Questa ricerca è sostenuta dalla Fondazione Nazionale delle Ricerche e il Ministero della Pubblica Istruzione di Singapore sotto il suo Centro di Ricerca di Eccellenza programma, lo Start-up Grants (M4330002.C70) da Nanyang Technological University, e ACRF Tier 2 (MOE2014-T2-2-172) da Ministero dell&#39;Istruzione, Singapore. Gli autori ringraziano Joey Yam Kuok Hoong per la partecipazione alla manifestazione di questo protocollo.

1. biofilm Coltivazione <ol><li> Preparazione di ceppi batterici <ol><li> 1 giorno prima coltivazione biofilm, preparare colture batteriche planctonici inoculando 2 ml di adeguato mezzo di crescita di coltura batterica congelato. Utilizzare Luria Broth-mezzo (10 g L -1 NaCl, 10 g di estratto di lievito L -1 e 10 g L -1 triptone) per mucoide P. aeruginosa e le sue Δ pel e Δ PSL mutanti difettosi. Incubare una notte a 200 rpm condizioni scuotendo 37 ° C e. Diluire culture durante la notte ad una OD 600 di 0,40 usando uno spettrofotometro. </li><li> Assemblare configurazione cella di flusso, che è stato descritto in precedenza 14, preferibilmente su una stazione mobile o carrello che può essere portato nella camera microscopio imaging la cella di flusso senza smontaggio. </li><li> Preparare terreno di coltura sterile in 2 L o 5 bottiglie L per ogni cella di flusso. Utilizzare minimo medio M9 (48 mm Na 2 HPO 4, 22 mmKH 2 PO 4 19 mM NH 4 Cl, 9 mM NaCl, 2 mM MgSO 4, e 0,1 mM CaCl 2) integrato con 0,04% di glucosio (peso / volume) e 0,2% (peso / volume) Casamino acidi) per P. aeruginosa flusso biofilm cellulari. </li><li> Microsfere Aliquota fluorescenti in provette da microcentrifuga e spin down in H 2 O sterile per 5 min in centrifugare a 9,391.2 g. Rimuovere il surnatante e risospendere in 1 ml di mezzo di crescita per rimuovere sodio azide (disinfettante) prima di aggiungere al mezzo di coltura in 2 L o 5 bottiglie L medie. NOTA: I formati differenti di microsfere sono disponibili in diverse concentrazioni e devono essere diluiti di conseguenza per le istruzioni. Ad esempio, disperdere 120 ml di 1,0 micron microsfere di diametro, 15 ml di 0,5 micron microsfere diametro e 0,96 ml di 0,2 micron di diametro microsfere in 2 litri di terreno di crescita per dare 2,18 × 10 6 microsfere ml -1 per ogni dimensione delle particelle. </li><li> Fissare medio bottiglia upstrisma di cella di flusso e consentire terreno di coltura di fluire attraverso tutta l&#39;installazione. Interrompere il flusso e iniettare culture durante la notte nelle camere diluito cella di flusso. Consentire batteri per attaccare al substrato coprioggetto per 1 ora prima di continuare il flusso di terreno di coltura ad una portata di circa 5,5 x 10 -3 m sec -1, o 8 rpm con una pompa peristaltica. NOTA: I biofilm di solito sono cresciuti a temperatura ambiente (25 ° C) per 3-7 giorni. </li><li> In alternativa, per la messa a cultura statica, diluire culture durante la notte a 100 volte in provette da microcentrifuga con l&#39;aggiunta di 10 ml di cultura durante la notte per 1 ml di terreno di coltura con particelle. Aumentare la concentrazione di particelle nel mezzo di crescita per la cultura statica di circa 500 volte rispetto a quella utilizzata per biofilm di flusso (ad esempio lavare e risospendere 600 ml di 1,0 micron sfere di diametro in 10 ml di terreno di coltura) come biofilm cella di flusso sono costantemente rifornito con particelle ma le culture non sono statiche. <ol><li> Aggiungere 2001; l di cultura durante la notte diluito con particelle alle camere di 8 scivoli pure. Incubare a 37 ° C in condizioni statiche. Biofilm sono generalmente coltivate per 1 giorno. Sostituire crescita media spesi quotidianamente con terreno di coltura fresco con particelle se biofilm viene coltivato per più di 1 giorno. </li></ol></li></ol></li></ol> 2. Microscopia <ol><li> Il giorno 3 e 5, spegnere flusso dalla pompa peristaltica e portare configurazione cella di flusso nella camera microscopia. Tubo morsetto vicino all&#39;entrata e all&#39;uscita delle camere cella di flusso per prevenire drift (un errore sistematico causa da cambiamenti nell&#39;ambiente) dal flusso. Posizionare la cella di flusso sul palco del microscopio di un microscopio in posizione verticale. NOTA: utilizzare un microscopio invertito per l&#39;imaging di pozzetti. </li><li> Utilizzare microscopia a fluorescenza con obiettivo 40x olio per prendere i video di moto microsfere incorporate nel biofilm in varie località (microcolonie e strati indifferenziate piatto) e in diversi giorni (giorno 3 e 5) perindagini temporali e spaziali. Prendere brevi video con una maggiore frequenza di fotogrammi per indagare gli eventi che si verificano all&#39;interno scale temporali brevi (ad esempio 1,5-3 numero minimo di video su frame rate di 25-50 fotogrammi al secondo per la dinamica veloce), e più a lungo video con frame rate inferiore