J.O.B. and A.M.R. were supported by predoctoral fellowships from the National Science Foundation.

NOTA: per questo protocollo, si sono concentrati sulla analizzando frequenza stromule nell&#39;epidermide di N. benthamiana lascia. Diverse linee transgeniche stabili sono stati generati che può essere utilizzato per questo scopo, tra cui 35S PRO: FNRtp: EGFP 13 e NRIP1: Cerulean 6. Entrambe queste linee mostrano robusta espressione di fluorofori nello stroma dei cloroplasti delle foglie cresciute in un&#39;ampia gamma di condizioni. In alternativa, fluorofori cloroplasti mirati possono essere transitoriamente espressi in N. benthamiana utilizzando Agrobacterium trasformazioni 13. Questo è meno ideale che le linee transgeniche, dal momento che le infiltrazioni tumefaciens inducono alcune risposte di difesa basali in N. benthamiana e le interazioni con Agrobacterium possono alterare la frequenza stromule nella foglia 14, potenzialmente complicando l&#39;interpretazione dei risultati. Infine, di visualizzare formazione stromule in vitro,cloroplasti possono essere estratti da tutte le specie vegetali, utilizzando sia fluorofori geneticamente codificati o un colorante fluorescente, come descritto nella sezione 5 di seguito. 9,15 NOTA:. Metodi dettagliati per la coltivazione delle piante sono stati precedentemente descritti 16 In breve, crescere N. piante benthamiana in 4 vasi &quot;riempite con qualsiasi miscela di suolo professionale che fornisce un buon drenaggio. Rivestimento piantine con una cupola di plastica per i primi 10-14 giorni di tempo per fornire un ambiente umido per la germinazione. aggiungere qualsiasi combinazione di fertilizzanti di serie seguendo le istruzioni del produttore per 14- giorno-vecchi impianti. crescere le piante sotto la luce bianca, con ~ 100 micromol fotoni m -2 sec -1 intensità della luce. piante acquatiche regolarmente. 1. Preparazione campioni di foglie per la visualizzazione NOTA: le dinamiche Stromule sono influenzati da ferendo 8, in modo preparazione dei tessuti deve essere condotta immediatamente prima della visualizzazione stromules mediante microscopia confocale. Idealmente, un campione deve essere visualizzato con 15 min dopo la rimozione dalla pianta. <ol><li> Tagliare una piccola sezione della foglia con una lama di rasoio molto tagliente. Utilizzare sempre la stessa regione della foglia di coerenza, ed essere certi di visualizzare la stessa superficie (adaxial o abaxial) in tutti gli esperimenti. </li><li> Subito dopo il taglio della sezione di foglia, immergere la sezione foglia in una siringa da 5 ml senza ago riempita con acqua, rimuovere l&#39;aria dalla siringa, e applicare un vuoto coprendo l&#39;orifizio siringa con un dito e tirando lo stantuffo. <ol><li> Rilasciare lo stantuffo delicatamente per evitare di danneggiare la sezione di foglia. Ripetere questo due o tre volte, o fino a quando la maggior parte dell&#39;aria è stato rimosso e la foglia sembra essere verde intenso. NOTA: Questo passaggio rimuove l&#39;aria nella foglia che può interferire con accurata visualizzazione dei stromules. </li></ol></li><li> Aggiungere una goccia d&#39;acqua su un vetrino e posizionare la sezione foglia su questo calo. Quindi, aggiungere un altro drop di acqua all&#39;inizio della foglia, e posizionare un vetrino sulla foglia. Se ci sono bolle d&#39;aria, toccare delicatamente il vetrino fino a quando non vengono rimossi. Il vetrino è ora pronto per la microscopia, e dovrebbe essere visualizzato immediatamente. </li></ol> 2. Visualizing Stromules con confocale microscopia a fluorescenza <ol><li> Con luce trasmessa, concentrarsi su un campo di vista vicino al centro della sezione del foglio (lontano dalle cellule danneggiate dalla lama di rasoio). Utilizzare un obiettivo 20X modo che molti cloroplasti possono essere visualizzate contemporaneamente. Salvare un&#39;immagine con luce trasmessa in modo che i tipi di cellule possono essere distinti in seguito, se necessario. </li><li> Commutare la sorgente di illuminazione