 냉각 </li><li> pipettor과 한천 솔루션 1 ML를 수집하고 천천히 챔버 슬라이드 (랩 - 테크 II 쏠 수 # 1.5 독일어 Coverglass 시스템) 내부의 커버 유리에 적용됩니다. 솔루션 which이 너무 뜨겁거나 접착제 또는 균열 유리가 다 녹을 수 있습니다 너무 빨리 적용됩니다. </li><li> 즉시 한천 솔루션의 두 번째 ML를 추가합니다. 한천 미디어는 이제 완전히 슬라이드 챔버의 바닥을 커버해야합니다. 이 최소한의 미디어 수분을 유지하고 가스 확산을 허용 × 4 5 일짜리 저장 영양소를 포함 식물 자엽에서 개발을 지원하기에 충분한 것입니다. </li><li> 챔버가있는 benchtop에 차가운 슬라이드 커버. 술병이 단단히 덮여있다 경우, 솔루션은 저장 및 나중에 사용하기 위해 재가열 될 수 있습니다. </li></ol> 3. 시드 해부 <ol><li> 4 ° C 저장소에서 무균 종자의 튜브를 제거합니다. </li><li> 해부 현미경의 무대에 종이 조직을 놓고 물을 축축하게하다. 부드러운 표면을 생성 할 조직을 조정합니다. 조직은 해부에 대한 씨앗을 안정하는 데 사용됩니다. </li><li> 관에서 조직 Pipet 20-30 종자를 뷰의 해부 범위의 필드에 넣어주의 것. </li><li> 날카로운 집게 (Roboz, # 5 생물 팁을)를 사용신중하게 묘목 내부에서 씨앗 코트를 제거합니다. 바깥 쪽과 안쪽 모두 integuments를 제거해야합니다. </li><li> 때 이미지 떡잎, 그것은 hypocotyl와 잔뿌리를 제거하는 유용합니다. Cotyledons은이 프로토콜에 따라 며칠 동안 개발하기에 충분한 영양소가 포함되어 있습니다. 그대로하면 hypocotyl은보기의 시간 경과 필드의 떡잎을 이동 크게 바르게하고 길어합니다. hypocotyl 무료로 cotyledons 썰 메스를 사용합니다. </li><li> microtube 또는 이와 유사한에, 물에 익숙해 해부 cotyledons을 잡아. </li><li> cotyledons의 원하는 수에 도달 할 때까지 3.4-3.6를 반복합니다. 15-20 성공적으로 dissections는 설치하기 전에 권장합니다. </li></ol> 4. 상공 회의소 슬라이드에 장착 Cotyledons <ol><li> 작은 (18mm 2) 커버 슬립, 한천 매체를 통해 잘라 챔버 슬라이드의 유리베이스에서 전체 층을 엘리베이터를 사용합니다. </li><li> Pipet이 버티고에서 물을 최소로 cotyledons를 해부해제 한천 층 아래에​​있는 챔버 슬라이드, 튜브에. </li><li> 부드럽게 cotyledons에 한천을 낮 춥니 다. 초과 물이있는 경우, cotyledons을 방해하지 않도록주의하는 것 조직으로 거리에 즐겨보십시오. 마운트가 완료되면 표본은 더 이상 쉽게 이동할 없습니다. </li><li> 챔버 슬라이드과 현미경 단계에 이동 슬라이드 커버를 놓습니다. </li></ol> 5. 시간 경과 영상 <ol><li> 이미지가 원하는 형광 (들)에 역 공 촛점 현미경을 설정합니다. 사용 LSM700 매개 변수 : GFP, 440-530 nm의에서 필터 밴드 패스 488 나노 미터 및 수집에 여기에, RFP, 570-610 nm의에서 필터 밴드 패스 555 나노 미터 및 수집에 여기에. </li><li> 손해에 대해 마운트 된 표본을 검사합니다. 그대로의 위치를​​ 프로그램이 제대로는 현미경 소프트웨어에 cotyledons를 장착. 선 (禅) 2009 년 : 20x 목적이있는 떡잎에 초점와 챔버 슬라이드의 센터로 목표를 이동합니다. 