Gli autori desiderano ringraziare Javier E. Garcia e Lene Grønkjær per l&#39;assistenza tecnica e Merete K. Raarup per le discussioni fruttuose. Questo lavoro è stato finanziato dalla Fondazione Aarhus University Research e Simon Spies Fondazione.

Il protocollo sperimentale è stato esaminato e approvato dal Comitato Etico di Aarhus County (M-20.100.032). 1. confocale microscopica calibrazione del Raziometrico Dye <ol><li> Per l&#39;acquisizione delle immagini, utilizzare un microscopio invertito confocale equipaggiato con un incubatore, un obiettivo ad immersione dell&#39;acqua apertura 63X / 1.2-numerica, una linea laser 543 nm e un rivelatore META. </li><li> Preparare tampone HEPES soluzioni madre (50 mm, portata a pH 4.5-8.5 in passi di unità 0,1 pH). Dispensare 100 ml di ogni soluzione nei pozzetti di una chiara-bottom piastra a 96 pozzetti per la microscopia a fluorescenza. </li><li> Indossare guanti di nitrile quando si maneggia il colorante raziometrica C-SNARF-4. Preparare una 1 mM soluzione madre del colorante in dimetilsolfossido. Aggiungere 5 ml di soluzione madre di ogni pozzetto con tampone HEPES. Posizionare la piastra a 96 pozzetti sul microscopio. </li><li> Accendere il microscopio. Aprire il software microscopio. Clicca i seguenti pannelli: Acquisire → Laser; Acquisire → MICRO; Acquisire → Config; Acquisire → Scan; Acquisire → Stage. Riscaldare l&#39;incubatore a 37 ° C. </li><li> Accendere la linea laser 543 nm cliccando sul laser a 543 nm e il tasto &quot;On&quot; nella finestra &quot;Controllo Laser&quot;. Scegliere l&#39;obiettivo di immersione in acqua apertura 63X / 1.2-numerico nella finestra &quot;Microscope Control&quot;. </li><li> Impostare il rilevatore META di monitorare simultaneamente la fluorescenza all&#39;interno 576- a 608 nm (verde) e 629- a 661 nm intervalli di (RED) ( &quot;Controllo Configurazione&quot; → &quot;ChS&quot;). Regolare la potenza del laser ( &quot;Controllo Configurazione&quot; → &quot;eccitazione&quot;). Impostare il foro stenopeico per produrre uno spessore fetta ottica di 1,6 micron ( &quot;Scan Control&quot; → &quot;Pinhole&quot;). </li><li> Acquisire un&#39;immagine di ogni soluzione tampone HEPES, 5 micron sopra il fondo di vetro della piastra a 96 pozzetti. Nota: Appena il piano focale è situato sotto il fondo di vetro, nessuna luce fluorescente può essere vistosullo schermo. Dopo ogni terza immagine, impostare la potenza del laser a zero e prendere una immagine di sfondo sottrazione. </li><li> Eseguire l&#39;esperimento di calibrazione in triplice copia (1,2-1,7). </li><li> Determinare l&#39;intensità della fluorescenza media e deviazione standard in tutte le immagini rosse e verdi. <ol><li> Calcolare il rapporto R ed errore standard della media, S R, per ogni immagine in base alle equazioni (1) e (2) (1) <img alt="Equation1" src="/files/ftp_upload/53622/53622equation1.jpg" /> (2) <img alt="Equation2" src="/files/ftp_upload/53622/53622equation2.jpg" /> g, r, s g e s r sono le medie e le deviazioni standard nei rispettivi immagini verde e rosso. b g, b R, S bg e s br sono i valori corrispondenti per le immagini di sfondo. n <em&gt; 2 è il numero di pixel imaged. </li><li> Tracciare i rapporti calcolati per ciascun valore di pH dei tre esperimenti di calibrazione replica in un diagramma e costruire una curva montato da questa serie di punti dati (vale a dire utilizzando il software SigmaPlot 13). Fare una funzione matematica dalla curva montato in grado di convertire i rapporti in valori di pH 10. </li></ol></li></ol> 2. Raccolta di In Situ Grown dentale biofilm Campioni <ol><li> Selezionare volontari che soddisfano i criteri di inclusione ed esclusione rilevanti per lo studio. Fare impressioni alginato di loro arcata dentaria superiore e inferiore. Fare modelli del cast da queste impressioni e produzione di una stecca