Yazarlar, onun tavsiye ve Exeter Üniversitesi bio-Tesis Exeter Üniversitesi&#39;nden Prof. Nicholas Smirnoff teşekkür etmek istiyorum. Finansman İngiltere Biyoteknoloji ve Biyolojik Bilimler Araştırma Konseyi (hibe referans BB/E002682/1) tarafından sağlanmıştır.

Aşağıda verilen protokol Arabidopsis thaliana yaprakları bir infiltratif montaj aracı olarak PFD kullanarak için basit bir yöntem açıklanır, ama biz, bu yöntem görüntüleme hava zengin dokular istenilen uygulamaların çeşitli kullanılıyor olabilir tahmin ediyoruz . 1. PFD montaj yaprak örnekleri <ol><li> Polidimetilsiloksan gaz-geçirgen bir conta (PDMS Carolina Gözlem Jel) ile bir mikroskop lamı hazırlayın. PDMS viskoelastik bir polimer ve deneysel gereksinimlerine uygun bir oda sağlamak için kalıp olabilir. </li><li> Hava ile PFD dengeye. Bu hava ile köpüren veya hava dolu bir şişede PFD küçük hacimli bir sallayarak elde edilebilir. </li><li> Durusu açık bir Petri kabı içine PFD ve bütün bir fide veya 5 dakika PFD eksize yaprak yüzer. Doku (Şekil 2 (a)), yarı saydam, vitrifiye doku anımsatan haline gelmelidir . Yapraklar PFD, bağımlı maruz kalmadan önce daha koyu veya daha açık bir şekilde görünebilir.aydınlatma koşulları ve doku yaşına göre kullanılır. </li><li> PDMS odasına hava dengelenmiş PFD ile doldurun ve dikkatle PDMS odasına kuluçka Petri kabı doku örnekleri aktarmak. Deneysel gereksinimlerine göre lamel ve görüntü ile slayt Seal. </li><li> Not: PFD de bir lamel üzerine inşa açık bir oda veya bir perfüzyon odasında iyi bir performans sergilediğini ve silikon gres ile uyumlu. Erirken PFD teflon bileşenleri ile uyumlu değildir. </li></ol> 2. Temsilcisi Sonuçlar: PFD-inkübe yaprak örneklerinin mikroskobik muayene, hava sahasındaki çoğunluğu Şekil 2 (b) PFD askıya rekombinant GFP ile sular altında hava sahasındaki gösterir sular altında olduğunu gösterir . Yaprak PFD ile inkübasyon üzerine sular altında olduğu açıktır, ama hava o zaman zaman ceplerinde kalır. Su, bu koşullar altında bir yaprak sel değildir. Numune LSCM inkübe ve monte<html> ir, su ya da, PFD (Şekil 3) görüntüleme derinliğinde su ve hava üzerinde bir avantaj verdiğini PFD gösterir . Şekil 3&#39;te verilen örnekte, hava, su ve PFD monte Arabidopsis cytosolically lokalize Venüs, YFP 11 35S promotör altında yapısal olarak ifade edilir bir varyantı ifade yaprakları gösterir . PFD ve su karşılaştırırken görüntüleme derinliği yaklaşık 2 kat artış görebilirsiniz. PFD kullanarak görülen kesin iyileşme sayısal diyafram ile lens ve kullanılan doku tipi değişir, ancak dikkat edilmelidir. PFD tedavi fide sitoplazmik akışı ve kök saç uzama, sağlıklı bitkiler, her gösterge gördük. Buna ek olarak Fv / Fm, 12, bitkilerin fotosentez işleyen bir ölçüsüdür görüntülemede kullanılan zaman çizelgesi üzerinde tolere edilebilir sınırlar içinde kalmasını 2 göstermiştir. 3394fig1.jpg &quot;/&gt; Şekil 1. Bitki yaprak karmaşıklığı ve optik etkileri A. anatomik özellikleri gösteren Şematik gösterim thaliana yaprak optik set-up ile ilgili. Kullanılan kısaltmalar obj. = Objektif lens, imm. = Daldırma sıvısı, cov. = Lamel, mnt. = Mountant, kesme. = Manikür, reklam. ep. = Adaxial epidermis, st. = Stoma gözenek, sp. = Süngerimsi mezofil = hava sahasını, dostum. = Palisad mezofil, fi = vasküler paket, reklam. ep. = Adaxial epidermis. Hücre duvarları siyah çizgilerle gösterilir. <img alt="Şekil 2." src="/files/ftp_upload/3394/3394fig2.jpg" /> Şekil 2. PFD kolayca Arabidopsis yaprakları nüfuz (A) hava, su inkübe veya 5 dakika için PFD ve Leica MZ16F mikroskop (Leica Microsystems (UK) Ltd, Milton Keynes, Birleşik Krallık) ile Leica DCF3000FX dijital kamerayı kullanarak görüntülü bırakır. Vallahi logosu asetat film baskılı ve fro aydınlatmalım aşağıda bir ışık kutusu. Pozlama süresi 89 ms ve tüm görüntüleri toplanan ve aynı şekilde işlendi. Ölçek çubuklar 2 mm temsil eder. (B) PFD in vivo olarak rekombinant yeşil floresan proteininin (GFP) saflaştırılmış hava sahasındaki sınırlandırır taşıma. GFP yanlış renkli yeşil ve kırmızı klorofil mezofil hücreleri sınırlandırıyor oto-floresan, göstermek için kullanılır. (Şekil 2 (b) izni ve tüm teknik detayları Littlejohn ark ile çoğaltılamaz 2010 2). Ölçek bar, 25 m. temsil <img alt="Şekil 3." src="/files/ftp_upload/3394/3394fig3.jpg" /> Şekil 3. PFD yaprakları konfokal görüntüleme artırır LSCM sağlam A. thaliana cytoplasmically lokalize Venüs floresans (yeşil) ve klorofil otofloresans (kırmızı) gösteren görüntüler, hava, su ya da PFD görüntülü bırakır. Dalgaboyu 514 nm ve floresans emisyon Venüs için 604 nm ile 518 nm kaydedildi ve 657 nmKloroplast için 679 nm. Her panel, 5 mikron aralıklarla alınan 11 konfokal görüntüleri Z-yığından alınan tek bir konfokal bölümünde. Bu ek filmleri üretmek için kullanılan 1 mikron aralıklarla elde edilen 59 görüntü tam bir Z-yığını çıkarıldı. Derinlik ölçümleri epidermal yüzeye göre verilmiştir. Aynı şekilde işlendi Temsilcisi görüntüler, gösterilmiştir. Deneysel mikroskop ayarları gibi ayrıntılar, Littlejohn ve ark. 2010 2 bulunanlara aynıdır . Scalebars 20 mikron temsil eder.