취득 모드에서 0.5 줌을 변경하고 프레임 크기 &lt;48x48 픽셀 / em&gt;는. 위치에 따라 위치 확인란 및 스캔 개요 이미지 ... 버튼을 선택한 다음, 30-35로 수평 및 수직 타일 번호를 향상시킬 수 있습니다. 스캔이 완료되면 관찰하기 위해 각 떡잎의 특성 항목 탭 및 장소 십자선 아래에 위치 버튼을 클릭합니다. </li><li> 모든 samples.In 선 (禅) 2009 년 자엽의 표피를 포함한 Z 시리즈를 설정합니다 : Z-스택 확인란을 클릭합니다. 위치 목록 아래의 각 위치를 선택하고 버튼 이동을 누릅니다. 떡잎의 표피의 맨면에 집중하고 Z-스택 아래의 설정 첫 번째 버튼을 클릭합니다. 표피가 유용하게 볼 수있는에서 가장 낮은 위치에 집중하고 세트 마지막 버튼을 클릭합니다. 설정하는 최선의 Z-슬라이스의 두께를 보여줍니다하는 최적의 옆에있는 버튼을 클릭합니다. 이러한 설정은 모든 샘플을 포괄 확인하기 위해 각 떡잎을 확인, Z 매개 변수가 개인에 대해 설정 될 수 없습니다선 (禅) 2009 년 idual 위치. </li><li> 원하는 해상도와 길이의 시계열을 설정합니다. 3 일간 15-30 분 간격 표피 세포 분열의 단백질 역학에 효과적입니다. 이 간격은 필요에 따라 변경 될 수 있습니다. 선 (禅) 2009 년 : 시간 시리즈 확인란을 클릭합니다. 시간 시리즈에서 텍스트 필드에 슬라이더 또는 입력을 사용하여 원하는 이미지 번호를주기를 설정합니다. 30 일 간격을 설정하고 분을 선택 풀다운 메뉴를 사용합니다. </li><li> xyzt 다중 위치 스캔을 시작합니다. 위치의 번호가 스캔 사양에 의해 제한해야합니다, 총 스캔 시간이 원하는 간격보다 클 경우, 시간 지점이 정확하지 않습니다. 이미지 GFP 및 RFP는 59에서 30 분 간격으로 Z-슬라이스가 시스템을 사용할 때 여섯 동시 입장의 최대 가능합니다. 선 (禅) 2009 년 : 변경 프레임 크기 원하는 해상도와 시작 실험을 클릭합니다. </li></ol> 6. 비디오 편집 <ol><li> 공 촛점 데이터 파일에서 최대 강도 등 TIFF와 같은 무손실 형식의 이미지 파일을 각 시간 / 위치 조합과 수출의 Z-투영합니다. 파일 이름은 영화으로 결합하는 소프트웨어에 대한 위치 당 하나의 시리즈 (예를 들어, 극지-GFP_0001_pos1 등 극지-GFP_pos1_0001, 극지-GFP_pos1_0002되지 않음)에 있어야합니다. 선 (禅) 2009 년 : 사용 복사 : 후 별도의 파일로, 최대 강도 프로젝션으로 위치를 분할 할 수있는 처리 탭 아래 하위 집합입니다. (: - 시리즈 수출, 전체 해상도 이미지 창 파일) 마지막으로, TIFF로 내보낼 수 있습니다. </li><li> bUnwarpJ 매크로 9 일 (: 등록 : bUnwarpJ 플러그인)를 실행하여 연속 이미지를 정렬 피지 7,8을 사용합니다. 모노로 등록 모드를 변경합니다. 모든 고급 옵션은 기본 값을 사용할 수 있습니다. 오랜 시간 경과 시퀀스를 들어, 다운로드 및 설치 매크로 &quot;아핀 + 일관된 탄성 2D 이미지 등록&quot;을 자동으로 일련의 이미지에 bUnwarpJ을 적용됩니다, 그리고 개방 Y파일을 사용하여 Google 데이터 : 가져 오기 : IT를 사용하는 이미지 시퀀스. 대걸레의 랜드 마크 추출 및 실행 선택 해제합니다. </li><li> 정렬 이미지의 순서를 열고 원하는 비디오 포맷 (AVI 좋은 선택이 될 것입니다)에 저장 피지 / ImageJ 또는 QuickTime Pro를 사용합니다. </li></ol>

