저자는 그의 조언과 Exeter에의 Bioimaging 시설의 대학은 Exeter의 대학의 교수 니콜라스 노프 감사하고 싶습니다. 기금은 영국의 생명 공학 및 생물 과학 연구 협의회 (부여 BB/E002682/1 참조)에 의해 제공되었다.

아래 Arabidopsis thaliana 프로토콜은 단풍의 infiltrative 장착 매체로 PFD 사용하기위한 간단한 방법을 설명하지만이 방법은 공기 이미징이 풍부한 조직이 원하는됩니다 다양한 어플 리케이션에 사용할 수 것으로 예상. 1. PFD의 마운트 잎 샘플 <ol><li> polydimethylsiloxane의 가스 투과 가스켓 (PDMS, 캐롤라이나 전망대 젤)과 현미경 슬라이드를 준비합니다. PDMS는 점탄성 폴리머이며 실험적인 요구 사항에 맞는 챔버를 제공하기 위해 성형 수 있습니다. </li><li> 공기와 PFD 평형. 이것은 공기와 버블링 또는 공기 주입 병에 PFD의 작은 볼륨을 흔들어에 의해 달성있을 수 있습니다. </li><li> 가만히 따르다가 오픈 페트리 접시에 PFD 및 전체 묘목 또는 5 분 PFD에 excised 잎을 떠. 조직 (그림 2 (A)), 반투명 vitrified 조직의 연상이 될 것입니다. 나뭇잎이 PFD, depen에 노출보다 어두운 또는 밝은 나타날 수 있습니다조명 조건과 조직의 나이에 땡 사용됩니다. </li><li> 공기 equilibrated PFD와 PDMS 챔버를 입력하고 조심스럽게 PDMS 챔버에 부화 페트리 접시에서 조직 샘플을 전송할 수 있습니다. 실험 요구 사항에 따라 coverslip와 이미지로 슬라이드를 밀봉합니다. </li><li> 참고 : PFD 또한 coverslip에 만들어진 오픈 챔버 또는 재관류 챔버에서 잘 수행하고 실리콘 그리스와 호환됩니다. 그것이 그들을 녹이고으로 PFD는 테플론 구성 요소와 호환되지 않습니다. </li></ol> 2. 대표 결과 : PFD - incubated 잎 샘플을 현미경으로 검사 airspaces의 대부분은. 그림 2 (B)는 PFD에서 일시 중지 재조합 GFP와 홍수 airspaces을 보여줍니다 침수 것을 보여줍니다. 그것은 나뭇잎이 PFD와 부화시 홍수는 것을 분명하지만, 공기의 가끔 주머니가 남아있을 수 있습니다. 물은 이러한 조건 하에서 잎을 홍수하지​​ 않습니다. 샘플 LSCM는 incubated과에 마운트<html> IR, 물 또는 PFD 가능한 영상의 깊이에 물과 공기를 통해 장점 (그림 3) 준 PFD 보여줍니다. 그림 3에 주어진 예제는 공기, 물, PFD 탑재 Arabidopsis가 cytosolically화된 비너스, 35S 프로 모터하에 constitutively 표현 YFP 11 변종을 표현하는 단풍 보여줍니다. PFD과 물을 비교했을 때 우리는 영상의 깊이 약 2 배 증가를 볼 수 있습니다. 이것은 PFD를 사용하는 경우 본 정확한 개선이 렌즈 및 사용 조직의 유형의 수치 조리개에 따라 다릅니다 것을 그러나, 주목하여야한다. 우리는 PFD 치료 모종의 세포질 스트리밍 및 루트 헤어 연신율, 건강한 식물의 각 지표를 볼 수있다. 또한 우리는 FV / FM, 식물 12 광합성 기능의 측정은 이미지에 사용된 timescales 이상 참을 한도 내에서 유지 2 나타났습니다. 3394fig1.jpg &quot;/&gt; 그림 1. 식물 잎의 복잡 및 광학 의미 A.의 해부 학적 특징을 보여주는 Diagrammatical 표현 설정 광학 관련 thaliana의 잎. 사용되는 약어는 OBJ 있습니다. = 객관적 렌즈, imm. = 침지 오일, cov. = coverslip, mnt. = mountant, 컷. = 표피, 광고. EP. = adaxial 표피, 성. = stomatal 기공, SP. = 해면 mesophyll로 = 공기 공간, 친구. = 방어벽의 mesophyll, VB = 혈관 다발, 광고. EP. = adaxial 표피. 세포 벽은 검은 선으로 표시됩니다. <img alt="그림 2." src="/files/ftp_upload/3394/3394fig2.jpg" /> 그림 2. PFD는 쉽게 Arabidopsis 치르곤 (A) 공기, 물에서 incubated 5 분 PFD와 Leica MZ16F 현미경 (Leica 마이크로 시스템즈 (영국) (주), 밀튼 케인즈 -, 영국)와 Leica DCF3000FX 디지털 카메라를 사용하여 몇 군데 잎. 주피터 로고는 아세테이트 필름에 인쇄된 이리저리 조명했다가벼운 상자 아래 해요. 노출 시간은 89 MS했고 모든 이미지는 수집 동일하게 처리되었습니다. 스케일 바는 2mm를 나타냅니다. (B) PFD는 재조합 녹색 형광 단백질 (GFP)을 정화하는 생체내에 airspaces을 delimits 들고. GFP는 거짓 색 녹색이며, 빨간색은 엽록소 mesophyll 세포를 delimits 자동 형광을 표시하는 데 사용됩니다. (그림 2 (B) 허가 및 Littlejohn 외에서 사용 가능한 모든 기술적 세부 재현. 2010 2). 스케일 바는 25 미터를 나타냅니다 <img alt="그림 3." src="/files/ftp_upload/3394/3394fig3.jpg" /> 그림 3. PFD는 나뭇잎에 공촛점 이미징을 향상 A. 그대로의 thaliana의 cytoplasmically화된 비너스 형광 (녹색)과 엽록소의 autofluorescence (적색)을 보여주는 LSCM 이미지는 공기, 물 또는 PFD에서 몇 군데 떠납니다. 여기 파장은 514 nm의 형광 방출했고이 비너스의 604 nm의 518 nm의에서 녹음되었으며에서 657 nm의chloroplasts에 대한 679 NM. 각 패널은 5 μm의 간격으로 취득한 11 공촛점 이미지의 Z - 스택에서 가져온 단일 공촛점 섹션입니다. 이들은 보조 영화를 생산하는 데 사용되었습니다 1 μm의 간격에서 획득한 59 이미지의 전체 Z - 스택에서 추출한되었습니다. 깊이 측정은 표피 표면에 상대적으로 부여됩니다. 동일 처리되었습니다 대표 이미지는, 표시됩니다. 이러한 현미경 설정으로 실험 내용은 Littlejohn 외., 2010 2 찾은 것과 동일합니다. Scalebars 20 μm의를 나타냅니다.

