We are grateful to B. Stevenson for technical assistance and Dr Marc R. Knight for providing Aequorin transgenic line. This work was funded by the National Institutes of Health Grant R01 GM059138.

FAS Sistemini Kullanma 1. Aequorin Tabanlı Ca 2 Görüntüleme <ol><li> Lüminesans görüntüleme için fide hazırlayın. % 0.01 Triton-100 içeren% 10 çamaşır suyu çözeltisi ile Aequorini Arabidopsis bitkileri tohumları sterilize edin. Tam kuvvet MS (Murashige ve Skoog temel tuz karışımı),% 1 sükroz ve% 1.2 agar içeren bir kare plaka (ızgara ile 10 x 10 cm kare Petri kabı,) hakkında steril tohum ekmek. Dikey olarak 2 gün boyunca 4 ° C&#39;de (Şekil 1A) tabakalaşma sonra bir büyütme odası içinde yer plakalar. </li><li> Bir film üzerine fidan aktarın. Plaka üzerinde büyüyen 7-10 günlük fidelerin üzerine bir yapışkan film (Şekil 1B) yerleştirin. Yavaşça fidan filmin (Şekil 1C) uygun olmasını sağlamak için elle filmi itin. Fideler filme bağlanmış kalacak, böylece yavaşça filmin sıyrılması (Şekil 1D ve 1E) </li><li> Cofactor, fide inkübe edin. A yerleştiriniçeren kare plaka (10 x 10 cm) üzerine dhered fide 2 ug, 15 ml / ml sa-CTZ su (coelenterazine). Gece boyunca (Şekil 1F), 4 saat boyunca oda sıcaklığında inkübe fide. </li><li> Lüminesans görüntüleme için hazırlayın. Iki parça oluşturan, h-CTZ çözeltiden filmi çıkarın ve orta aşağı kesti. Fideler iki farklı plakalarda yüz ile filmin her parça yerleştirin. 5 dakika boyunca karanlıkta plakaları bırakın. </li><li> Lüminesans görüntü kazanır. Karanlıkta, aşağıdaki lüminesans görüntüleme sistemi (Şekil 1G) evresine üzerinde birbirine iki tabak yerleştirin. Hemen eş zamanlı olarak plakalara uyaranlara çözeltisi 20 ml ilave edilmesiyle görüntüler elde. </li><li> Lüminesans görüntüleri analiz. Tüm ışıldama görüntüler için aynı görüntü aralığını seçin. Faiz (ROI) bölgesini kırpın ve JPEG dosyaları (Şekil 2A) olarak görüntüler oluşturmak. Alternatif olarak, SPE olarak formatındaki dosyalar görüntüleri dışa ve içe aktardıktanİmageJ görüntü analizi yazılımı. ROI ortalama gri değerinin hesaplanmasında ayarlama ölçümleri. ROI alanının aynı boyutunu seçin ve çubuk grafikler (Şekil 2B) olarak ortalama gri ve mevcut verileri ölçmek. </li></ol> 2. Canlı Hücre Eşodaklı Ca 2 Görüntüleme <ol><li> Konfokal görüntüleme için fide hazırlayın. % 0.01 Triton ihtiva eden% 10 çamaşır suyu solüsyonu ile case12 Arabidopsis bitkileri tohumları sterilize edin. Tam kuvvet MS tuzları,% 1 sükroz ve% 1.2 agar ihtiva eden bir plaka üzerinde steril tohum ekmek. 