Name: measuring spatial and temporal ca2+ signals in arabidopsis plants
Uploaded: 2014-09-02T14:00:00
Description: Watch this Scientific Journal Video about Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants at JoVE.com

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.

1. aequorin Based Ca 2 + Imaging Brug FAS systemet <ol><li> Forbered stiklinger til luminescens billeddannelse. Steriliser frø af Arabidopsis planter udtrykker aequorin med 10% blegemiddel indeholdende 0,01% Triton-100. Sår sterile frø på en kvadratisk plade (10 x 10 cm kvadrat petriskål med gitter) indeholdende fuld styrke MS (Murashige og Skoog Basal Salt Blanding), 1% saccharose og 1,2% agar. Placer pladerne lodret i en vækst kammer efter stratificering ved 4 ° C i 2 dage (figur 1A).</stro...

<ol>
	<li>Kudla, J., Batistic, O., Hashimoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signals:+the+lead+currency+of+plant+information+processing.">Calcium signals: the lead currency of plant information processing.</a> Plant Cell. 22 (3), 541-563 (2010).</li><li>Pan, Z., Zhao, Y., Zheng, Y., Liu, J., Jiang, X., Guo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+for+screening+Arabidopsis+mutants+with+disordered+abiotic+stress-induced+calcium+signal.">A high-throughput method for screening Arabidopsis mutants with disordered abiotic stress-induced calcium signal.</a> J. Genet. Genomics. 39 (5), 225-235 (2012).</li><li>Ranf, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defense-related+calcium+signaling+mutants+uncovered+via+a+quantitative+high-throughput+screen+in+Arabidopsis+thaliana.">Defense-related calcium signaling mutants uncovered via a quantitative high-throughput screen in Arabidopsis thaliana.</a> Mol. Plant. 5 (1), 115-130 (2012).</li><li>Zhu, X., Feng, Y., Liang, G., Liu, N., Zhu, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aequorin-based+luminescence+imaging+reveals+stimulus-+and+tissue-specific+Ca2++dynamics+in+Arabidopsis+plants.">Aequorin-based luminescence imaging reveals stimulus- and tissue-specific Ca2+ dynamics in Arabidopsis plants.</a> Mol. Plant. 6 (2), 444-455 (2013).</li><li>Knight, M. R., Campbell, A. K., Smith, S. M., Trewavas, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+plant+aequorin+reports+the+effects+of+touch+and+cold-shock+and+elicitors+on+cytoplasmic+calcium.">Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium.</a> Nature. 352 (6335), 524-526 (1991).</li><li>Van der Luit, A. H., Olivari, C., Haley, A., Knight, M. R., Trewavas, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+calcium+signalling+pathways+regulate+calmodulin+gene+expression+in+tobacco.">Distinct calcium signalling pathways regulate calmodulin gene expression in tobacco.</a> Plant Physiol. 121 (3), 705-714 (1999).</li><li>Johnson, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+oscillations+of+cytosolic+and+chloroplastic+free+calcium+in+plants.">Circadian oscillations of cytosolic and chloroplastic free calcium in plants.</a> Science. 269 (5232), 489-503 (1995).</li><li>Knight, H., Trewavas, A. J., Knight, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+calcium+signaling+in+Arabidopsis+involves+two+cellular+pools+and+a+change+in+calcium+signature+after+acclimation.">Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation.</a> Plant Cell. 8 (3), 489-503 (1996).</li><li>Logan, D. C., Knight, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+and+cytosolic+calcium+dynamics+are+differentially+regulated+in+plants.">Mitochondrial and cytosolic calcium dynamics are differentially regulated in plants.</a> Plant Physiol. 133 (1), 21-24 (2003).</li><li>Mehlmer, N., Parvin, N., Hurst, C. H., Knight, M. R., Teige, M., Vothknecht, U. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+toolset+of+aequorin+expression+vectors+for+in+planta+studies+of+subcellular+calcium+concentrations+in+Arabidopsis+thaliana.">A toolset of aequorin expression vectors for in planta studies of subcellular calcium concentrations in Arabidopsis thaliana.</a> J. Exp. Bot. 63 (4), 1751-1761 (2012).</li><li>Kiegle, E., Moore, C. A., Haseloff, J., Tester, M. A., Knight, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-type-specific+calcium+responses+to+drought,+salt+and+cold+in+the+Arabidopsis+root.">Cell-type-specific calcium responses to drought, salt and cold in the Arabidopsis root.</a> Plant J. 23 (2), 267-278 (2000).</li><li>Palmer, A. E., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+calcium+signaling+using+genetically+targetable+fluorescent+indicators.">Measuring calcium signaling using genetically targetable fluorescent indicators.</a> Nat. Protoc. 1 (3), 1057-1065 (2006).