acrilico nella mandibola. Progettare la stecca con flange acrilici buccali collegati da un filo ortodontico linguale che permette al volontario di mordere normale occlusione 12. </li><li> Recessioni Drill nelle flange buccali della stecca acrilico (Figura 1 </strong&gt;) Con l&#39;aiuto di frese acriliche dentali per consentire l&#39;inserimento di lastre di vetro per la raccolta biofilm. La profondità delle recessioni deve essere di almeno 1,5 mm, mentre la larghezza e la lunghezza delle recessioni possono variare a seconda del numero di lastre di vetro da inserire. </li><li> Per la raccolta biofilm, lastre su misura non fluorescenti di vetro (4 x 4 x 1 mm 3) con una rugosità superficiale di graniglia 1200 al fine di imitare il modello di colonizzazione su smalto naturale 11. </li><li> Sterilizzare le lastre di vetro in autoclave prima del montaggio. Montare le lastre di vetro con cera collante nelle depressioni nelle flange buccali di ciascun lato leggermente incassati alla superficie della superficie acrilica per proteggere il biofilm da forze di taglio esercitate dal movimento delle guance 11. Nota: Il numero di lastre di vetro disposte in recessione può variare tra 3 e 14, a seconda dello scopo dello studio. </li><li> Inserire l&#39;apparecchio in bocca del volontario. Istruire il VolunTeer di mantenere l&#39;apparecchio intra-orale per tutto il periodo sperimentale. Istruire il volontario per immagazzinare l&#39;apparecchio in un contenitore fermo ortodontico con un pezzo di tessuto carta bagnata (per mantenerlo umido) a temperatura ambiente durante lo spazzolamento dei denti e l&#39;assunzione di cibo e bevande diverse dall&#39;acqua. Istruire il volontario a non toccare le flange acrilici buccali con le lastre di vetro durante l&#39;inserimento e la rimozione dell&#39;apparecchio. Nota: Il periodo sperimentale può variare a seconda dello scopo dello studio (un giorno a diverse settimane). </li><li> Rimuovere accuratamente le lastre di vetro dalla macchina al termine del periodo sperimentale. Rimuovere la cera adesiva intorno le lastre con un coltello e trasferirli con un paio di pinzette per un contenitore chiuso, il biofilm rivolta verso l&#39;alto, fino all&#39;analisi microscopica. Tenere il contenitore umido con fazzoletto di carta bagnata. Eseguire l&#39;imaging pH entro poche ore dalla raccolta biofilm. </li></ol> 3. biofilm pH Imaging <ol><li> Prepararesoluzione salivare aggiungendo ditiotreitolo alla saliva raccolta secondo il metodo di de Jong et al. 13. Titolare la soluzione salivare a pH 7.0 e aggiungere il glucosio ad una concentrazione di 0,4% (peso / volume). Dispensare 100 microlitri per biofilm da analizzare in un 96-pozzetti con fondo di vetro per la microscopia. Aggiungere 5 ml di tintura raziometrico per pozzetto. </li><li> Posizionare la piastra a 96 pozzetti sul palco microscopio. Accendere il microscopio e la linea laser 543 nm. Riscaldare l&#39;incubatore a 37 ° C. Utilizzare le stesse impostazioni microscopio come per la calibrazione del colorante (vedere i passaggi 1,5-1,6). Attendere per 30 minuti, fino a quando la piastra a 96 pozzetti ha raggiunto la temperatura di lavoro. </li><li> Pick up una o più lastre di vetro con un insieme sottile di pinzette e metterli nei pozzetti saliva pieno, una lastra per pozzetto, con i biofilm rivolti verso il basso. </li><li> Acquisire le immagini singole ( &quot;Scan Control&quot; → &quot;single&quot;) o Z-stack ( &quot;Scan Control&quot; → &quot;Start&quot;) spanning la profondità dei biofilm in diversi settori. Per acquisire z-stacks scegliere il numero di fette di essere ripreso ( &quot;Scan Control&quot; → &quot;Impostazioni Z&quot; → &quot;Num fette&quot;) e segnare la posizione z per la prima e l&#39;ultima fetta nel software del microscopio ( &quot;Scan Control &quot;→&quot; Impostazioni Z &quot;→&quot; Mark First &quot;,&quot; Mark last &quot;). Nota: Z-stack con una profondità massima di 75 micron possono essere acquisite con un buon contrasto tra le aree extracellulari e intracellulari. </li><li> Per seguire variazioni di pH in un campo di vista microscopico nel corso del tempo, marcare la xy posizione nel software del microscopio ( &quot;stage e di controllo messa a fuoco&quot; → &quot;Mark Pos&quot;) e prendere immagini ripetute in momenti successivi ( &quot;Scan Control&quot; → &quot; Singolo&quot;). Regolarmente prendere le immagini con la potenza del laser impostato a zero per sottrazione del fondo. </li></ol> 4. Analisi Digital Image <ol><li> per export le immagini microscopiche come file TIF, usano l&#39;esportazione di file batch del software microscopio ( &quot;Macro&quot; → &quot;Batch File Export&quot;). Selezionare i file da esportare e salvare le immagini del canale rosso e verde in cartelle separate come TIF-files ( &quot;Start Batch Export&quot;). Rinominare i file in entrambe le cartelle dando loro numeri sequenziali. </li><li> Importare la serie di immagini rosso e verde in software come Daime (analisi di immagine digitale in ecologia microbica) 14. Segmento le immagini del canale verde con soglie scelti individualmente luminosità (segmento → → segmentazione automatica di soglia personalizzate). Scegliere le soglie di luminosità con cura (tipicamente tra 20 e 80), in modo che tutti i batteri (più luminosa della matrice extracellulare), ma non la matrice saranno riconosciuti come oggetti durante la segmentazione. Verificare visivamente che le aree riconosciute come oggetti corrispondono bene alla biomassa batterica. </li><li> Trasferire il livello di oggetto del g segmentatoimmagini reen canale alle immagini dei canali rosso corrispondenti (segmento → Trasferimento strato oggetto). Utilizzare la funzione Editor oggetti di rifiutare e cancellare tutti gli oggetti nelle immagini del canale rosso e verde. Ora solo la matrice extracellulare è lasciato nelle immagini biofilm. Esportare la serie immagine elaborata sotto forma di file TIF. </li><li> Importare la serie di immagini in ImageJ (http://rsb.info.nih.gov/ij; v.1.47). Determinare l&#39;intensità media di fluorescenza nelle immagini di sfondo scattate con il laser spento (Analisi → Istogramma). Sottrarre il fondo adeguato dalle immagini rosso e verde (processo → Math → Sottrai). </li><li> Ancora in ImageJ, dividere la serie di immagini verde (G1) da solo (processo → Immagine calcolatrice). Quindi moltiplicare la serie immagine risultante (G2) con la serie di immagini verde (G1). Questo produrrà una serie di immagini (G3), dove NaN viene assegnato a tutti i pixel che appartengono a zone che sono state riconosciute come oggetti in Daime. Procedere in tlui stesso modo con la serie rosso immagine (R1 / R1 = R2; R2 x R1 = R3). Nota: Poiché la biomassa batterica è stato rimosso dalle immagini nel passaggio 4.3, l&#39;intensità fluorescente è 0 in queste aree. Fase 4.5 è necessario convertire il valore 0 per NaN, che permette di calcolo del rapporto nel passo 4.6. </li><li> Applicare il filtro &#39;Mean&#39; (processo → → Filtri Media; raggio: 1 pixel) per compensare il rumore del rivelatore. Dividere la serie immagine verde dalla serie di immagini rosso (processo → Immagine calcolatrice). Ciò si traduce in un rapporto verde / rosso per ogni pixel rimanente nello spazio extracellulare delle immagini. Utilizzare falsa colorazione per la rappresentazione grafica dei rapporti nelle immagini (Tabelle Immagine → Lookup). Calcolare il rapporto medio per ogni immagine (Analisi → Istogramma). </li><li> Convertire i rapporti di verde / rosso a valori di pH in base alla funzione montato sotto 1.9.2). Nota: Un esempio per dati di calibrazione e la curva a muro può essere visto in Schlafer etal 2015 11. </li></ol>