<ol>
	<li>Inoue, S., Pawley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foundations+of+Confocal+Scanned+Imaging+in+Light+Microscopy.">Foundations of Confocal Scanned Imaging in Light Microscopy.</a> Handbook of Biological Confocal Microscopy, 3rd. , 1-16 (2006).</li><li>Littlejohn, G. R., Gouveia, J. D., Edner, C. S. m. i. r. n. o. f. f., N, ., Love, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfluorodecalin+substantially+improves+confocal+depth+resolution+in+air-filled+tissues.">Perfluorodecalin substantially improves confocal depth resolution in air-filled tissues.</a> New. Phytologist. 186, 1018-1025 (2010).</li><li>Sargent, J. W., Seffl, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+perfluorinated+liquids.+Fed.">Properties of perfluorinated liquids. Fed.</a> Fed. Proc. 29, 1699-1703 (1970).</li><li>Sch&#246;nherr, J., Bukovac, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetration+of+stomata+by+liquids.">Penetration of stomata by liquids.</a> Plant Physiol. 49, 813-819 (1972).</li><li>Davies, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+ventilation.">Liquid ventilation.</a> Paediatr. Child. Health. 35, 434-437 (1999).</li><li>Shaffer, T. H., Wolfson, M. R., Greenspan, J. S., Hoffman, R. E., Davis, S. L., Clark, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+ventilation+in+premature+lambs:+uptake,+biodistribution+and+elimination+of+perfluorodecalin+liquid.">Liquid ventilation in premature lambs: uptake, biodistribution and elimination of perfluorodecalin liquid.</a> Reprod. Fertil. Dev. 8, 409-416 (1996).</li><li>Crafoord, S., Larsson, J., Hansson, L. J., Carlsson, J. O., Stenkula, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+perfluorocarbon+liquids+in+vitreoretinal+surgery.">The use of perfluorocarbon liquids in vitreoretinal surgery.</a> Acta. Ophthalmol. Scand. 73, 442-445 (1995).</li><li>Lowe, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+blood:+synthetic+substitutes+from+fluorinated+compounds.">Engineering blood: synthetic substitutes from fluorinated compounds.</a> Tissue Eng. 9, 389-399 (2003).</li><li>Wardrop, J., Edwards, C. M., Lowe, K. C., Davey, M. R., Power, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+responses+of+plant+protoplasts+to+culture+with+oxygenated+perfluorocarbon.">Cellular responses of plant protoplasts to culture with oxygenated perfluorocarbon.</a> Adv. Exp. Med. Biol. 428, 501-505 (1997).</li><li>Sukumaran, N. P., Quamme, H., Weiser, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+fluorocarbons+to+study+freezing+in+plant+tissues.">Use of fluorocarbons to study freezing in plant tissues.</a> Plant Physiol. 50, 632-634 (1972).</li><li>Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nat. Biotechnol. 20, 87-90 (2002).</li><li>Baker, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorophyll+fluorescence:+a+probe+of+photosynthesis+in+vivo.">Chlorophyll fluorescence: a probe of photosynthesis in vivo.</a> Annu. Rev. Plant. Biol. 59, 89-113 (2008).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이것은 공기 주입 또는 광학 복잡한 조직의 현미경을 향상시킬 수있는 간단하고 사용하기 쉬운 기술입니다. 우리는 기술이 몇 가지 강력한 장점을 가지고 있으며, 우리는 그것이 공기 풍부한 조직에 관한 생물 학적 질문을 명료하게하다하는 데 사용될 수 있기를 바랍니다 것으로 나타났습니다. 예를 들어, 그것은 식물 mesophyll이나 폐에 병원체 공격의 연구에 대한 자연 선택이 될 것입니다. 우리는 또한 기술의 한계를 알고 있습니다. 우리는 더 굴절률 정합 작업 있으며, 긴 timescale 실험시 현미경의 다른 모드 및 perfluorocarbon mountant 보충과 함께 사용합니다. 우리는 또한 PFCs의 주요 장점 중 하나는, 즉 생물학적으로 불활성 flipside가되고, 그렇게, 인식하고 있습니다. PFCs는 쉽게 또한 쉽게 이러한 호르몬, 약물, 그리고 다른 작은 분자 또는 이온과 같은 관심의 화합물을 전달하는 데 사용할 수 없다는 것을 의미 생물 학적 분자를 분해하지 않습니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> Perfluorodecalin </td><td> F2 화학 </td><td> N / A </td><td> 주문 전화. </td></tr><tr><td> 캐롤라이나 관측 젤 </td><td> 생물 블레이드 </td><td> 13-2700 </td><td></td></tr></tbody></table>