</li><li>Souslova, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+fluorescent+protein-based+Ca2++sensors+with+increased+dynamic+range.">Single fluorescent protein-based Ca2+ sensors with increased dynamic range.</a> BMC. Biotechnol. 7, 37-46 (2007).</li><li>Tanaka, K., Swanson, S., Gilroy, S., Stacey, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+nucleotides+elicit+cytosolic+free+calcium+oscillations+in+Arabidopsis.">Extracellular nucleotides elicit cytosolic free calcium oscillations in Arabidopsis.</a> Plant Physiol. 154 (2), 705-719 (2010).</li><li>Iwano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine-tuning+of+the+cytoplasmic+Ca2++concentration+is+essential+for+pollen+tube+growth.">Fine-tuning of the cytoplasmic Ca2+ concentration is essential for pollen tube growth.</a> Plant Physiol. 150 (3), 1322-1334 (2009).</li><li>Sieberer, B., Chabaud, M., Timmers, A., Monin, A., Fournier, J., Barker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nuclear-targeted+Cameleon+demonstrates+intranuclear+Ca2++spiking+in+Medicago+truncatula+root+hairs+in+response+to+rhizobial+nodulation+factors.">A nuclear-targeted Cameleon demonstrates intranuclear Ca2+ spiking in Medicago truncatula root hairs in response to rhizobial nodulation factors.</a> Plant Physiol. 151 (3), 1197-1206 (2009).</li><li>Loro, G., Drago, I., Pozzan, T., Lo Schiavo, F., Zottini, M., Costa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+Cameleons+to+different+subcellular+compartments+reveals+a+strict+cytoplasmic/mitochondrial+Ca2++handling+relationship+in+plant+cells.">Targeting of Cameleons to different subcellular compartments reveals a strict cytoplasmic/mitochondrial Ca2+ handling relationship in plant cells.</a> Plant J. 71 (1), 1-13 (2012).</li><li>Krebs, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRET-based+genetically+encoded+sensors+allow+high-resolution+live+cell+imaging+of+Ca2++dynamics.">FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca2+ dynamics.</a> Plant J. 69 (1), 181-192 (2012).</li><li>Bonza, M. C., Loro, G., Behera, S., Wong, A., Kudla, J., Costa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyses+of+Ca2++accumulation+and+dynamics+in+the+endoplasmic+reticulum+of+Arabidopsis+root+cells+using+a+genetically+encoded+Cameleon+sensor.">Analyses of Ca2+ accumulation and dynamics in the endoplasmic reticulum of Arabidopsis root cells using a genetically encoded Cameleon sensor.</a> Plant Physiol. 163 (3), 1230-1241 (2013).</li><li>Costa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2O2+in+plant+peroxisomes:+An+in+vivo+analysis+uncovers+a+Ca2++dependent+scavenging+system.">H2O2 in plant peroxisomes: An in vivo analysis uncovers a Ca2+ dependent scavenging system.</a> Plant J. 62 (5), 760-772 (2010).</li><li>Zhu, X., Caplan, J., Mamillapalli, P., Czymmek, K., Dinesh-Kumar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+endoplasmic+reticulum+calcium+ATPase+in+innate+immunity-mediated+programmed+cell+death.">Function of endoplasmic reticulum calcium ATPase in innate immunity-mediated programmed cell death.</a> EMBO J. 29 (5), 1007-1018 (2010).</li><li>Roszik, J., Lisboa, D., Sz&#246;llosi, J., Vereb, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+intensity-based+ratiometric+FRET+in+image+cytometry--approaches+and+a+software+solution.">Evaluation of intensity-based ratiometric FRET in image cytometry--approaches and a software solution.</a> Cytometry A. 75 (9), 761-767 (2009).</li><li>Kotlikoff, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+Ca2++indicators:+using+genetics+and+molecular+design+to+understand+complex+physiology.">Genetically encoded Ca2+ indicators: using genetics and molecular design to understand complex physiology.</a> J. Physiol. 578 (Pt 1), 55-67 (2007).</li><li>Tian, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neural+activity+in+worms,+flies+and+mice+with+improved+GCaMP+calcium+indicators.">Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.</a> Nat methods. 6 (12), 875-881 (2009).</li><li>Akerboom, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+GCaMP+calcium+indicator+for+neural+activity+imaging.">Optimization of a GCaMP calcium indicator for neural activity imaging.</a> J Neurosci. 32 (40), 13819-13840 (2012).</li></ol>

Vi har vist et FAS system til registrering af den rumlige-temporale Ca 2 + svar fra Arabidopsis frøplanter. Dette FAS Ca2 + optagelse system giver en simpel, følsom og robust fremgangsmåde, der kan tilpasses til måling af Ca2 + dynamik udløst af forskellige stimuli foruden de abiotisk stress stimuli, der er præsenteret her. Ved hjælp af dette system, kan vi nemt sammenligne vævs- eller stimuli-specifikke rumlige-tidsmæssige Ca 2 + dynamik på helplanteniveau. Den høj...