a un laser con opportuni filtri di eccitazione ed emissione per il fluoroforo utilizzato. Ad esempio, eccitare GFP con un laser 488 nm e raccogliere emissioni tra 500 nm e 525 nm. <ol><li> Utilizzando il software del microscopio, selezionare il canale GFP e fare clic per regolare l&#39;apertura a foro stenopeico al 1 Aiunità ry. Selezionare scansione laser, per avviare la visualizzazione del campione, e quindi fare clic sul canale GFP per regolare l&#39;intensità del laser e il guadagno del rivelatore per ridurre al minimo la potenza del laser, mentre ancora la visualizzazione chiaramente stromules. Una volta che queste impostazioni sono state determinate, tenerli il più coerente possibile in tutti gli esperimenti. </li></ol></li><li> Preparare un esperimento impili z che raccoglierà una serie di immagini attraverso l&#39;epidermide foglia nel campo visivo. <ol><li> Per la z impili, includere tutti cloroplasti epidermiche nel campo di vista per evitare distorsioni (per esempio, se cloroplasti vicino alla superficie del foglio hanno più stromules in condizioni testate). </li><li> Prendere immagini a l&#39;intervallo massimo possibile che includerà tutti stromules ma ridurre al minimo il tempo necessario per una z impili. Ad esempio, se 1 unità Airy è equivalente ad una sezione confocale a fuoco che è 2 micron profondo, riprendere un&#39;immagine ogni 2 micron è probabile ideale. NOTA: Il epidermi foglias è una superficie irregolare, per cui la profondità totale del z impili varierà a seconda del campione; la maggior parte -Pile z richiederanno 10-20 immagini, ma possono includere più. </li><li> Regolare la velocità di scansione e risoluzione dell&#39;immagine come necessario per assicurarsi che la z impili viene raccolto rapidamente (tipicamente entro 5-10 min, se possibile). Salvare la z impili per una successiva analisi. </li></ol></li></ol> Processing 3. Immagine <ol><li> Determinare la frequenza stromule utilizzando qualsiasi software di analisi delle immagini. Per questo protocollo, si consiglia di utilizzare ImageJ, che è a disposizione del pubblico dal National Institutes of Health (http://imagej.nih.gov/ij/), o l&#39;aggiornamento popolare di ImageJ, Fiji (http://fiji.sc / Fiji). </li><li> Unire la z impili in un&#39;unica immagine utilizzando la sporgenza massima intensità nel software. </li><li> Identificare e contare manualmente tutti i cloroplasti nell&#39;immagine. </li><li> Per ogni cloroplasto, determinare visivamente se uno o più stromulessono visti che si estende dal cloroplasto in immagine z unito impili. Per esempi, vedi Figura 1. NOTA: Poiché le funzioni biologiche di stromules non sono noti, ogni, stroma-riempite, prolungamento tubolare sottile dal cloroplasto può essere considerato un &quot;stromule&quot;, indipendentemente dalla lunghezza. Come regola generale, un&#39;estensione stroma-riempita dal cloroplasto è un stromule se è inferiore a circa 1 micron di diametro lungo la maggior parte della sua lunghezza 7. Assicurarsi che una persona analizza tutte le immagini per controllare le differenze soggettive nel determinare se un cloroplasto ha un stromule. Un secondo ricercatore deve verificare in modo indipendente le frequenze stromule per ridurre ulteriormente pregiudizi soggettivi. </li></ol> 4. Disegno sperimentale e campionamento NOTA: Stromule frequenza è molto variabile tra le foglie, ma diversi rapporti suggeriscono che c&#39;è una piccola variazione nella frequenza stromule all&#39;interno di un individuale 9,17 foglia. <ol><li> Calcolare la frequenza foglio stromule da un unico campo di vista di una foglia. Eliminare la foglia, e considerare questo un campione indipendente. Non considerare campi separati di vista da una foglia come campioni indipendenti. NOTA: Non vi è ampio consenso sul modo migliore di misurare le variazioni nell&#39;attività stromule, e fino a quando la funzione di stromules è più chiaramente definito, i ricercatori dovrebbero continuare ad essere creativi nel quantificare la dinamica stromule. Per