<ol>
	<li>Hunter, R. C., Beveridge, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+pH-sensitive+fluoroprobe+(C-SNARF-4)+for+pH+microenvironment+analysis+in+Pseudomonas+aeruginosa+biofilms.">Application of a pH-sensitive fluoroprobe (C-SNARF-4) for pH microenvironment analysis in Pseudomonas aeruginosa biofilms.</a> Appl. Environ. Microbiol. 71 (5), 2501-2510 (2005).</li><li>Takahashi, N., Nyvad, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caries+ecology+revisited:+microbial+dynamics+and+the+caries+process.">Caries ecology revisited: microbial dynamics and the caries process.</a> Caries Res. 42 (6), 409-418 (2008).</li><li>Schlafer, 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=pH+landscapes+in+a+novel+five-species+model+of+early+dental+biofilm.">pH landscapes in a novel five-species model of early dental biofilm.</a> PLoS. One. 6 (9), e25299 (2011).</li><li>von Ohle, O. 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=Real-time+microsensor+measurement+of+local+metabolic+activities+in+ex+vivo+dental+biofilms+exposed+to+sucrose+and+treated+with+chlorhexidine.">Real-time microsensor measurement of local metabolic activities in ex vivo dental biofilms exposed to sucrose and treated with chlorhexidine.</a> Appl. Environ. Microbiol. 76 (7), 2326-2334 (2010).</li><li>Revsbech, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+microbial+communities+with+electrochemical+microsensors+and+microscale+biosensors.">Analysis of microbial communities with electrochemical microsensors and microscale biosensors.</a> Methods Enzymol. 397, 147-166 (2005).</li><li>Vanhoudt, P., Lewandowski, Z., Little, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iridium+oxide+pH+microelectrode.">Iridium oxide pH microelectrode.</a> Biotechnol. Bioeng. 40 (5), 601-608 (1992).</li><li>Franks, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+strategy+for+three-dimensional+real-time+imaging+of+microbial+fuel+cell+communities:+monitoring+the+inhibitory+effects+of+proton+accumulation+within+the+anode+biofilm.">Novel strategy for three-dimensional real-time imaging of microbial fuel cell communities: monitoring the inhibitory effects of proton accumulation within the anode biofilm.</a> Energy &amp; Environmental Science. 2 (1), 113-119 (2009).</li><li>Hidalgo, G., 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+tomographic+fluorescence+imaging+of+pH+microenvironments+in+microbial+biofilms+by+use+of+silica+nanoparticle+sensors.">Functional tomographic fluorescence imaging of pH microenvironments in microbial biofilms by use of silica nanoparticle sensors.</a> Appl. Environ. Microbiol. 75 (23), 7426-7435 (2009).</li><li>Vroom, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depth+penetration+and+detection+of+pH+gradients+in+biofilms+by+two-photon+excitation+microscopy.">Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy.</a> Appl. Environ. Microbiol. 65 (8), 3502-3511 (1999).</li><li>Bender, G. R., Sutton, S. V., Marquis, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid+tolerance,+proton+permeabilities,+and+membrane+ATPases+of+oral+streptococci.">Acid tolerance, proton permeabilities, and membrane ATPases of oral streptococci.</a> Infect. Immun. 53 (2), 331-338 (1986).</li><li>Schlafer, 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=Ratiometric+imaging+of+extracellular+pH+in+bacterial+biofilms+using+C-SNARF-4.">Ratiometric imaging of extracellular pH in bacterial biofilms using C-SNARF-4.</a> Appl. Environ. Microbiol. 81 (4), 1267-1273 (2015).</li><li>Dige, I., Nilsson, H., Kilian, M., Nyvad, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+identification+of+streptococci+and+other+bacteria+in+initial+dental+biofilm+by+confocal+laser+scanning+microscopy+and+fluorescence+in+situ+hybridization.">In situ identification of streptococci and other bacteria in initial dental biofilm by confocal laser scanning microscopy and fluorescence in situ hybridization.</a> Eur. J Oral Sci. 115 (6), 459-467 (2007).</li><li>de Jong, M. H., van der Hoeven, J. S., van OS, J. H., Olijve, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+oral+Streptococcus+species+and+Actinomyces+viscosus+in+human+saliva.">Growth of oral Streptococcus species and Actinomyces viscosus in human saliva.</a> Appl. Environ. Microbiol. 47 (5), 901-904 (1984).</li><li>Daims, H., Lucker, S., Wagner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=daime,+a+novel+image+analysis+program+for+microbial+ecology+and+biofilm+research.">daime, a novel image analysis program for microbial ecology and biofilm research.</a> Environ. Microbiol. 8 (2), 200-213 (2006).</li><li>Liu, Y. L., Nascimento, M., Burne, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+toward+understanding+the+contribution+of+alkali+generation+in+dental+biofilms+to+inhibition+of+dental+caries.">Progress toward understanding the contribution of alkali generation in dental biofilms to inhibition of dental caries.</a> Int. J Oral Sci. 4 (3), 135-140 (2012).</li></ol>