2 gün boyunca 4 ° C&#39;de tabakalaşma sonra bir büyütme odası içinde dikey olarak yer plakalar. </li><li> Kurulum Görüntüleme Chambers <ol><li> Bir slayt ve lamel (Chamber A) kullanarak bir görüntüleme odasının birleştirin. Slayt ortasında üzerine su batırılmış pamuk parçası yerleştirin. Daha sonra su ile ıslatılmış pamuk üst plaka üzerine bir ya da iki 5 günlük fidan aktarın. Bir lamel, An her köşesine kil küçük parçalar sopalamel ve slayt arasındaki boşluğu (hazne) (Şekil 3A, sol panel) oluşturmak için fidan üstüne d yer lamel. Bir tane 1 ml&#39;lik şırınga polietilen tüp (0.58 mm çapında) bir ucunu ve bölmenin hemen bitişik borunun diğer ucuna yerleştirin. Bant (Şekil 3A, sağ panel) ile yerine boru tutun. </li><li> Pleksiglas odasını (Karain B) kullanılarak bir görüntüleme odasının birleştirin. <ol><li> Pleksiglas odasının her tarafında kanallar içine kaplanmış tüpler iki polietilen borular (0.58 mm çap) etrafında silikon gres ince bir tabaka yayıldı ve basın (Şekil 3B paneli bıraktı). Tüp oluklar içeren haznenin yüzeyi üzerine silikon ince bir yağ tabakası yayıldı ve hazne ağzı (kesilmiş) bir tarafını kapatmak için yağ içine bir lamel basın. </li><li> Odasına plakasından beş günlük fidelerden aktarın ve üst o su ile ıslatılmış pamuk parçası yerleştirinfide f. Odanın diğer yüzeyine silikon gres yayıldı ve mühür üstünde başka bir lamel basın. Bir tane 1 ml&#39;lik şırınga tüplerin biri ucunu. Başka boru açık (Şekil 3B sağ panel) ucunu bırakın. </li></ol></li><li> Bir odacıklı cam kapak (Chamber C) kullanarak bir görüntüleme odası hazırlayın. % 70 etanol ile odacıklı cam kapak Sterilize ve kuru kadar kaput üzerinde bırakın. 0.6 ml veya, sırasıyla, bir 2-çukurlu odanın havuzlarında veya bir 8-çukurlu odasına (Şekil 3C sağ panel),% 1 sükroz ve% 0.5 Phytagel içeren tam kuvvet MS ortamı, 0.2 ml ekleyin. Sterilize tohumlar ve berrak bir jel ince bir tabakasını ihtiva eden bölmeli bir kapak camı doğrudan tohum ekmek ve bunları 5 gün boyunca (Şekil 3C) için dikey olarak büyümesine izin. </li></ol></li><li> Konfokal mikroskop kullanılarak görüntü kazanır. , Buz col yavaş yavaş 150 mM NaCl yaklaşık 200 ul enjekte, tuz, soğuk ya da peroksit gibi stres uyaranları, uygulamak içinhemen önce sırasıyla görüntü almak ya da hafifçe 2 veya 8-çukurlu bölmenin oyuğuna uyarıcı çözeltisi 500 ul veya 100 ul için bölme (Oda A veya B) içine akar, su ya da 1mM H2O 2 çözeltisi . Yakalama görüntüleri hemen 20X su daldırma lens (Sayısal açıklık 0.75) ile ters Nikon A1R odaklı lazer tarama mikroskobu kullanarak uyaran çözümü uyguladıktan sonra. Sırasıyla 550 nm, ve 512 x 512 piksel çözünürlükte - 488 nm ve 500 uyarma ve emisyon dalga boyları ile 4 sn aralıklarla görüntüleri bir zaman serisi toplayın. </li><li> Görüntü Analizi Nikon Elements kullanarak, ROI&#39;ler ilgi her hücrenin (veya alan) etrafında çizildi. Her ROI içerisindeki toplam yoğunluğu Zaman Ölçümü iletişim kutusunu (İmageJ yerine kullanılabilir) kullanılarak zamanla ölçüldü. Toplam yoğunluğu ölçümleri ihraç ve DataGraph işlenmiştir. Ca 2 + başak genlik inci gibi piklerinde eksi istirahat şiddeti, süresi olarak tanımlandıİki sivri bitişik başak zirveleri arasındaki zaman aralığı olarak başlatılması ve bir başak tamamlanması ve dönem arasında e saati. -test aracı karşılaştırmak için kullanıldı; t. </li></ol>