fare un confronto tra letteratura, tuttavia, norme comuni di segnalazione devono essere utilizzati in aggiunta agli approcci più creativi. Come minimo, segnalare sempre la frequenza di cloroplasti che hanno uno o più stromules. </li><li> Determinare la frequenza dei cloroplasti che hanno stromules in un campo di vista. Utilizzare ImageJ per identificare tutti i cloroplasti in un campo di vista. Quindi, per ogni cloroplasto, determinare se il cloroplasto è formato almeno un stromule. Rapporto frequenza stromule come Percentage di cloroplasti con stromule. NOTA: Alcuni ricercatori trasformano la percentuale, per esempio con la trasformazione arcoseno, prima di riportare le frequenze stromule; Anche se questa è una opzione per l&#39;analisi statistica, l&#39;arcoseno di frequenza di stromule non è un valore intuitivo, e non ha bisogno di essere riportato nel testo principale o figure di uno studio. <ol><li> Come approccio alternativo, contare il numero totale di stromules, e dividerlo per il numero totale di cloroplasti. NOTA: Nella maggior parte dei casi, questo produrrà risultati simili per avvicinarsi 4.2; può sovrastimare la frequenza stromule, tuttavia, se un solo cloroplasto ha formato molti stromules. Nell&#39;esempio mostrato in risultati rappresentativi, ad esempio, diversi cloroplasti hanno due o più stromules, aumentando il numero di stromules per cloroplasto al 40/87 (rispetto a una frequenza stromule di 33/87). </li><li> In alternativa, determinare la lunghezza stromule anziché frequenza stromule. La lunghezza può essere misuratain ImageJ tracciando il stromule con lo strumento linea a mano libera. NOTA: Dal momento che il ruolo biologico di stromules rimane sconosciuta, non è chiaro che la lunghezza stromule colpisce la loro funzione. Relazione significative differenze di lunghezza stromule se osservati, ma inoltre segnalare le frequenze stromule (come descritto in 4.2). NOTA: la frequenza Stromule può essere molto variabile tra le foglie diverse alle stesse condizioni di crescita. Pertanto, anche se la determinazione della frequenza stromule può richiedere molto tempo, gli esperimenti devono essere attentamente progettati per includere campioni di grandi dimensioni. Utilizzando set di dati dal nostro laboratorio, abbiamo determinato che un campione di almeno 16 piante per ogni trattamento è di solito sufficientemente potente per determinare se vi è una differenza statisticamente significativa di almeno una variazione di 1,5 volte in frequenza stromule tra due condizioni (con il campione α = 0.05 e β = 0,80). </li></ol></li><li> L&#39;analisi statistica dei risultati. <ol><li> Per confrontare il meun frequenze stromule di due trattamenti, utilizzare il test t di Welch (noto anche come il test t test o t heteroscedastic supponendo che la varianza disuguale). NOTA: Questo test non è potente come test t di Student convenzionale, ma il test t di Welch è vantaggioso perché non si assume che la varianza nella distribuzione di frequenza stromule è simile tra i trattamenti. NOTA: Dal momento che la frequenza stromule è una &quot;proporzione&quot;, la sua distribuzione campionaria è previsto per essere binomio più che normale, che potrebbero analisi statistiche teoricamente skew se le dimensioni del campione sono molto bassi o se la frequenza stromule è molto basso (vicino allo 0%) o molto alta (vicina al 100%). Alcuni report hanno usato trasformazioni arcoseno prima analisi statistiche, che ha lo scopo di trasformare una distribuzione binomiale di una distribuzione normale, e, quindi, di soddisfare i requisiti standard di test t di Student o ANOVA. In pratica, tuttavia, untrasformazioni rcsine hanno poco o nessun effetto sulla analisi statistica, e pertanto non sono raccomandati. Invece, ripetere gli esperimenti di aumentare la dimensione del campione, che consentirà di aumentare la potenza statistica e ridurre gli errori di interpretazione dei dati. </li><li> Qualunque sia l&#39;approccio statistico utilizzato, assicurarsi di segnalare con