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 Ca2 + Imaging Utilisation du système FAS <ol><li> Préparer les semis pour l&#39;imagerie de luminescence. Stériliser graines de plantes d&#39;Arabidopsis exprimant Aequorin avec une solution d&#39;eau de Javel à 10% contenant 0,01% de Triton-100. Semez les graines stériles sur une plaque carrée (10 x 10 cm de plat carré de Pétri avec grille,) contenant la pleine force MS (Murashige et Skoog basale sel Mélange), 1% de saccharose et 1,2% d&#39;agar. Placer les plaques verticalement dans une chambre de croissance après stratification à 4 ° C pendant 2 jours (Figure 1A). </li><li> Transférer les plants sur un film. Placer un film adhésif (figure 1B) sur la partie supérieure de 7-10 jours de culture des plantes de semis de la plaque. Poussez doucement le film par la main pour s&#39;assurer que les plants adhèrent au film (figure 1C). Retirer le film doucement pour que les plants restent collés au film (figure 1D et 1E) </li><li> Incuber les plants avec cofacteur. Placer la unesemis dhered sur la plaque carrée (10 x 10 cm) contenant 15 ml de 2 pg / ml h-CTZ (coelentérazine) dans l&#39;eau. Incuber les plants à la température ambiante pendant 4 heures à une nuit (figure 1F). </li><li> Préparez-vous pour l&#39;imagerie de luminescence. Retirer le film de la solution h-CTZ et le couper par le milieu, formant deux pièces. Placez chaque morceau de film avec des plants face vers le haut en deux plaques différentes. Laisser les plaques à l&#39;obscurité pendant 5 min. </li><li> Acquérir les images de luminescence. Dans l&#39;obscurité, placer les deux plaques côte à côte sur la scène du système d&#39;imagerie par luminescence (figure 1G). Acquérir des images immédiatement après l&#39;ajout de 20 ml de solution de stimuli à la fois les plaques. </li><li> Analyser les images de luminescence. Choisir la même plage d&#39;affichage de toutes les images de luminescence. Recadrage de la région d&#39;intérêt (ROI) et de générer les images que les fichiers JPEG (figure 2A). Sinon, exporter les images sous forme de fichiers au format SPE et les importer dans leImageJ logiciel d&#39;analyse d&#39;image. Mesures établies pour le calcul de la valeur de gris moyen de retour sur investissement. Sélectionnez la même taille de la zone ROI et de mesurer les données de gris et moyennes présents sous forme de graphiques à barres (figure 2B). </li></ol> 2 Live Cell Imaging confocale Ca2 + <ol><li> Préparer les semis pour l&#39;imagerie confocale. Stériliser graines de plantes d&#39;Arabidopsis exprimant cas12 avec une solution d&#39;eau de Javel à 10% contenant 0,01% de Triton. Semez les graines stériles sur une plaque contenant résistance plein sels MS, 1% de saccharose et 1,2% d&#39;agar. Placer les plaques verticalement dans une chambre de croissance après stratification à 4 ° C pendant 2 jours. </li><li> Chambres d&#39;imagerie de configuration <ol><li> Assembler une chambre d&#39;imagerie utilisant une lame et lamelle (Chambre A). Placez un morceau de coton imbibé d&#39;eau sur le milieu de la diapositive. Puis transférer un ou deux 5 jours plantules âgées de la plaque sur le dessus de la laine de coton imbibée d&#39;eau. Collez les petits morceaux d&#39;argile à chaque coin d&#39;une lamelle, uned lieu lamelle sur le dessus des plants de créer un espace (chambre) entre la lamelle et toboggan (figure 3A, panneau de gauche). Raccorder une extrémité du tube de polyéthylène (diamètre 0,58 mm) pour une seringue de 1 ml et placer l&#39;autre extrémité du tube immédiatement adjacente à la chambre. Tenir le tube en place avec du ruban adhésif (figure 3A, panneau de droite). </li><li> Assembler une chambre d&#39;imagerie utilisant une chambre en plexiglas (Chambre B). <ol><li> Étendre une mince couche de graisse de silicone autour de deux tubes en polyéthylène (diamètre 0,58 mm) et appuyez sur les tubes revêtus dans les canaux de chaque côté de la chambre de plexiglas (figure 3B gauche panneau). Étendre une mince couche de graisse de silicone sur la surface de la chambre qui contient les rainures du tube et appuyez sur une lamelle dans la graisse pour sceller un côté de l&#39;ouverture de la chambre (découper). </li><li> Transférer un vieux semis de cinq jours à partir de la plaque dans la chambre et placer un morceau de coton imbibé d&#39;eau sur le joint hautf le semis. Etaler de la graisse silicone sur l&#39;autre surface de la chambre, et appuyez sur une autre lamelle sur le dessus pour sceller. Connecter l&#39;extrémité de l&#39;un des tubes à une seringue de 1 ml. Laissez la fin de l&#39;autre tube ouvert (panneau de droite figure 3B). </li></ol></li><li> Préparer une chambre d&#39;imagerie utilisant un couvercle en verre chambré (chambre C). Stériliser le couvercle en verre chambré avec 70% d&#39;éthanol et laisser sur le capot jusqu&#39;à ce que sec. Ajouter 0,6 ml ou 0,2 ml de milieu MS de force complet contenant 1% de saccharose et 0,5% phytagel dans chaque puits d&#39;une chambre à 2 ou bien une chambre à 8 puits (Figure 3C panneau de droite), respectivement. Stériliser les semences et semer les graines directement dans un verre de couverture chambré contenant une fine couche de gel transparent et laisser pousser verticalement pendant 5 jours (figure 3C). </li></ol></li><li> Acquérir des images à l&#39;aide d&#39;un microscope confocal. Pour appliquer des stimuli de stress, tels que le sel, le froid ou le peroxyde, injecter lentement environ 200 pi de 150 mM de NaCl, glace-cold eau ou de 1 mM de H 2 O 2 dans la chambre (chambre A ou B) juste avant l&#39;acquisition d&#39;images, ou ajouter doucement 500 ul ou 100 ul d&#39;une solution de stimulation pour le puits d&#39;une chambre 2 ou 8 puits, respectivement . Les images de capture, immédiatement après application de la solution de stimulus en utilisant un Nikon A1R confocal à balayage laser microscope inversé avec un objectif à immersion dans l&#39;eau 20x (ouverture numérique de 0,75). Recueillir une série temporelle d&#39;images à 4 secondes d&#39;intervalle avec excitation et d&#39;émission des longueurs d&#39;onde de 488 nm et de 500 à 550 nm, respectivement, et à une résolution de pixel de 512 x 512. </li><li> Analyse de l&#39;image L&#39;aide de Nikon éléments, ROI ont été établis autour de chaque cellule (ou région) d&#39;intérêt. L&#39;intensité totale dans chaque ROI a été mesurée au cours du temps en utilisant la boîte de dialogue Mesure du temps (ImageJ pourrait être utilisé à la place). Nombre de mesures d&#39;intensité ont été exportées et traitées dans Datagraph. Pointe l&#39;amplitude Ca a été définie comme l&#39;intensité de pointe moins l&#39;intensité de repos, la durée comme etemps de e entre le début et la fin d&#39;un pic, et la période de l&#39;intervalle de temps entre les pics de pointes adjacentes de deux pointes. -test T a été utilisé pour comparer les moyennes. </li></ol>