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The authors have nothing to disclose.

Biz Arabidopsis fidelerinin mekânsal-zamansal Ca 2 + yanıt kayıt için FAS sistemi göstermiştir. Bu FAS Ca 2 + kayıt sistemi, CA burada sunulmuştur abiyotik stres uyarılara ek olarak çeşitli uyaranlar tarafından tetiklenen 2 + dinamiğini ölçmek için adapte edilebilir bir basit, hassas ve güçlü bir yaklaşım sağlamaktadır. Bu sistemi kullanarak, kolayca bütün bitki düzeyinde doku ya da uyaranlara özgü mekânsal-zamansal Ca 2 + dinamiklerini karşılaş...

Bitki hücresi, hücre hareketlerini koordine sinyalizasyonu ile çevreye göre. Çevresel uyarılara tepki olarak sinyal olayını erken hücre, bir geçici Ca 2 + artıştır. Serbest Ca2 + konsantrasyonu içindeki desen ya da geçici bir artış imzası, genliği, sıklığı ve süresi ile karakterize edilir. Farklı uzay-zamansal Ca 2 + imzalar farklı hücresel aktiviteler 1 düzenler. Sıcak, soğuk, tuz, kuraklık, ışık, veya bitki hormonları gibi belirli uyaranlara, ince ayar olabilir membran lokalize Ca 2 + kanalları ve / veya taşıyıcıların uzay-zamansal etkinliği, belirli Ca 2 + imzalar sonuçlanır. Ca 2 + taşıyıcıları, iyi karakterize edilmiş olmasına rağmen, küçük bitkilerde 1 Ca 2 + kanalları moleküler kimlikleri ve işlevleri hakkında bilinmektedir. Uyaranlara stres değiştirilmiş Ca 2 + tepki ile mutantlar için genetik ekranlar etkili olabilir yaklCa 2 + imzaları oluşturan bileşenleri belirlemek için oach. Son zamanlarda birçok Aequorin göre Ca +2 algılama sistemleri patojen saldırısı ve abiyotik stres 2-4 yanıt bileşenleri sinyalizasyon 2 + Ca genetik ekranlar kolaylaştırmak geliştirilmiştir. Aequorin ilk 1990&#39;ların başında 5 CA bitkilerde 2+ sinyalleri algılamak için kullanıldı. O zamandan beri, Aequorin Ca-2 belirli bir hücre izlemek için kök 8 tonoplast sitoplazma 5, 6 çekirdeği, kloroplastlar 7, 9 mitokondri ve stroma 10, yanı sıra farklı hücre tipleri gibi farklı hücresel bölümlerin, hedeflendi + sinyalleri 11. Aequorin tabanlı Ca 2 + ölçümleri uyaranlara stres hücrelerin bir nüfusun mekansal ve zamansal Ca 2 + tepkilerini ortaya çıkarmak. Bununla birlikte, çoğu durumda, tek hücreler Ca 2 + tepkileri vardır unsynchroniyanıt doku 4 zed. Bu nedenle, Aequorin Ca 2 + kayıt mutlaka bireysel hücrelerin Ca 2 + sinyalini bildirmiyor. Son yıllarda, genetik olarak kodlanmış floresan proteini (FP), yüksek çözünürlüğe sahip hücre içi Ca2 + sinyali incelemek için kullanılmıştır, CA böyle sarı cameleon (YÇS) 12 ve 13 olarak CASEs12 2 + göstergelerini tabanlı. YÇS flüoresan rezonans enerji transferi (FRET) CFP ihtiva eden Ca2 + göstergeleri tabanlı ve YFP proteini kalmodulin ve kalmodulin-bağlanma peptidi M13 etmeye dayanır 2 + Ca ile bağlantılı varyantları bulunmaktadır. Ca2 bağlanan kalmodülin olarak bu şekilde daha yüksek enerji transferi (FRET geliştirilmiş) ile sonuçlanan, birbirine CFP ve YFP getiren, yapısal bir değişiklik olur. CFP sinyal yoğunluklarının için YFP oranı olarak yaklaşık olarak hesaplanan zaman, FRET düzeyi, hücre içi Ca2 + dinamiklerini göstermektedir. Çeşitli SK versiyonları tesislerinde kullanılmaktadır. YC3.6 t oldusitozolde 14,15, çekirdeğin 16, mitokondri 17 ve plazma zarı 18 ve YC4.6 ve D4ER için argeted ER 15,19 hedefleyen edildi ve D3cpv proksisomlara 20 hedeflenmiştir. YÇS sentezleyen transgenik bitkiler, farklı hücre tiplerinin farklı hücresel bölmeleri içinde Ca 2 + dinamiklerinin canlı hücre görüntüleme sağlar. DAVALAR (muhtemelen Ca lcium se Nsor) bir kalmodulin ve kalmoduline bağlayıcı peptid M13 barındıran tek dairesel sırası büyük floresan proteinleri (cpFPs) vardır. Ca 2 + bağlanma üzerine, olgu floresan yoğunluğundaki bir artışa yol açan, konformasyonel değişiklikler yapılabilir. Kasanın floresan tepkisi ve Ca +2 konsantrasyonu arasında korelasyon hücre içi Ca + 2 dinamikleri kantitatif olarak ölçüldü sağlar. Case12 varyant 12 kat Ca 2 + -saturated formları floresan artmıştır. N benthiminana bitkilerin geçici olarak İfadeessing Case12 veya stabil bir şekilde eksprese eden Örneği 12 Arabidopsis bitkileri koruma ve abiyotik stres 4,21 Ca 2 sinyalini incelemek için kullanılmıştır. , Veya dehidrasyon strese patojen saldırısına yanıt hücrelerinde asenkron mekansal ve zamansal Ca 2 + salınımlar Case12 göre Ca 2 + görüntüleme ile ortaya konmuştur. Burada, Arabidopsis fide 2+ dinamikleri Ca özel ve sitozolik konfokal görüntüleme ve nükleer Ca mendil ve uyaranlara Aequorin göre lüminesans görüntülemesi için ayrıntılı talimatlar sunmak FAS Vaka 12. Lüminesans görüntüleme ifade Arabidopsis kök hücrelerinde 2+ dinamikleri bozulmamış bitki ya da burada açıklanmayan dokularda stres ile indüklenen Ca2 + dinamiklerini analiz etmek için ya da 2 + sinyaller Ca etkisiyle değişmiş stres mutantlar için mutajenize bitki popülasyonlarını Arabidopsis taranması için adapte edilebilir. Canlı hücre Ca 2 + görüntüleme kurulum Ca analiz adapte olabilirCa 2 + Diğer göstergeler kullanılarak farklı bölmeler içinde subcelluar veya farklı hücre tiplerinde 2 + dinamiği.