a simple method for imaging arabidopsis leaves using perfluorodecalin as an infiltrative imaging medium

생물 학적 샘플로 깊이 고해상도 이미지를 획득의 문제는 크게 1 인정받고 있습니다. 이러한 추가적인 문제가 세포와 airspaces 사이의 생물 학적 조직과 광학 시스템의 나머지 부분 사이의 세포 구성 요소 사이의 굴절률에 여러 전환에서 발생하는 식물 잎이나 척추 폐의 해면 mesophyll으로 공기 주입 조직에. 또한, 굴절률의 불일치는 형광단 여기 및 형광 현미경에서 신호 방출의 감쇄로 이어집니다. 우리는 광학 레이저의 파워에 손상을 증가 resorting 않고, 깊이에서 공촛점 현미경 (LSCM) 샘플 이미지를 스캐닝 레이저를 개선하고 최소의 생리적 영향이있다 infiltrative 영상 매체로 여기 perfluorocarbon의 응용 프로그램 perfluorodecalin (PFD)를 설명합니다. 우리는 그것의 결과로 광학 Arabidopsis thaliana 복잡 잎 조직과 함께 PFD의 사용을 위해 프로토콜을 설명구조 (그림 1). PFD이 사용 세에 적합하게 속성을 가지고 있습니다. PFD의 굴절률 (1.313)은 물 (1.333)의와 비교하고 공기 (1.000)보다 cytosol (약 1.4)의에 가까운 것입니다. 또한, PFD가 아닌 형광등, 즉시 가능하며 무독성이다. 그것이 홍수하실 수 있습니다 stomatal 침투 4,에 대한 - PFD의 낮은 표면 장력 (19 dynes cm -1)는 물 (72 dynes cm -1)의보다 낮은 것이며, 또한 한도 (30 다인의 cm -1 25) 다음과 같습니다 잠재적으로 파괴적인 진공이나 계면 활성제의 응용 프로그램없이 해면 mesophyll의 airspaces. 마지막으로 결정적인 PFD는 CO 2와 가스 교환이 때문에 시료에 생리적인 영향을 최소화, 홍수 조직에 유지 수 있습니다 O 2, 분해에 대한 큰 용량을했습니다. 이 속성은 부분 액체 호흡과 폐 infla를 포함 다양한 어플 리케이션에 사용되고있다기 5,6, 수술 7, 인공 혈액 8, 성장의 산소 미디어 9, 식물 10 얼음 결정 형성 연구. 현재는 라이브 공촛점 이미징을위한 물 또는 수성 버퍼에 조직을 마운트하는 것이 일반적이다. 우리는 마운트 매체로 PFD의 사용은 기존의 관행에 대한 개선을 나타내는 및 이미징에 대한 라이브 잎 전체 샘플의 간단한 준비를 허용 생각합니다.