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관심 없음 충돌이 선언 없습니다.

이 시간 경과 공 촛점 기술은 휘황 태그 단백질 표현과 극 및 기타 동적으로 변화하는 단백질의 경우 자신의 기능의 올바른 이해에 매우 중요합니다 Arabidopsis 떡잎의 표피의 개별 셀의 국산화의 길이 방향 연구를 할 수 있습니다. 이전 지속 시간 경과 영상은 Arabidopsis 루트 곰팡이 감염 11 분열 조직의 성장 5,12을 검사하는 데 사용하지만, 자엽의 표피를 추가하면이 기?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> Bacto 한천 </td><td> BD </td><td> 214010 </td><td></td></tr><tr><td> 한 챔버 슬라이드 </td><td> Nunc (열 과학) </td><td> 15536​​0 </td><td> 또는 두 챔버 (155379) </td></tr><tr><td> 레이저 스캐닝 공 촛점 현미경 </td><td> Zeiss </td><td> LSM700 </td><td> 선 (禅) 2009 소프트웨어 </td></tr><tr><td> 20x 목적 렌즈 </td><td> Zeiss </td><td> 420650-9901 </td><td> NA 0.8 계획 - 고차 색지움 </td></tr><tr><td> 현미경을 해부 </td><td> 벤츠 (국가) </td><td> 431TBL </td><td> 아래에서 켜집니다 </td></tr><tr><td> # 5 포셉, 생물학 팁 </td><td> Roboz 외과 악기 </td><td> RS-4978 </td><td> 매우 좋은 팁중요합니다 </td></tr></tbody></table>

long-term, high-resolution confocal time lapse imaging of arabidopsis cotyledon epidermis during germination

발달 순서를 통해 세포 행동의 생체 역학의 이미징은 조직 패턴의 메커니즘을 이해하기위한 강력한 기술이 될 수 있습니다. 동물의 개발 과정, 주요 세포 증식 및 패터닝 이벤트는 매우 빠르게 발생합니다. 예를 들어, Caenorhabditis의 elegans에서 애벌레의 몸 계획에 필요한 모든 세포 분열은 7 mitotic주기 1, 수정을 한 후 6 시간 이내에 완료되며, Drosophila의 embryogenesis의 16 살 이상 mitoses 미만이 24 시간 2에 발생합니다. 반면, 공장 개발하는 동안 세포 분열은 일반적으로 하루 3,4,5의 순서에 속도가 느린 있습니다. 이 독특한 도전과 세포 분열 및 식물 organogenesis 동안 차별화 이벤트의 동적 동작을 문서화 장기 라이브 영상에 대한 필요성을 부과. Arabidopsis 표피는 조사 신호, 세포의 운명, 그리고 식물의 개발을위한 훌륭한 모델 시스템입니다. 떡잎에서이 조직이 구성되어 공기와 균일하게 분포 stomata 사이 사이 방수 포장 세포, 밸브을 열고 가스 교환 및 물 손실을 제어 할 가깝습니다. 이 stomata의 적절한 간격은 기능에 중요합니다, 그들의 개발은 비대칭 부서 및 조직 표피 (그림 1) 생산하는 세포 분화 단계의 순서를 따릅니다. 이 프로토콜은 개발 며칠 동안 세포와 표피의 단백질의 관찰을 할 수 있습니다. 이 시간 프레임은 줄기 세포 부서 및 stomata와 표피 포장 세포 등의 표피 세포의 분화의 정확한 문서를 수 있습니다. 형광 단백질은 세포 분열과 분화 과정 중에 역학을 평가하기 위해 관심 단백질에 융합 할 수 있습니다. 이 기술은 일에 stomatal - 계보 (Lineage) 세포의 확산 단계에서, 우리는 소설 단백질, 극지 6 현지화를 이해 할 수 있습니다이 비대칭 분열 이벤트 및 부서가 발생 직전 세포 피질의 특성 영역에 움직임을 이전 셀에 표시됩니다 전자 Arabidopsis의 떡잎의 표피. 이미지 등록 및 능률적 인 동영상은 쉽게 시간이 지남에 따라 변경으로 동적 단백질 지역화와 세포 유형을 시각화하는 공개 도메인 소프트웨어를 사용하여 생성 할 수 있습니다.