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measuring spatial and temporal ca2+ signals in arabidopsis plants

Gelişimsel ve çevresel ipuçları ca bitki hücrelerinde 2+ dalgalanmaları. Stimulus özgü mekânsal-zamansal Ca 2 + desenler Ca +2 sinyal kaskadlarını başlatmak hücresel Ca +2 bağlayıcı proteinler tarafından algılanır. Ancak, biz yine de belirli Ca 2 + sinyalleri üretilir uyaran nasıl hakkında biraz biliyorum. Ca 2 + sinyalinin özgünlüğü belirli bir uyarana cevap Ca 2 + kanalları ve / veya taşıyıcıların faaliyetlerinin sofistike regülasyonuna bağlı olabilir. Bu hücresel bileşenlerini belirlemek ve onların işlevlerini anlamak için, doku ve hücresel düzeydeki Ca 2 + sinyalleri hassas ve sağlam kayıt izin sistemleri kullanmak çok önemlidir. Farklı hücre bölmeleri hedefleyen genetik olarak kodlanmış Ca 2 + göstergeleri hücresel Ca 2 + sinyalleri canlı hücre konfokal görüntüleme için bir platform sağladı. Burada talimat tarifİki Ca 2 + algılama sistemlerinin kullanımı için s: FAS (film yapıştırıcı fidanları) ışıldama Ca 2 + görüntüleme ve case12 tabanlı canlı hücre konfokal floresan Ca 2 + görüntüleme bazlı Aequorin. Case12 kullanarak canlı hücre konfokal görüntüleme yüksek çözünürlükte sitozolik ve nükleer Ca 2 + sinyalleri aynı anda taranmasını sağlayan ise FAS sistemini kullanarak Lüminesans görüntüleme, doku seviyesinde mekansal ve zamansal Ca 2 + sinyalleri basit, sağlam ve duyarlı bir algılama sağlar.

Mannitol, NaCI ve H2O 2, sırasıyla, kurutma, tuz ve oksidatif stres uyarıcılar temsil etmek üzere kullanılmıştır. Ağır metal iyonu Cu2 +, bu üç stres uyaranları herhangi biri ile sinerji içinde kontrol etmek için, Cu2 + varlığında ya da yokluğunda her bir uyarana Ca 2 + tepkiyi. Şekil 2&#39;de gösterildiği gibi, FASB lüminesans görüntüleme Arabidopsis fide dehidrasyon, tuz ve oksidatif stres farklı tepki ortaya koymuştur. 1 mM <sub...