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Monitoraggio microscopico di pH biofilm fornisce diversi vantaggi, rispetto ad elettrodo o microelettrodi misurazioni 4-6. tecniche microscopiche permettono di determinare il pH con una risoluzione spaziale elevata e consentire catturare gradienti di pH orizzontali e verticali in biofilm senza disturbare il biofilm meccanicamente. I precedenti tentativi di monitoraggio del pH microscopico, tuttavia, non sono riusciti a distinguere tra il pH extracellulare e intracellulare nei biofilm 1,7,9. A causa...

Il metodo qui descritto permette di monitorare pH extracellulare in biofilm dentali nell&#39;intervallo tra 4,5 e 7, utilizzando il colorante raziometrica seminaphthorhodafluor-4F 5- (e-6) di acido carbossilico (C-SNARF-4) in combinazione con microscopio confocale a scansione laser e analisi di immagine digitale. Il colorante fluorescente impiegata è sensibile al pH e visualizza un cambiamento nella sua emissione fluorescente a seconda dello stato di protonazione. L&#39;emissione fluorescente dei picchi molecola protonata a 580 nm, e l&#39;emissione della molecola deprotonata a 640 nm 1. Il rapporto tra le intensità di emissione fluorescente in due finestre di rilevazione comprendente i due picchi di emissione (576 - 608 nm ei 629 - 661 nm) riflette quindi pH nella fase liquida, indipendentemente dalla concentrazione di colorante. Con un pK a di ~ 6,4 colorante è adatto per visualizzare pH in ambienti moderatamente acide. PH in biofilm batterici è di fondamentale importanza per tutti i processi metabolici.Nel caso di biofilm dentali, pH nella matrice extracellulare è il fattore di virulenza chiave per lo sviluppo della carie dentaria. Lunghi periodi con un basso pH al comando di interfaccia biofilm-dente per rallentare la demineralizzazione dello smalto di fondo 2. A causa della complessa architettura tridimensionale di biofilm, metaboliti, tra cui gli acidi organici, non sono distribuiti uniformemente in tutto il biofilm. Altamente e meno microambienti acidogeni possono essere trovati in prossimità spaziale 3. Per decenni, i gradienti di pH verticali in biofilm sono stati registrati con l&#39;aiuto di microelettrodi 4-6. Mentre offrono una buona risoluzione spaziale a causa della loro dimensione piccola punta, non sono particolarmente adatti per monitorare i gradienti orizzontali. Inoltre, l&#39;inserimento dell&#39;elettrodo disturba il biofilm meccanicamente. tecniche microscopiche a fluorescenza quantitative offrono il vantaggio di visualizzare variazioni di pH nei diversi settori di un biofilm, senza interferire meccanicance. Diversi campi microscopici di vista possono essere scelti liberamente e ripreso più volte per periodi prolungati 1,7-9. Tuttavia, quando si interpretano immagini microscopiche biofilm, è importante distinguere tra la fluorescenza derivante dalla biomassa microbica e fluorescenza derivante dallo spazio extracellulare. In condizioni acide, pH all&#39;interno delle cellule batteriche è diverso da pH nella matrice extracellulare, come i batteri trasportano attivamente protoni attraverso la membrana cellulare a scapito di adenosina trifosfato 10. Nel contesto della carie dentale, pH batterica intracellulare non ha un impatto diretto sullo smalto sottostanti, mentre bassi valori di pH extracellulare porta alla demineralizzazione. Una media di pH nelle immagini microscopiche che contengono entrambe le aree prive di batteri e batteri porta a risultati errati. L&#39;uso di