<ol>
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The authors have nothing to disclose.

Nous avons démontré un système FAS pour l&#39;enregistrement de la spatio-temporelle Ca2 + réponse des plants d&#39;Arabidopsis. Cette 2+ système d&#39;enregistrement FAS Ca offre une approche simple, sensible et robuste qui pourrait être adapté pour mesurer Ca2 + dynamique déclenchée par divers stimuli, en plus des stimuli de stress abiotiques qui sont présentés ici. Grâce à ce système, nous pouvons comparer facilement tissulaires ou des stimuli spécifiques spatio-tempore...

La cellule de plante répond à l&#39;environnement par l&#39;intermédiaire d&#39;une signalisation qui coordonne les actions cellulaires. Événement de signalisation en réponse à des stimuli de l&#39;environnement d&#39;une cellule début est une augmentation transitoire de Ca2 +. Le modèle, ou la signature d&#39;une augmentation transitoire de la concentration de Ca2 + libre est caractérisé par son amplitude, la fréquence et la durée. Signatures Ca2 + spatio-temporelles distinctes régissent différentes activités cellulaires 1. Stimuli spécifiques, tels que la chaleur, le froid, le sel, la sécheresse, la lumière, ou des hormones végétales, peuvent affiner l&#39;activité spatio-temporelle de la membrane localisée canaux Ca2 + et / ou transporteurs, entraînant spécifiques Ca2 + signatures. Bien que Ca2 + transporteurs ont été bien caractérisés, on sait peu sur l&#39;identité et les fonctions moléculaires des canaux de Ca dans les plantes 1. Écrans génétiques pour les mutants avec altéré Ca2 + réponse au stress stimuli peuvent être une appr efficaceOach pour identifier les composants qui composent Ca de signatures. Ca2 + systèmes de détection récemment fondé plusieurs Aequorin ont été développés qui facilitent écrans génétiques pour Ca2 + composants de signalisation en réponse à l&#39;attaque d&#39;agents pathogènes et stress abiotique 2-4. A été utilisé pour la première Aequorin pour détecter des signaux Ca2 + dans les usines dans les années 1990 5. Depuis lors, l&#39;aequorine a été ciblée pour différents compartiments cellulaires, tels que le cytoplasme 5, le noyau 6, les chloroplastes 7, tonoplastique 8, les mitochondries 9, et le stroma 10, ainsi que de différents types de cellules dans la racine de surveiller cellule spécifique Ca 2 + 11 signaux. Les mesures sur la base de Ca de l&#39;aequorine révèlent le Ca2 + réponse spatiale et temporelle d&#39;une population de cellules pour souligner stimuli. Cependant, dans la plupart des cas, les réponses du Ca de cellules individuelles sont unsynchronized dans le tissu de répondre 4. Par conséquent, l&#39;enregistrement de Aequorin Ca ne rend pas nécessairement le signal Ca2 + dans des cellules individuelles. Au cours des dernières années, la protéine fluorescente génétiquement codé (FP) à base de Ca2 + indicateurs, tels que cameleon jaune (centres jeunesse) 12 et 13 cas12 ont été utilisés pour étudier la signalisation Ca2 + avec une résolution subcellulaire élevé. Centres jeunesse sont le transfert d&#39;énergie de fluorescence par résonance (FRET) à base d&#39;indicateurs de Ca2 +, contenant CFP et YFP variants lié par le liant le Ca2 + et calmoduline de la protéine de peptide liant la calmoduline M13. Calmodulin subit un changement de conformation telle qu&#39;elle se lie au Ca2 +, ce qui apporte CFP et YFP rapprocher, ce qui entraîne un transfert d&#39;énergie accrue (FRET accru). Le niveau de FRET avec le temps, plus ou moins calculé comme le rapport de la YFP à la PCP intensités de signal, traduit la dynamique intracellulaire de Ca2 +. Plusieurs versions YC ont été utilisés dans des plantes. YC3.6 était targeted vers le cytosol 14,15, 16 noyau, les mitochondries 17, et la membrane plasmique 18, et YC4.6 et D4ER ont été ciblés à l&#39;urgence 15,19, et D3cpv a été visé aux peroxysomes 20. Les plantes transgéniques exprimant des centres jeunesse permettent l&#39;imagerie des cellules vivantes de Ca2 + dynamique au sein de différents compartiments cellulaires de différents types de cellules. Cas (probablement Ca lcium soi RNDS) sont des protéines simples circulairement permutées fluorescentes (cpFPs) abritant une calmoduline et le peptide liant la calmoduline M13. Lors de la liaison à Ca2 +, les cas sont soumis à des changements de conformation, conduisant à une augmentation de l&#39;intensité de fluorescence. La corrélation entre la réponse de fluorescence de la CAS et concentration de Ca2 + intracellulaire de Ca2 + permet dynamique à mesurer quantitativement. La variante cas12 a 12 fois fluorescence accrue dans les Ca2 + formes saturés. N. plantes benthiminana transitoire expresser cas12 ou de plantes d&#39;Arabidopsis exprimant de façon stable Case 12 ont été utilisés pour étudier la signalisation Ca2 + dans la défense et stress abiotique 4,21. Asynchrone spatiale et temporelle Ca2 + oscillations dans les cellules répondant à une attaque pathogène, ou au stress de déshydratation ont été révélés avec la société cas12 Ca2 + imagerie. Ici, nous présentons des instructions détaillées pour Aequorin imagerie de luminescence en fonction de stimuli et tissulaire spécifique Ca2 + dynamique en plants d&#39;Arabidopsis, et pour l&#39;imagerie confocale de cytosolique et nucléaire Ca 2 + dynamique dans les cellules d&#39;Arabidopsis profondes qui expriment Cas 12: Luminescence imagerie du SAF pourrait être adapté pour analyser le stress induit par Ca2 + dynamique dans les plantes ou les tissus ne sont pas décrits ici intacts, ou pour dépister les populations de plantes d&#39;Arabidopsis mutagénisées des mutants avec le stress a induit des troubles de Ca2 + signaux. L&#39;installation d&#39;imagerie Ca 2 + des cellules vivantes pourrait être adapté pour analyser Ca2 + dynamique au sein des différents compartiments de subcelluar ou dans différents types de cellules à l&#39;aide de Ca2 + autres indicateurs.