Plantecellen reagerer til miljøet via signalering, som koordinerer cellehandlinger. En tidlig cellesignalerende begivenhed i respons på stimuli af miljøet er en forbigående Ca2 + stigning. Mønstret eller underskrift af en forbigående stigning i fri Ca2 +-koncentration er kendetegnet ved sin amplitude, frekvens og varighed. Tydelige spatio-temporale Ca 2 + signaturer regulerer forskellige cellulære aktiviteter 1. Specifikke stimuli, såsom varme, kulde, salt, tørke, lys eller plante hormoner, kan finjustere spatio-temporale aktivitet af membran-lokaliserede Ca 2 + kanaler og / eller transportvirksomheder, hvilket resulterer i specifikke Ca 2 + underskrifter. Selvom Ca 2 + transportører er blevet godt karakteriseret, er lidt om de molekylære identiteter og funktioner af Ca 2 + kanaler i planter 1. Genetiske skærme for mutanter med ændret Ca2 + reaktion på stress stimuli kan være en effektiv ca.oach til at identificere de komponenter, der udgør Ca 2 + signaturer. For nylig flere aequorin baserede Ca 2 + detection systemer er blevet udviklet, der letter genetiske skærme for Ca 2 + signalering komponenter som reaktion på patogen angreb og abiotisk stress 2-4. Aequorin blev første gang brugt til at detektere Ca 2 +-signaler i planter i begyndelsen af 1990&#39;erne 5. Siden da har aequorin blevet målrettet til forskellige cellulære rum, såsom cytoplasmaet 5 kerne 6, chloroplaster 7, tonoplast 8 mitokondrier 9 og stroma 10, såvel som til forskellige celletyper i roden at overvåge cellespecifik Ca 2 + signaler 11. Aequorin baserede Ca2 + målinger afslører den rumlige og tidsmæssige Ca 2 + svar af en population af celler til at understrege stimuli. Men i de fleste tilfælde Ca2 + reaktioner enkeltceller er unsynchroniZed i reagerer væv 4. Derfor er aequorin Ca2 + optagelse ikke nødvendigvis rapportere Ca 2 +-signalet i de enkelte celler. I de seneste år er genetisk kodet fluorescerende protein (FP) -baseret Ca2 + indikatorer, såsom gul cameleon (ycs) 12 og CASEs12 13 er blevet brugt til at studere Ca 2 +-signalering høj subcellulær opløsning. YCS er fluorescensresonansenergioverførsel (FRET) -baseret Ca 2 + indikatorer, som indeholder den fælles fiskeripolitik og YFP varianter forbundet af Ca 2 + -bindende protein calmodulin og calmodulin-bindende peptid M13. Calmodulin undergår en konformationsændring da det binder sig til Ca 2 +, hvilket bringer den fælles fiskeripolitik og YFP tættere sammen, hvilket resulterer i øget energioverførsel (forstærket FRET). Niveauet FRET over tid, beregnes omtrent som forholdet mellem YFP til FFP signalintensiteter afspejler intracellulær Ca2 + dynamik. Adskillige YC versioner er blevet anvendt i planter. YC3.6 var targeted til cytosolen 14,15, kerne 16, mitokondrier 17, og plasmamembranen 18 og YC4.6 og D4ER var rettet til skadestuen 15,19, og D3cpv var målrettet til de peroxisomer 20. Transgene planter, der udtrykker ycs tillader live-cell imaging af Ca 2 + dynamik inden for forskellige cellulære rum i forskellige celletyper. Sager (formodentlig Ca lcium selv nsor) er enkelt cirkulært permuteret fluorescerende proteiner (cpFPs) indeholdende et calmodulin og calmodulin-bindende peptid M13. Ved binding til Ca2 +, tilfælde undergår konformationsændringer, hvilket fører til en forøgelse af fluorescens intensitet. Korrelationen mellem taskens fluorescens respons og Ca 2 + koncentration tillader intracellulær Ca 2 + dynamik, der skal måles kvantitativt. Den Case12 variant har 12 gange øget fluorescens i Ca2 + Mættede former. N. benthiminana planter transient EXPREssing Case12 eller Arabidopsis planter stabilt udtrykker sag 12 blev brugt til at studere Ca 2 + signalering i forsvar og abiotisk stress 4,21. Asynkron rumlige og tidslige Ca 2 + svingninger i celler reagerer på patogen angreb, eller dehydrering stress er blevet afsløret med Case12 baserede Ca 2 + billeddannelse. Her præsenterer vi detaljerede instruktioner til aequorin baseret luminescens billeddannelse af vævs- og stimuli specifik Ca 2 + dynamik i Arabidopsis kimplanter, og konfokal billeddannelse af cytosol og nuklear Ca 2 + dynamik i Arabidopsis rod celler, der udtrykker sag 12. Luminescence billeddannelse af FAS kan tilpasses til at analysere stress-induceret Ca2 + dynamik i intakte planter eller væv ikke er beskrevet her, eller til at screene mutageniserede Arabidopsis plante populationer mutanter med ændret stress induceret Ca2 +-signaler. Den levende celle Ca 2 + imaging setup kunne tilpasses til at analysere Ca2 + dynamik inden for forskellige subcelluar rum eller i forskellige celletyper ved hjælp af andre Ca 2 + indikatorer.