attenzione la strategia di campionamento, la dimensione del campione, test statistico, e il valore di p per ogni esperimento. NOTA: Come con altri campi della biologia, un valore di p inferiore a 0,05 può essere considerato &quot;statisticamente significativo&quot;, anche se i ricercatori dovrebbero certamente riportare il valore p preciso ottenuto. Se p è leggermente superiore a 0.05, aumentando la dimensione del campione con due o tre esperimenti possono aiutare a chiarire se la differenza osservata è riproducibile e statisticamente significativa. </li></ol></li></ol> 5. Estrazione cloroplasti Intact di visualizzare Stromule Dynamics NOTA: Diversi metodisono stati utilizzati per isolare cloroplasti da foglie, comprendente un protocollo leggermente diverso in un recente studio sulla formazione stromule in vitro 15. Il protocollo descritto di seguito utilizza un metodo relativamente semplice che non cede campioni cloroplasti biochimicamente puri, ma ha invece isolare una grande quantità di intatte, cloroplasti sani 9,18. <ol><li> Preparare tampone di estrazione a freddo: 50 mm Hepes NaOH, 330 mm sorbitolo, 2 mM EDTA, 1,0 mm MgCl 2 e 1,0 mm MnCl 2. Regolare il pH a 6,9 con NaOH e HCl, e conservare in frigorifero prima dell&#39;uso. NOTA: Anche se non è necessario, utilizzando tamponi freddi e mantenere i campioni in ghiaccio, quando possibile, produrrà una percentuale maggiore di cloroplasti intatti. </li><li> Preparare buffer di isolamento: 50 mm Hepes NaOH, 330 mm sorbitolo, 2 mM EDTA, 1,0 mm MnCl 2, 1,0 mm MgCl2, 10 mM KCl, e 1,0 mm di NaCl. Regolare il pH a 7,6 con NaOH e HCl e conservare in frigorifero prima dell&#39;uso. </li><li> Se le foglie non sonoesprimendo un fluoroforo stromale geneticamente codificato, preparare carboxyfluorescein diacetato soluzione (CFDA): Soluzione 50 mM CFDA in dimetilsolfossido (DMSO) (2,3 mg CFDA per 1,0 ml DMSO). NOTA: L&#39;esatta concentrazione non è critica. Mantenere CFDA magazzino al buio in ogni momento. Conservare piccole aliquote di 50 mm CFDA a -20 ° C. </li><li> Per isolare cloroplasti, rimuovere ~ 5-10 g di foglie di diverse piante e sciacquare brevemente in acqua fredda. Trasferire immediatamente a 50 ml tampone di estrazione a freddo. foglie Grind utilizzando un frullatore con diversi impulsi brevi. Filtrare su due o tre strati di garza per rimuovere i detriti foglia, dividere i cloroplasti estratti in due 50 ml provette da centrifuga e centrifugare per 1 min a 750 x g. </li><li> Eliminare il surnatante e risospendere le cloroplasti verdi nel buffer di isolamento 10 ml. Centrifugare ancora per 1 min a 750 xg, scartare il surnatante e cloroplasti risospendere in tampone isolamento per portare il volume finale a 5 ml. </li><li> Trasferimento 201, l dei cloroplasti a una diapositiva, coprire con un vetrino, e cominciare la microscopia. NOTA: cloroplasti da piante transgeniche che esprimono proteine ​​fluorescenti plastid mirati sono ora pronti per la visualizzazione al microscopio a fluorescenza confocale. </li><li> Stain cloroplasti da piante che non esprimono proteine ​​fluorescenti plastid mirati di visualizzare stromules. <ol><li> Aggiungere 5 ml di 50 mm CFDA magazzino alle 5 ml cloroplasti-sospese in un tampone di isolamento. Portare la concentrazione finale di 50 mM. </li><li> Consentire cloroplasti di incubare per 5 minuti, e poi trasferire una piccola aliquota (~ 50 ml) a una diapositiva, coprire con un vetrino, e cominciano la microscopia. </li><li> Utilizzare un set di filtri FITC o GFP. Utilizzare un obiettivo 20X di visualizzare molti cloroplasti simultaneamente, o un obiettivo più alto di visualizzare un unico cloroplasto isolato. NOTA: CFDA sarà fluorescenza all&#39;interno dello stroma dei cloroplasti intatti. </li><li> Se vi è una forte fluorescenza di fondo, lavare il chloroplasts di nuovo in 10 ml di tampone di isolamento dopo la 5 min incubazione con CFDA, centrifugare per 1 min a 750 xg, scartare il surnatante, e risospendere in 5 ml di tampone di isolamento. </li></ol></li></ol>