Watch this Scientific Journal Video about Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants at JoVE.com

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca 2 + sinyalizasyon tesislerinde çeşitli biyolojik süreçleri düzenler. Burada mekansal ve zamansal Ca kaynaklı abiyotik stres izlemek için yaklaşımlar sunmak Arabidopsis hücre ve genetik olarak kodlanmış Ca 2 + göstergeleri Aequorin veya Case12 kullanarak dokularda 2+ sinyaller.

Ölçme Mekansal ve zamansal Ca<sup&gt; 2+</supArabidopsis Bitkilerde&gt; Sinyalleri

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

measuring-spatial-and-temporal-ca2-signals-in-arabidopsis-plants

Research

JoVE Journal

Biology

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

12.0K Görüntüleme.  Purdue University.  Ca 2 + sinyalizasyon tesislerinde çeşitli biyolojik süreçleri düzenler. Burada mekansal ve zamansal Ca kaynaklı abiyotik stres izlemek için yaklaşımlar sunmak Arabidopsis hücre ve genetik olarak kodlanmış Ca 2 + göstergeleri Aequorin veya Case12 kullanarak dokularda 2+ sinyaller. 

Video: Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

Devre Biyolojik Membranlar Modelleme ve Aksonlar Elektrik Sinyalleri Ölçme: Öğrenci Laboratuvar Egzersizler

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

Biyoloji

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

Ice-Cap: Büyüyen Bir Yöntem Arabidopsis</emYüksek Verim Genotiplendirme için 96-iyi Tabaklar ve Domates Tesisleri>

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

Hücre içi Ca Ölçme<sup&gt; 2 +</supDört Teknikleri ile İnsan rm&gt; Değişiklikler: Konvansiyonel Florometre, Akış Florometre, Sitometrisi ve Tek Hücre Görüntüleme Durduruldu

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Dinamik Ca Otomatik Analizi<sup&gt; 2 +</supGörüntü Dizilerinde&gt; Sinyaller

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Görüntüleme Yerel Ca<sup&gt; 2+</sup&gt; Kültürlenmiş memeli hücrelerinde sinyaller

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

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.

Floresan Biyoalgılayıcı kullanarak böcek besleme sırasında gerçek zamanlı Vivo kayıt Arabidopsis kalsiyum sinyallerin

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

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

Kendisinin geçici eşleme ve parçacık analiz teknikleri intrasellüler Ca sinyal2 + in Situ ölçmek için uygulamaları

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

Bitkilerde Mekansal ve Zamansal Dokuya Özgü Gen Ekspresyonu Analizi için Lazer Yakalama Mikrodiszesi RNA-Dizileme

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. Upon wounding of a leaf, an increase in cytosolic calcium ion concentration (Ca2+ signal) occurs at the wound site. This signal is rapidly transmitted to undamaged leaves, where defense responses are activated. Our recent research revealed that glutamate leaking from the wounded cells of the leaf into the apoplast around them serves as a wound signal. This glutamate activates glutamate receptor-like Ca2+ permeable channels, which then leads to long-distance Ca2+ signal propagation throughout the plant. The spatial and temporal characteristics of these events can be captured with real-time imaging of living plants expressing genetically encoded fluorescent biosensors. Here we introduce a plant-wide, real-time imaging method to monitor the dynamics of both the Ca2+ signals and changes in apoplastic glutamate that occur in response to wounding. This approach uses a wide-field fluorescence microscope and transgenic Arabidopsis plants expressing Green Fluorescent Protein (GFP)-based Ca2+ and glutamate biosensors. In addition, we present methodology to easily elicit wound-induced, glutamate-triggered rapid and long-distance Ca2+ signal propagation. This protocol can also be applied to studies on other plant stresses to help investigate how plant systemic signaling might be involved in their signaling and response networks.

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

Arabidopsis'te Lokal ve Sistemik Yara Sinyallerinin Geniş Alan, Gerçek Zamanlı Görüntülenmesi

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. ...

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

Arabidopsis ve diğer bitkilerin külleme enfeksiyonu fenotiplerini doğru bir şekilde değerlendirmek için kolay ve esnek bir aşılama yöntemi

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...