<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>

Çıkar çatışması ilan etti.

Bu hava ile doldurulmuş veya optik karmaşık dokuların mikroskopi artırmak için basit ve kullanımı kolay bir tekniktir. Biz tekniği, bazı güçlü avantajlara sahiptir ve biz bu hava zengin dokulara ait biyolojik soruları aydınlatmak için kullanılan olacağını umuyoruz göstermiştir. Örneğin, bitki mezofil ya da akciğerlerde patojen saldırı çalışmaları için doğal bir seçim olacaktır. Ayrıca tekniğin sınırlılıkları farkında. Biz daha iyi refraktif indeksi eşleşen üzerinde çalışıyoruz, uzun zaman ölçeğinde deneyler sırasında mikroskopi diğer modları ve perflorokarbon mountant ikmal kullanın. Ayrıca PFC en önemli avantajlarından biri, yani biyolojik olarak tesirsiz bir flipside olsa da, tanımak. PFC, ayrıca hormonlar, ilaçlar, ve diğer küçük moleküller veya iyonlar gibi ilgi bileşikleri kolayca sunmak için kullanılabilir olduğunu göstermektedir biyolojik moleküllerin kolayca çözülür.

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Perfluorodecalin </td><td> F2 Kimyasallar </td><td> N / A </td><td> Sipariş vermek için telefon. </td></tr><tr><td> Carolina Gözlem Jel </td><td> Biyolojik Bıçaklar </td><td> 13-2700 </td><td></td></tr></tbody></table>