<ol>
	<li>Koussevitzky, 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=Signals+from+chloroplasts+converge+to+regulate+nuclear+gene+expression.">Signals from chloroplasts converge to regulate nuclear gene expression.</a> Science. 316 (5825), 715-719 (2007).</li><li>Burch-Smith, T. M., Brunkard, J. O., Choi, Y. G., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PNAS+Plus:+Organelle-nucleus+cross-talk+regulates+plant+intercellular+communication+via+plasmodesmata.">PNAS Plus: Organelle-nucleus cross-talk regulates plant intercellular communication via plasmodesmata.</a> Proc. Natl. Acad. Sci. USA. 108 (51), E1451-E1460 (2011).</li><li>Stonebloom, S., Brunkard, J. O., Cheung, A. C., Jiang, K., Feldman, L., Zambryski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+states+of+plastids+and+mitochondria+differentially+regulate+intercellular+transport+via+plasmodesmata.">Redox states of plastids and mitochondria differentially regulate intercellular transport via plasmodesmata.</a> Plant Physiol. 158 (1), 190-199 (2012).</li><li>Nomura, 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=Chloroplast-mediated+activation+of+plant+immune+signalling+in+Arabidopsis.">Chloroplast-mediated activation of plant immune signalling in Arabidopsis.</a> Nat. Commun. 3, 926 (2012).</li><li>Avenda&#241;o-V&#225;zquez, A. -. O., 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=An+uncharacterized+apocarotenoid-derived+signal+generated+in+&#950;-carotene+desaturase+mutants+regulates+leaf+development+and+the+expression+of+chloroplast+and+nuclear+genes+in+Arabidopsis.">An uncharacterized apocarotenoid-derived signal generated in &#950;-carotene desaturase mutants regulates leaf development and the expression of chloroplast and nuclear genes in Arabidopsis.</a> Plant Cell. 26 (June), 2524-2537 (2014).</li><li>Caplan, J. 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=Chloroplast+stromules+runction+during+innate+immunity.">Chloroplast stromules runction during innate immunity.</a> Dev. Cell. 34 (1), 45-57 (2015).</li><li>Hanson, M. R., Sattarzadeh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromules:+recent+insights+into+a+long+neglected+feature+of+plastid+morphology+and+function.">Stromules: recent insights into a long neglected feature of plastid morphology and function.</a> Plant Physiol. 155 (4), 1486-1492 (2011).</li><li>Gray, J. C., 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=Plastid+stromules+are+induced+by+stress+treatments+acting+through+abscisic+acid.">Plastid stromules are induced by stress treatments acting through abscisic acid.</a> Plant J. 69 (3), 387-398 (2012).</li><li>Brunkard, J., Runkel, A., Zambryski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplasts+extend+stromules+independently+and+in+response+to+internal+redox+signals.">Chloroplasts extend stromules independently and in response to internal redox signals.</a> Proc. Natl. Acad. Sci. USA. 112 (32), 10044-10049 (2015).</li><li>Dupree, P., Pwee, K. -. H., Gray, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+photosynthesis+gene-promoter+fusions+in+leaf+epidermal+cells+of+transgenic+tobacco+plants.">Expression of photosynthesis gene-promoter fusions in leaf epidermal cells of transgenic tobacco plants.</a> Plant J. 1 (1), 115-120 (1991).</li><li>Charuvi, D., Kiss, V., Nevo, R., Shimoni, E., Adam, Z., Reich, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain+and+loss+of+photosynthetic+membranes+during+plastid+differentiation+in+the+shoot+apex+of+Arabidopsis.">Gain and loss of photosynthetic membranes during plastid differentiation in the shoot apex of Arabidopsis.</a> Plant Cell. 24 (3), 1143-1157 (2012).</li><li>Chiang, Y. -. 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=Functional+characterization+of+the+GATA+transcription+factors+GNC+and+CGA1+reveals+their+key+role+in+chloroplast+development,+growth,+and+division+in+Arabidopsis.">Functional characterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development, growth, and division in Arabidopsis.</a> Plant Physiol. 160 (1), 332-348 (2012).</li><li>Schattat, M., Barton, K., Baudisch, B., Kl&#246;sgen, R. B., Mathur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastid+stromule+branching+coincides+with+contiguous+endoplasmic+reticulum+dynamics.">Plastid stromule branching coincides with contiguous endoplasmic reticulum dynamics.</a> Plant Physiol. 155 (4), 1667-1677 (2011).</li><li>Erickson, J. 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=Agrobacterium-derived+cytokinin+influences+plastid+morphology+and+starch+accumulation+in+Nicotiana+benthamiana+during+transient+assays.">Agrobacterium-derived cytokinin influences plastid morphology and starch accumulation in Nicotiana benthamiana during transient assays.</a> BMC Plant Biol. 14 (1), 127 (2014).</li><li>Ho, J., Theg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formation+of+stromules+in+vitro+from+chloroplasts+isolated+from+Nicotiana+benthamiana.">The formation of stromules in vitro from chloroplasts isolated from Nicotiana benthamiana.</a> PLoS One. 11 (2), e0146489 (2016).</li><li>Brunkard, J. O., Burch-Smith, T. M., Runkel, A. M., Zambryski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+plasmodesmata+genetics+with+virus-induced+gene+silencing+and+an+Agrobacterium-mediated+GFP+movement+assay.">Investigating plasmodesmata genetics with virus-induced gene silencing and an Agrobacterium-mediated GFP movement assay.</a> Method. Mol. Biol. , 185-198 (2015).</li><li>Schattat, M. H., Kl&#246;sgen, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+stromule+formation+by+extracellular+sucrose+and+glucose+in+epidermal+leaf+tissue+of+Arabidopsis+thaliana.">Induction of stromule formation by extracellular sucrose and glucose in epidermal leaf tissue of Arabidopsis thaliana.</a> BMC Plant Biol. 11, 115 (2011).</li><li>Joly, D., Carpentier, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+intact+chloroplasts+from+spinach+leaves.">Rapid isolation of intact chloroplasts from spinach leaves.</a> Method. Mol. Biol. 684, 321-325 (2011).</li><li>Waters, M. T., Fray, R. G., Pyke, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromule+formation+is+dependent+upon+plastid+size,+plastid+differentiation+status+and+the+density+of+plastids+within+the+cell.">Stromule formation is dependent upon plastid size, plastid differentiation status and the density of plastids within the cell.</a> Plant J. 39 (4), 655-667 (2004).</li></ol>