altre macchie insieme con il colorante sensibile al pH per visualizzare la biomassa batterica e differenziare tra le aree extra ed intracellulari porta abil rischio di contaminazione fluorescente dello spazio extracellulare e false misurazioni 11. La presente manoscritto descrive pertanto l&#39;uso del colorante raziometrica in una duplice funzione; sia come marcatore pH e come una macchia batterica universale. Poiché il colorante è up-concentrato in cellule batteriche, la combinazione di confocale immagini microscopiche e un&#39;accurata procedura di analisi di immagine digitale consente la determinazione del pH extracellulare nel campo tra 4,5 e 7,0 in biofilm dentali sottili.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Zeiss LSM 510 META</td>
			<td style="width:305px;">Zeiss</td>
			<td style="width:243px;">N/A</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">C-Apochromat 63X water immersion objective</td>
			<td style="width:305px;">Zeiss</td>
			<td style="width:243px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">XL Incubator</td>
			<td style="width:305px;">PeCON</td>
			<td style="width:243px;">N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">SNARF-4F 5-(and-6)-Carboxylic Acid</td>
			<td style="width:305px;">Life Technologies</td>
			<td>S23920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Dimethyl sulfoxide</td>
			<td style="width:305px;">Life Technologies</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">HEPES</td>
			<td style="width:305px;">Life Technologies</td>
			<td>11344-041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Costar 96-well black clear-bottom plate</td>
			<td style="width:305px;">Fisher Scientific</td>
			<td>07-200-567</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:409px;">Custom-made glass slabs (4x4x1 mm; 1,200 grit)</td>
			<td style="width:305px;">Menzel</td>
			<td style="width:243px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Alginate impression material</td>
			<td style="width:305px;">GC Corporation</td>
			<td style="width:243px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Acrylic Adjusting Logic Sets/set of acrylic dental burs</td>
			<td style="width:305px;">Axis Dental</td>
			<td style="width:243px;">LS-906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Orthodontic retainer containers</td>
			<td style="width:305px;">Spark Medical Equipment Co., Ltd</td>
			<td style="width:243px;">SK-WDTC01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Sticky wax</td>
			<td style="width:305px;">Dentsply</td>
			<td style="width:243px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chewing paraffin wax&#160;</td>
			<td>Ivoclar Vivadent AG</td>
			<td style="width:243px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Dithiothreitol</td>
			<td style="width:305px;">Sigma Aldrich</td>
			<td>D0632</td>
			<td>Used during preparation of salivary solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">0.45 &#181;m and 0.2 &#181;m syringe filters</td>
			<td style="width:305px;">Sigma Aldrich</td>
			<td>CLS431220; CLS431219&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:409px;">daime</td>
			<td style="width:305px;">University of Vienna, Austria</td>
			<td style="width:243px;">http://dome.csb.univie.ac.at/daime</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">ImageJ</td>
			<td>NIH, Bethesda, Maryland, USA</td>
			<td style="width:243px;">http://imagej.nih.gov/ij/</td>
			<td></td>
		</tr>
	</tbody>
</table>