Watch this Scientific Journal Video about A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium at JoVE.com

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

우리는 infiltrative 설치 매체로 perfluorodecalin의 사용을 설명합니다. 이것이 영상의 깊이를 개선하기위한 간단한 방법입니다<em&gt; Arabidopsis thaliana</em최소한의 생리적 영향&gt; 잎 조직.

이미징을위한 간단한 방법 Arabidopsis는 Infiltrative 영상 매체로 Perfluorodecalin를 사용하여 잎

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

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

21.5K 조회수.  The University of Exeter. 우리는 infiltrative 설치 매체로 perfluorodecalin의 사용을 설명합니다. 이것이 영상의 깊이를 개선하기위한 간단한 방법입니다 Arabidopsis thaliana 잎 조직.

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

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing : 정확한 Genotyping을위한 간단한 방법

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

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생물학

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

전 비켜</em이미징 및 미세 애플 리케이션에 적합> 치킨의 배아 문화 시스템

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

의 세포 구체적인 분석<em&gt; Arabidopsis</em&gt; 형광 활성화 셀 정렬을 사용하여 잎

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time.

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

This protocol describes the setup of a light sheet microscope and its implementation for in vivo imaging of C. elegans embryos.

에 대한 간단한 라이트 시트 현미경 설정<em&gt; 토토에서</em&gt; 이미징<em&gt; C. 엘레</em&gt; 개발

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

공장에서 유체 Apoplastic의 추출을위한 침투-원심 분리 기술을 사용하여 잎<em&gt; 위 Phaseolus 심상</em&gt; 예를 들어

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

3 차원 히드로 겔의 이미지를 FRET

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do not allow for accurate interpretation of this interplay. Using a quantitative imaging-based approach, we present a high-content protocol for characterizing the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations to changes in environmental stimuli. We highlight our ability to distinguish between cell types based upon either fluorescence intensity or inherent morphology features depending on the application. This platform allows for a more comprehensive characterization of subpopulation response to perturbation while utilizing shorter time, smaller amounts of reagents, and lower likelihood of error than traditional cell biology assays. However, in some cases, cell populations may be difficult to identify and quantitate based on complex cellular features and will require additional troubleshooting; we highlight some of these circumstances in the protocol. We demonstrate this application using response to drug in a cancer model; however, it can easily be applied more broadly to other physiological processes. This protocol allows one to identify subpopulations within a co-culture system and characterize the particular response of each to external stimuli.

We have developed a novel protocol for studying heterocellular population dynamics in response to perturbations. This manuscript describes an imaging-based platform that produces quantitative datasets for simultaneous characterization of multiple cellular phenotypes of heterocellular populations in a robust manner.

정량적 이미징 기법을 이용한 이종 세포 집단의 차별화 및 특성 규명

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

애기 씨의 높은 처리 살 균에 대 한 표준화 된 방법

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Luciferase 보완성 이미징 분석 결과 Nicotiana benthamiana 잎 뚜렷이 결정 단백질 단백질 상호 작용 역동성에 대 한

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

이미징 FITC-dextran 규제 Exocytosis 위한 기자로 서

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...