The authors have nothing to disclose.

Quando si indaga stromules, tre importanti fattori devono essere considerati durante: (i) la manipolazione del tessuto vegetale deve essere ridotto al minimo, (ii) il sistema sperimentale deve essere mantenuta costante, e (iii) le strategie di campionamento deve essere attentamente pianificata per garantire robusta, i dati vengono analizzati riproducibili. Stromules sono molto dinamici: possono estendere e ritrarre velocemente davanti agli occhi di un osservatore al microscopio. Inoltre, la ...

Chloroplasts are dynamic organelles in plant cells responsible for photosynthesis and a host of other metabolic processes. Signaling pathways from the chloroplast also exert significant influence on plant physiology and development, coordinating plant responses to environmental stress, pathogens, and even leaf shape1-6. Recently, biologists have gained interest in a poorly understood aspect of chloroplast structure: stromules, very thin stroma-filled tubules that extend from the surface of the chloroplast7.
The biological functions of stromules remain unknown, although stromule frequency is known to vary in response to environmental stimuli7-9, and stromules may be capable of transmitting signaling molecules between organelles6. All types of plastids (not only the green, photosynthetic chloroplasts, but also clear leucoplasts, starch-filled amyloplasts, and pigmented chromoplasts, to name a few types of plastids) make stromules, and stromules are found in all land plant species that have been examined to date. Stromules can extend and retract dynamically, appearing or disappearing within seconds, or they can remain relatively stationary for long times. One of the major hurdles facing stromule biologists is that stromules are often studied using dramatically different methods, tissues, and species, making comparisons across the stromule biology literature difficult. Going forward, standard practices and thorough descriptions of the experimental systems used to study stromules will be critical to discovering the function of these ubiquitous features of chloroplast morphology.
Here we describe methods for visualizing stromule formation in the epidermal chloroplasts of Nicotiana benthamiana leaves. In the mesophyll, chloroplasts are densely packed into large, three-dimensional cells, which makes it difficult to accurately and rapidly visualize stromules by confocal microscopy. By contrast, epidermal cells are relatively flat, contain fewer chloroplasts, and are at the surface of the leaf, allowing for easy and rapid visualization of stromules. N. benthamiana is an ideal model system for these experiments because, unlike many plant species, all cells in the epidermis of N. benthamiana make chloroplasts10. In the epidermis of most plants, including Arabidopsis thaliana, only the stomatal guard cells have chloroplasts, while other epidermal cells have "leucoplasts", plastids that are clear, relatively amorphous, and nonphotosynthetic9,11,12. Thus, whereas a single field of view of an A. thaliana epidermis might show only a handful of chloroplasts in a pair of guard cells, a field of view of an N. benthamiana epidermis will include dozens or even hundreds of chloroplasts. All of the methods described here, however, can be modified to investigate other questions in stromule biology; for example, we have used the same approach to study leucoplast stromules of A. thaliana9.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hepes</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NaOH</td>
			<td style="width:71px;">Fischer-Scientific</td>
			<td style="width:119px;">S320-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sorbitol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">S1876</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EDTA</td>
			<td style="width:71px;">Fischer-Biotech</td>
			<td style="width:119px;">BP121</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MnCl2</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">221279</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MgCl2</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">M0250</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">KCl</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P3911</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NaCl</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9625</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laser Scanning Confocal Microscope&#160;</td>
			<td style="width:71px;">Carl Zeiss Inc</td>
			<td style="width:119px;">Model: LSM710</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Carboxyfluorescein diacetate (CFDA)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">21879&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:71px;">EMD</td>
			<td style="width:119px;">MX1458-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Waring blender</td>
			<td style="width:71px;">Waring&#160;</td>
			<td style="width:119px;">Model: 31BL92</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fiji</td>
			<td style="width:71px;">fiji.sc</td>
			<td style="width:119px;"></td>
			<td>Open-source software for analyzing biological images</td>
		</tr>
	</tbody>
</table>