<html> 이 방법으로 수집 된 정보를 시간 점의 집합은 그림 3에 표시됩니다. 셀 멤브레인은 RFP (PM-RB)와 GFP는 30 분 시간 척도에서 (극 :: 극지-GFP)을 기본 발기인에 따라 극 단백질에 6을 융합하고 있습니다으로 분류됩니다, 우리는 단백질 현지화의 변화와 함께 세포 분열을 참조하십시오 을 이전. 비대칭 셀 stomatal 계보 (Lineage) 형태의 부서 줄기 세포과 같은 추가 비대칭 분?...

Watch this Scientific Journal Video about Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination at JoVE.com

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

우리는 stomatal 차별화를 문서화, 개발 며칠 동안 표피의 공 촛점 이미징을위한 식물 cotyledons를 고정하기 위해 챔버 슬라이드 및 미디어를 사용하여 프로토콜을 설명합니다. 형광 태그 단백질은 세포 분열과 세포 형 차별화 중에 가능한 역할의 이해를 증가 표현과 subcellular 지방화에 의해 동적으로 추적 할 수 있습니다.

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 ...

long-term-high-resolution-confocal-time-lapse-imaging-arabidopsis

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Biology

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

13.7K 조회수.  University of Washington. 우리는 stomatal 차별화를 문서화, 개발 며칠 동안 표피의 공 촛점 이미징을위한 식물 cotyledons를 고정하기 위해 챔버 슬라이드 및 미디어를 사용하여 프로토콜을 설명합니다. 형광 태그 단백질은 세포 분열과 세포 형 차별화 중에 가능한 역할의 이해를 증가 표현과 subcellular 지방화에 의해 동적으로 추적 할 수 있습니다.

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

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

와이드 필드 현미경을 사용하여 장기 이미징 포유 동물 세포

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.  ...

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 ...

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

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

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, ...

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories can face is the development and attainment of these methods. Observation of phenotypes in a process of interest is a typical objective of research labs studying gene function and this is often achieved through image capture. A particular process that is amenable to observation using imaging approaches is the corrective growth of a seedling root that has been displaced from alignment with the gravity vector. Imaging platforms used to measure the root gravitropic response can be expensive, relatively low in throughput, and/or labor intensive. These issues have been addressed by developing a high-throughput image capture method using inexpensive, yet high-resolution, flatbed scanners. Using this method, images can be captured every few minutes at 4,800 dpi. The current setup enables collection of 216 individual responses per day. The image data collected is of ample quality for image analysis applications.

This protocol describes a process for rapid collection of images of Arabidopsis seedlings responding to a gravity stimulus using commercially-available flatbed scanners. The method allows for inexpensive, high-volume capture of high-resolution images amenable for downstream analysis algorithms.

애기 장대 루트 Gravitropic 응답의 고해상도 시간 경과 이미지를 수집하는 평판 스캐너를 사용하여

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

고해상도 시간 경과 이미징 및 생활 인간 제대 정맥 내피 세포의 미세 소관 역학의 자동 분석

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

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

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

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

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

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

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

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

유사 분열 동안 다기능 세포 주기 단백질의 역할을 연구 하기 Mitotic 세포 동기화와 고해상도 Confocal 현미경 검사 법

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. 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 ...

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

시간 경과 구조화 조명 현미경 검사법에 대한 미토콘드리아 핵의 특정 라벨링

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...

의 장기, 고해상도 공 촛점 시간 경과 영상

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