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		<tr height="21">
			<td height="21" style="height:21px;">1 mL syringe&#160;</td>
			<td style="width:172px;">VWR</td>
			<td style="width:175px;">53548-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">silicone grease&#160;</td>
			<td style="width:172px;">Beckman</td>
			<td style="width:175px;">335148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-well chambered cover glass&#160;</td>
			<td>Nalge Nunc international</td>
			<td>155379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">8-well chambered cover glass</td>
			<td>Nalge Nunc international</td>
			<td>155409</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">luminescence imaging system&#160;</td>
			<td>Princeton Instruments</td>
			<td style="width:175px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">inverted confocal laser-scanning microscope&#160;</td>
			<td>Nikon Instruments Inc.</td>
			<td style="width:175px;">N/A</td>
			<td style="width:184px;">Nikon A1R&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Imaging software</td>
			<td>Nikon Instruments Inc.</td>
			<td style="width:175px;">N/A</td>
			<td>Nikon Elements&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">ImageJ</td>
			<td style="width:172px;">NIH</td>
			<td style="width:175px;">N/A</td>
			<td>Public domain, Java-based image processing program</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DataGraph</td>
			<td>Visual Data Tools Inc</td>
			<td style="width:175px;">N/A</td>
			<td>DataGraph 3.1.1 is the newest version</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">coelenterazine</td>
			<td>NanoLight Technolgies</td>
			<td style="width:175px;">#301B NF-BCTZ-FB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">All purpose Bleach</td>
			<td style="width:172px;">Any local store</td>
			<td style="width:175px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Triton X-100</td>
			<td style="width:172px;">Fisher</td>
			<td style="width:175px;">BP151500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">MS salt</td>
			<td style="width:172px;">Phytotechnology Labs</td>
			<td style="width:175px;">M524-50L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">sucrose</td>
			<td style="width:172px;">VWR</td>
			<td style="width:175px;">BDH8029-12KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Agar</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:175px;">A1296-5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Phytagel</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:175px;">P8169-1KG</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring spatial and temporal ca2+ signals in arabidopsis plants

