Name: visualizing stromule frequency with fluorescence microscopy
Uploaded: 2016-11-23T14:00:00.000+00:00
Duration: 8 min 27 s
Description: Watch this Scientific Journal Video about Visualizing Stromule Frequency with Fluorescence Microscopy at JoVE.com

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>

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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><tbody><tr><td>Hepes</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>NaOH</td><td>Fischer-Scientific</td><td>S320-1</td><td></td></tr><tr><td>Sorbitol</td><td>Sigma-Aldrich</td><td>S1876</td><td></td></tr><tr><td>EDTA</td><td>Fischer-Biotech</td><td>BP121</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>221279</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M0250</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9625</td><td></td></tr><tr><td>Laser Scanning Confocal Microscope&amp;nbsp;</td><td>Carl Zeiss Inc</td><td>Model: LSM710</td><td></td></tr><tr><td>Carboxyfluorescein diacetate (CFDA)</td><td>Sigma-Aldrich</td><td>21879&amp;nbsp;</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>EMD</td><td>MX1458-6</td><td></td></tr><tr><td>Waring blender</td><td>Waring&amp;nbsp;</td><td>Model: 31BL92</td><td></td></tr><tr><td>Fiji</td><td>fiji.sc</td><td></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

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins.
Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

Il monitoraggio spaziale segregazione in superficie Colonizzare microbiche Popolazioni

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

Ricerca

Genetica

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

Lifetime Imaging di fluorescenza di rotori molecolari nelle cellule viventi

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Bioingegneria

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

Cellulare Imaging in diretta di Bacillus subtilis E Streptococcus pneumoniae Utilizzando automatico Time-lapse Microscopia

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Immunologia e infezioni

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 &#181;m x 500 &#181;m region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

Fluorescenza della riflessione interna totale ad alto consumo e microscopia della ricostruzione ottica stocastica diretta utilizzando un chip fotonico

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

Biochimica

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 ...

Biologia

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. 
Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series.

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Streptomyces are characterized by a complex life cycle that has been experimentally challenging to study by cell biological means. Here we present a protocol to perform fluorescence time-lapse microscopy of the complete life cycle by growing Streptomyces venezuelae in a microfluidic device.

Fluorescenza Time-lapse imaging del Complete<em&gt; S. venezuelae</em&gt; Ciclo di vita Uso di un dispositivo a microfluidi

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of ...

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

Strumenti microfluidici per sondare le interazioni fungine-microbiche a livello cellulare

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

La quantificazione temporale di espressione MAPK indotta in cellule di lievito singole

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

Metodo per l'etichettatura di trascrizioni in cellule individuali Escherichia coli per singola molecola di fluorescenza In Situ gli esperimenti di ibridazione

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

Visualizzazione Frequenza Stromule con microscopia a fluorescenza

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