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

Biyolojik örnekler derinliklerine yüksek çözünürlüklü görüntüler elde sorun yaygın olarak 1 kabul edilir . Süngerimsi mezofil hücreleri ve hava sahasındaki arasında ve biyolojik doku ve optik sistemin geri kalanı arasındaki hücresel bileşenleri arasındaki kırılma indisi çoklu geçişler, daha fazla zorluklar ortaya çıkan bitki yaprakları veya omurgalı akciğerler gibi hava dolu doku. Ayrıca, refraktif indeks uyumsuzlukları fluorofor uyarma ve floresan mikroskobu sinyal emisyon zayıflama neden olur. Biz perflorokarbon uygulama perfluorodecalin (PFD), optik, lazer güç zarar artar başvurmadan, derinlik konfokal mikroskobu (LSCM) örnek görüntüleme lazer tarama artırır ve fizyolojik etkilerini minimum 2 infiltratif bir görüntüleme aracı olarak burada açıklayın . Biz onun bir sonucu olarak optik karmaşık Arabidopsis thaliana yaprak dokusu ile PFD kullanımı için protokol tarifyapısı (Şekil 1). PFD 3 bu kullanım için uygun hale öznitelikleri bir numarası vardır. PFD kırılma indisi (1.313), su (1.333) ile karşılaştırılabilir ve sitoplazmada (yaklaşık 1.4) hava (1.000) daha yakındır. Buna ek olarak, PFD floresan olmayan, hazır ve toksik değildir. Sel sağlar stoma penetrasyon 4 - PFD düşük yüzey gerilimi (19 dynes cm-1), su (72 dynes cm-1) daha düşük ve aynı zamanda sınırı (30 dyne cm -1 25) altında potansiyel olarak yıkıcı bir vakum veya surfaktan uygulama olmadan süngerimsi mezofil hava sahasındaki. Son olarak ve en önemlisi, PFD, CO 2 ve O 2, böylece gaz değişimi örnek üzerinde fizyolojik etkileri en aza indirerek, sular altında doku muhafaza sağlar çözme için büyük bir kapasiteye sahiptir . Bu özellikler, kısmi sıvı solunum ve akciğer enflasyona içeren çeşitli uygulamalarda kullanılmaktadırTION 5,6 cerrahi, 7, 8 yapay kan, büyüme oksijenasyon medya 9, ve bitkiler buz 10 kristal oluşumu çalışmaları. Şu anda, canlı konfokal görüntüleme için su ya da sulu tampon doku monte etmek yaygındır. PFD montaj aracı olarak kullanımı, mevcut uygulamada bir iyileşmeyi temsil etmektedir ve görüntüleme için canlı bütün yaprak örnekleri basit bir hazırlık sağlar düşünün.

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

Biz perfluorodecalin infiltratif montaj aracı olarak kullanımı açıklanmaktadır. Bu görüntüleme derinliğini artırmak için basit bir yöntem.<em&gt; Arabidopsis thaliana</em&gt; Yaprak dokusu minimal fizyolojik etkiye sahip.

Görüntüleme için Basit Bir Yöntem Arabidopsis Infiltratif Görüntüleme Orta Perfluorodecalin kullanarak bırakır.

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 Görüntüleme.  The University of Exeter. Biz perfluorodecalin infiltratif montaj aracı olarak kullanımı açıklanmaktadır. Bu görüntüleme derinliğini artırmak için basit bir yöntem. Arabidopsis thaliana Yaprak dokusu minimal fizyolojik etkiye sahip.

Video: 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: Doğru Genotiplendirme için Basit Bir Yöntem

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

Biyoloji

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.

Bir Eski-ovo</emGörüntüleme ve Mikrocerrahi Uygulamaları için uygundur> Tavuk Embriyo Kültürü Sistemi

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.

Hücre Özgül Analizi<em&gt; Arabidopsis</em&gt; Floresans Aktif Hücre Ayırma kullanma Leaves

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.

Için bir Basit Işık Formu Mikroskop Kurma<em&gt; Toto yılında</em&gt; Gürüntülenmesi<em&gt; C. elegans</em&gt; Gelişme

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.

Tesisinden Akışkan apoplastik çıkarımı için infiltrasyonu-santrifüj Tekniği Kullanarak bırakır<em&gt; Phaseolus vulgaris</em&gt; Bir örnek olarak

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.

Üç boyutlu Hidrojeller içinde Görüntüleme 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.

Kantitatif Görüntüleme Teknikleri Kullanılarak Heterosellüler Popülasyonların Ayrımcılaştırılması ve Karakterizasyonu

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.

Yüksek işlem hacmi sterilizasyon Arabidopsis tohum için standart yöntem

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 tamamlayıcı Imaging tahlil Nicotiana benthamiana yaprakları Protein-protein etkileşim Dynamics geçici belirlenmesi için

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.

Düzenlenmiş ekzositozu için muhabir olarak FITC dextran görüntüleme

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