ratiometric imaging of extracellular ph in dental biofilms

Il pH di biofilm batterici sui denti è di importanza centrale per la carie dentale, una malattia con un&#39;alta prevalenza in tutto il mondo. Nutrienti e metaboliti non sono distribuite in modo uniforme in biofilm dentali. Un complesso gioco di assorbimento da e per la reazione con la materia organica nel biofilm riduce i percorsi di diffusione di soluti e crea pendenze di molecole reattive, tra cui gli acidi organici, in tutto il biofilm. Metodi microscopici fluorescenti quantitativi, come l&#39;imaging tempo di vita di fluorescenza o pH ratiometry, possono essere impiegati per visualizzare pH in diversi microambienti di biofilm dentali. pH ratiometry sfrutta una variazione del pH-dipendente nella emissione fluorescente di coloranti sensibili al pH. Calcolo del rapporto emissione a due diverse lunghezze d&#39;onda permette di determinare pH locale in immagini microscopiche, indipendentemente dalla concentrazione del colorante. Contrariamente a microelettrodi tecnica permette il monitoraggio entrambi gradienti di pH verticali ed orizzontali in tempo reale condisturbare meccanicamente il biofilm. Tuttavia, la cura deve essere presa per distinguere con precisione tra i compartimenti extra-intracellulari e del biofilm. Qui, il colorante raziometrica, seminaphthorhodafluor-4F 5- (e-6) di acido carbossilico (C-SNARF-4) è impiegato per monitorare pH extracellulare in in vivo biofilm dentali coltivate di composizione delle specie sconosciuta. Al momento l&#39;esposizione al glucosio il colorante è up-concentrato all&#39;interno di tutte le cellule batteriche nei biofilm; è quindi usato sia come una macchia batterica universale e come indicatore di pH extracellulare. Dopo l&#39;acquisizione di immagini al microscopio confocale, la biomassa batterica viene rimosso da tutte le immagini utilizzando il software di analisi di immagine digitale, che permette di calcolare esclusivamente pH extracellulare. pH ratiometry con il colorante raziometrico è particolarmente adatto per studiare pH extracellulare in biofilm sottili fino a 75 micron di spessore, ma è limitato al campo di pH tra 4,5 e 7,0.

Il metodo presentato consente pH extracellulare monitoraggio scende in differenti microambienti di biofilm dentali nel range di pH da 4,5 a 7 in tempo reale. Se le condizioni sperimentali sono scelti come descritto sopra, pH inizia a cadere in tutte le aree del biofilm poco dopo l&#39;esposizione al glucosio. Quando il pH in un biofilm gocce, cellule batteriche diventano visibili entro breve tempo (&lt;1 min), come il colorant...

Watch this Scientific Journal Video about Ratiometric Imaging of Extracellular pH in Dental Biofilms at JoVE.com

Ratiometric Imaging of Extracellular pH in Dental Biofilms

Imaging Raziometrico del pH extracellulare nel dentale biofilm

Un colorante raziometrico pH-sensibile è utilizzato in combinazione con microscopio confocale a scansione laser e analisi di immagine digitale per monitorare pH extracellulare in biofilm dentali in tempo reale.

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

ratiometric-imaging-of-extracellular-ph-in-dental-biofilms

Research

JoVE Journal

Immunology and Infection

9.9K Views.  Aarhus University.  Un colorante raziometrico pH-sensibile è utilizzato in combinazione con microscopio confocale a scansione laser e analisi di immagine digitale per monitorare pH extracellulare in biofilm dentali in tempo reale. 

Video: Ratiometric Imaging of Extracellular pH in Dental Biofilms

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Crescita Mycobacterium tuberculosis I biofilm

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Ricerca

Immunologia e infezioni

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

Quantificazione concomitante di cellulare e extracellulare componenti del biofilm

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

Misurare phagosome pH Raziometrico microscopia a fluorescenza

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

Imaging dei neutrofili phagosome e nel citoplasma utilizzo di un indicatore di pH Raziometrico

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

Stenosi della Vena Cava inferiore: un modello murino di trombosi venosa profonda

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

Purification of a High Molecular Mass Protein in Streptococcus mutans

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has been disrupted. Loss of function following gene disruption is subsequently restored by exogenous addition of the product of the disrupted gene. This helps to determine the function of the gene. A method previously described involves generating a gtfC gene-disrupted Streptococcus mutans strain. Here, an undemanding method is described for purifying the gtfC gene product from the newly generated S. mutans strain following the gene disruption. It involves the addition of a polyhistidine-coding sequence at the 3&#8242; end of the gene of interest, which allows simple purification of the gene product using immobilized metal affinity chromatography. No enzymatic reactions other than PCR are required for the genetic modification in this method. The restoration of the gene product by exogenous addition after gene disruption is an efficient method for determining gene function, which may also be adapted to different species.