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			<td>Visual Data Tools Inc</td>
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			<td>DataGraph 3.1.1 is the newest version</td>
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measuring spatial and temporal ca2+ signals in arabidopsis plants

Developmental og miljømæssige signaler inducerer Ca 2 + udsving i planteceller. Stimulus-specifikke rumlige-temporale Ca 2 + mønstre sanses af cellulære Ca 2 +-bindende proteiner, der initierer Ca 2 + signaleringskaskader. Men vi stadig ved meget lidt om, hvordan stimulus specifik Ca2 + signaler genereres. Specificiteten af en Ca 2 +-signal kan tilskrives den sofistikerede regulering af aktiviteterne i Ca 2 + kanaler og / eller transportvirksomheder som reaktion på en given stimulus. At identificere disse cellulære komponenter og forstå deres funktioner, er det afgørende at anvende systemer, der tillader en følsom og robust optagelse af Ca 2 +-signaler på både væv og cellulære niveauer. Genetisk indkodede Ca 2 + indikatorer, der er målrettet til forskellige cellulære rum har givet en platform for levende celle konfokal billeddannelse af cellulære Ca 2 +-signaler. Her beskriver vi instruktions for anvendelsen af to Ca 2 + detektionssystemer: aequorin baserede FAS (film selvklæbende frøplanter) luminescens Ca 2 + billedbehandling og case12 baseret levende celler konfokal fluorescens Ca 2 + billeddannelse. Luminescensdatering billeddannelse ved hjælp af FAS-systemet giver en enkel, robust og følsom påvisning af rumlige og tidslige Ca 2 + signaler på vævet niveau, mens levende celler konfokal billeddannelse ved hjælp Case12 giver samtidig påvisning af cytosol og nukleare Ca 2 +-signaler i en høj opløsning.

Mannitol, NaCl og H 2 O 2 blev anvendt som stedfortrædere for dehydrering, salt og oxidativt stress stimuli hhv. For at kontrollere om tungmetalion Cu2 + synergizes med nogen af disse tre stress stimuli, vi sammenlignede Ca2 + reaktionerne på hver stimuli i nærvær eller fravær af Cu2 +. Som vist i figur 2, afslørede FAS luminescens billeddannelse at Arabidoposis kimplanter reagerede forskelligt på dehydrering, salt og oxidativt stress. For ko...

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Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Måling rumlige og tidslige Ca<sup&gt; 2+</sup&gt; Signaler i Arabidopsis Planter

Ca 2 +-signalering regulerer forskellige biologiske processer i planter. Her præsenterer vi tilgange til overvågning af abiotisk stress induceret rumlige og tidslige Ca 2 +-signaler i Arabidopsis celler og væv ved hjælp af genetisk indkodede Ca 2 + indikatorer aequorin eller Case12.

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

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Biology

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 ...

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

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

Ice-Cap: En metode til Voksende Arabidopsis Og tomatplanter i 96-brønds plader for High-Throughput genotypebestemmes

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

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

Måling Intracellular Ca<sup&gt; 2 +</sup&gt; Ændringer i human Sperm med fire teknikker: Konventionel fluorometri, Stoppet Flow fluorometri, Flowcytometri og 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 ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Automatiseret analyse af dynamiske Ca<sup&gt; 2 +</sup&gt; Signaler i billedsekvenser

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

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Imaging Local Ca<sup&gt; 2+</sup&gt; Signaler i dyrkede pattedyrceller

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

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

Real-time In Vivo optagelse af Arabidopsis calciumsignaler under insekt fodring ved hjælp af en fluorescerende Biosensor

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

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

Anvendelser af Spatio-temporale kortlægning og partikel analyseteknikker til at kvantificere intracellulære Ca2 + signalering 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 ...

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

Laser-Capture Microdissection RNA-sekventering for rumlige og tidsmæssige vævsspecifikke genekspressionsanalyser i planter

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

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

Wide-Field, Real-Time Imaging af lokale og systemiske sårsignaler i 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. ...

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

En nem og fleksibel podningsmetode til nøjagtig vurdering af melduginfektionsfænotyper af Arabidopsis og andre planter

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