Les indices de développement de l&#39;environnement et induisent des fluctuations de Ca2 + dans les cellules végétales. spatio-temporelles de Ca2 + modèles spécifiques de stimulation sont détectées par cellulaires Ca2 + protéines de liaison qui initient Ca2 + cascades de signalisation. Cependant, nous en savons encore peu de choses sur la façon dont les signaux de stimulus Ca spécifique sont générés. La spécificité d&#39;un signal de Ca2 + peut être attribuée à la régulation sophistiquées de l&#39;activité des canaux et / ou des transporteurs de Ca en réponse à un stimulus donné. Pour identifier ces composants cellulaires et comprendre leurs fonctions, il est essentiel d&#39;utiliser des systèmes qui permettent un enregistrement sensible et robuste des signaux de Ca à la fois le tissu et niveaux cellulaires. Codés génétiquement Ca2 + indicateurs qui ciblent différents compartiments cellulaires ont fourni une plate-forme pour des cellules vivantes imagerie confocale de cellulaires Ca2 + signaux. Nous décrivons ici l&#39;instructions pour l&#39;utilisation de deux systèmes de Ca2 + de détection: aequorine base (FAS adhésives Les plants de film luminescence) Ca2 + et d&#39;imagerie des cellules vivantes cas12 fluorescence confocal basé imagerie de Ca2 +. imagerie de luminescence en utilisant le système FAS fournit une détection simple, robuste et sensible spatiales et temporelles des signaux Ca2 + au niveau des tissus, tandis que la cellule en direct imagerie confocale en utilisant cas12 permet la détection simultanée de cytosolique de Ca2 + et signaux nucléaires à haute résolution.

Mannitol, NaCl et H 2 O 2 ont été utilisés comme substituts pour la déshydratation, le sel et le stress oxydatif stimuli, respectivement. Pour vérifier si les ions de métaux lourds Cu 2 + en synergie avec l&#39;une de ces trois stimuli de stress, on a comparé la réponse de Ca2 + à chacune des stimuli de la présence ou de l&#39;absence de Cu 2 +. Comme le montre la Figure 2, le SAF luminescence imagerie a révélé que les plantules Arabido...

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 la signalisation régule divers processus biologiques dans les plantes. Ici, nous présentons des approches pour le suivi du stress abiotique induit Ca 2 + spatiale et temporelle des signaux dans les cellules et les tissus en utilisant les codés génétiquement Ca2 + indicateurs Aequorin ou cas12 Arabidopsis.

Mesure spatiale et temporelle Ca<sup&gt; 2 +</sup&gt; Signaux de plantes d&#39;Arabidopsis

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 vues.  Purdue University.  Ca la signalisation régule divers processus biologiques dans les plantes. Ici, nous présentons des approches pour le suivi du stress abiotique induit Ca 2 + spatiale et temporelle des signaux dans les cellules et les tissus en utilisant les codés génétiquement Ca2 + indicateurs Aequorin ou cas12 Arabidopsis. 

Vidéo: 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.

Modélisation des membranes biologiques avec des cartes de circuits et la mesure des signaux électriques dans les axones: Exercices de laboratoire étudiant

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

Recherche

Biologie

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: un procédé de croissance Arabidopsis Et plants de tomates dans des plaques à 96 puits pour génotypage à haut débit

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.

Mesure intracellulaire de Ca<sup&gt; 2 +</sup&gt; Les changements dans le sperme humain à l&#39;aide de quatre techniques: FLUOROMETRIE conventionnel, s&#39;est arrêté Fluorimétrie du flux, la cytométrie en flux et l&#39;imagerie cellulaire unique

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.

Analyse automatisée de dynamique Ca<sup&gt; 2 +</sup&gt; Signaux de séquences d&#39;images

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.

Imagerie Ca locale<sup&gt; 2+</sup&gt; Signaux de cellules de mammifères cultivées

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.

Enregistrement en temps réel In Vivo des signaux calciques Arabidopsis pendant la tétée insectes à l’aide d’un biocapteur Fluorescent

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.

Applications de cartographie spatio-temporelle et Techniques d’analyse des particules pour quantifier la signalisation intracellulaire Ca2 + 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

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.

Analyse de l’ARN de microdissection à capture laser pour analyse d’expression génique spécifique aux tissus spatiaux et temporels chez les plantes

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.

Imagerie en temps réel à large champ des signaux de plaies locales et systémiques chez 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

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.

Une méthode d’inoculation simple et flexible pour évaluer avec précision les phénotypes d’oïdium d’Arabidopsis et d’autres plantes

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