visualizing stromule frequency with fluorescence microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Questo protocollo è stato utilizzato per visualizzare la frequenza stromule di giorno e di notte nei cotiledoni di giovane N. piantine benthamiana. Fette da una pila Z sono state fuse in un&#39;unica immagine (Figura 1A). Per scopi visivi, che l&#39;immagine è stata poi desaturata e invertito in modo che appare stroma nero (Figura 1B). I cloroplasti sono stati etichettati o come non avere stromules (asterisco verde) o che han...

Watch this Scientific Journal Video about Visualizing Stromule Frequency with Fluorescence Microscopy at JoVE.com

Visualizing Stromule Frequency with Fluorescence Microscopy

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Visualizzazione Frequenza Stromule con microscopia a fluorescenza

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

visualizing-stromule-frequency-with-fluorescence-microscopy

Research

JoVE Journal

Biology

8.0K Views.  USDA. Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Video: Visualizing Stromule Frequency with Fluorescence Microscopy

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

Visualizzazione di Live Drosophila Gliali-giunzione neuromuscolare con tinture fluorescenti

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

Ricerca

Biologia

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

Visualizzazione singola complessi molecolari In Vivo Utilizzo avanzato microscopia a fluorescenza

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Visualizzazione singola molecola di DNA replica con microscopia a fluorescenza

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Time-lapse imaging di mitosi dopo la trasfezione di siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

Elettrosaldabili cellula visualizzate con microscopia a fluorescenza

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Quantitativa vivo cellulare-microscopio a fluorescenza Analisi di lievito Fission

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

Visualizzare batteri nei nematodi usando la microscopia a fluorescenza

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Analisi rapida ed esplorazione di microscopia a fluorescenza Immagini

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

Analisi di autofagia in<em&gt; Penicillium chrysogenum</em&gt; Utilizzando Starvation Pads in combinazione con microscopia a fluorescenza

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...