Purification of a High Molecular Mass Protein in Streptococcus mutans

Described here is a simple method for the purification of a gene product in Streptococcus mutans. This technique may be advantageous in the purification of proteins, especially membrane proteins and high molecular mass proteins, and can be used with various other bacterial species.

Purificazione di una proteina di massa molecolare elevata in Streptococcus mutans

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 &#181;m or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

The protocol describes the cultivation of cross-kingdom biofilms consisting of Candida albicans and Streptococcus mutans and presents a confocal microscopy-based method for the monitoring of extracellular pH inside these biofilms.

Monitoraggio del pH extracellulare nei biofilm tra regni incrociati utilizzando la microscopia Confocal

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

Chiarire e imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

Supervised Machine Learning for Semi-Quantification of Extracellular DNA in Glomerulonephritis

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular deoxyribonucleic acid (ecDNA) is released during different forms of cell death including apoptosis, necrosis, necroptosis, neutrophil extracellular traps (NETs) and pyroptosis. Measurement of this cell death is time consuming with several different biomarkers required to identify the different biochemical forms of cell death. Measurement of ecDNA is generally conducted in serum and urine as a surrogate for renal damage, not in the actual target organ where the pathological injury occurs. The current difficulty in investigating ecDNA in the kidney is the lack of methods for formalin fixed paraffin embedded tissue (FFPE) both experimentally and in archived human kidney biopsies. This protocol provides a summary of the steps required to stain for ecDNA in FFPE tissue (both human and murine), quench autofluorescence and measure the ecDNA in the resulting images using a machine learning tool from the publicly available open source ImageJ plugin trainable Weka segmentation. Trainable Weka segmentation is applied to ecDNA within the glomeruli where the program learns to classify ecDNA. This classifier is applied to subsequent acquired kidney images, reducing the need for manual annotations of each individual image. The adaptability of the trainable Weka segmentation is demonstrated further in kidney tissue from experimental murine anti-MPO glomerulonephritis (GN), to identify NETs and ecMPO, common pathological contributors to anti-MPO GN. This method provides objective analysis of ecDNA in kidney tissue that demonstrates clearly the efficacy in which the trainable Weka segmentation program can distinguish ecDNA between healthy normal kidney tissue and diseased kidney tissue. This protocol can easily be adapted to identify ecDNA, NETs and ecMPO in other organs.

Extracellular DNA (ecDNA) released during cell death is proinflammatory and contributes to inflammation. Measurement of ecDNA at the site of injury can determine the efficacy of therapeutic treatment in the target organ. This protocol describes the use of a machine learning tool to automate measurement of ecDNA in kidney tissue.

Apprendimento automatico supervisionato per la semi-quantificazione del DNA extracellulare in Glomerulonephritis

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular ...

Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy

In recent years, the field of macropinocytosis has grown rapidly. Macropinocytosis has emerged as a central mechanism by which innate immune cells maintain organismal homeostasis and immunity. Simultaneously, and in contrast to its homeostatic role, it can also drive various pathologies, including cancer and viral infections. Unlike other modes of endocytosis, the tools developed for studying the maturation of macropinosomes remain underdeveloped. Here the protocol describes newly developed tools for studying the redox environment within the lumen of early and maturing macropinosomes. Methodologies for using ratiometric fluorescence microscopy in assessing the pH, production of reactive oxygen species, and the degradative capacity within the lumen of individual macropinosomes in live cells are described. Single organelle measurements offer the advantage of revealing spatiotemporal heterogeneity, which is often lost with population-based approaches. Emphasis is placed on the basic principles of dual fluorophore ratiometric microscopy, including probe selection, instrumentation, calibration, and single-cell versus population-based methods.

We describe protocols for measuring pH, oxidative events, and protein digestion in individual macropinosomes in live cells. An emphasis is placed on dual-fluorophore ratiometric microscopy and the advantages it offers over population-based techniques.