per indagare gli eventi nel tempo più lungo scale (per esempio 15-30 min video su frame rate di 2,5-5 fotogrammi / sec per le dinamiche più lente). NOTA: Un video contiene tipicamente 5-10 particelle, ed è tipicamente 1,0-2,5 GB di dimensione del file. Microcolonie più grandi possono contenere più particelle. Prendere 5-10 video per ogni categoria (per esempio 5-10 video di diversi microcolonie e di luoghi diversi strati indifferenziate piatti). Il biofilm ha un&#39;architettura eterogenea che può essere costituito da microcolonie, canali, spazi vuoti e strati indifferenziate piane. </li><li> Salvare i video in formato appropriato che possono essere letti da Fiji / ImageJ (ad esempio CZI, tif). </li><li> Controllare i video per la deriva scorrendo vid finitoeo e osservando che le particelle non si muovono nella stessa direzione simultaneamente. Drift può essere causato dal movimento z-fase, correnti nella cella di flusso, squilibrio di tavolo antivibrante, la pressione dell&#39;aria o variazioni di temperatura. Correggere minore alla deriva con tecniche di post-elaborazione di solito forniti con il software microscopio. Eliminare tutti i video in cui la deriva non possono essere corretti. Registrare i seguenti parametri, che sono tenuti nel corso delle particelle Monitoraggio Analisi: Risoluzione (px / um), numero di fotogrammi, Durata. </li><li> Rimuovere cella di flusso da palco microscopio e riavviare pompa peristaltica per continuare a coltivare il biofilm. </li></ol> 3. Analisi delle particelle Monitoraggio <ol><li> Ottenere traiettorie delle particelle utilizzando il plug-in TrackMate in software open source (ImageJ). Scarica Fiji a <a href="http://fiji.sc/Fiji">http://fiji.sc/Fiji.</a> Aprire il video con le Figi e sotto il menu Plugins, selezionare Monitoraggio e TrackMate. </li><li> Controllare o regolare le impostazioni in the suggerendo finestra impostazioni di calibrazione iniziali per abbinare i parametri video come registrato al punto 2.4. </li><li> Rileva particelle TrackMate utilizzando ImageJ. <ol><li> Seleziona &#39;LoG detector&#39;, ingresso diametro delle particelle e valore di soglia (ad esempio, 1000). Scorrere il video, mentre cliccando sul pulsante &#39;Anteprima&#39; per verificare che i cerchi viola seguono le particelle di tutto il video. Regolare i valori di diametro e di soglia, se necessario: aumentare il valore di soglia, se cerchi viola figurare, nello spazio vuoto. Ridurre il valore di soglia, se vengono rilevati particelle non abbastanza. Fare clic sul pulsante accanto a completare il rilevamento delle particelle. </li><li> Fare clic sul pulsante accanto quando sono presentati con la possibilità di aggiungere o rimuovere le particelle individuate nell&#39;ambito di &#39;soglia iniziale&#39;, &#39;Selezionare una vista&#39; e &#39;selezionare un filtro&#39; finestre di continuare senza regolazione se il riconoscimento iniziale di particelle è stato soddisfacente. </li><li> Seleziona &#39;LAP inseguitore Semplice&#39; nel optisulla barra di &#39;Selezionare un metodo tracker&#39; finestra come le particelle si muovono raramente fuori fuoco in biofilm e sono facilmente monitorati. Utilizzare i valori di distanza massima suggerita o basso di collegamento e gap-chiusura, e fare clic su Avanti. </li><li> Controllare che il monitoraggio delle particelle è soddisfacente scorrendo il video per vedere che le particelle seguono le tracce disegnate da TrackMate, e che non ci siano tracce indesiderate o mancanti. Passa le varie fasi di filtraggio. Passare alla finestra finale &#39;Seleziona un&#39;azione&#39;. Seleziona &#39;brani Esporta file XML&#39; dal menu a discesa e fare clic su &#39;Esegui&#39;. Scegliere una cartella in cui salvare le tracce. NOTA: Ci sono vari programmi disponibili nel pubblico dominio per l&#39;analisi delle traiettorie delle particelle e l&#39;uso di microrheology. msdanalyzer e TrackArt sono esempi di programmi di analisi di particelle di monitoraggio che vengono eseguite in ambiente Matlab. </li></ol></li><li> Preparare Matlab per importare le traiettorie delle particelle nei file xml selezionando nel menu diMatlab File&gt; Imposta percorso ... e Aggiungi cartella disponibili gli script con il pacchetto Fiji. </li><li> Per analizzare le traiettorie delle particelle con msdanalyzer, scaricare il collegamento al file zip o il file tar.gz at <a href="http://www.mathworks.com/matlabcentral/fileexchange/40692-mean-square-displacement-analysis-of-particles-trajectories">http://www.mathworks.com/matlabcentral/fileexchange/40692-mean-square-displacement-analysis-of-particles-trajectories</a> . <ol><li> Estrarre la cartellamsdanalyzer 15 e rilasciarlo in una cartella che appartiene al percorso Matlab (ad esempio C: Documents and Settings &lt;nome utente&gt; Documenti Matlab). Avviare Matlab e avviare l&#39;analizzatore digitando: ma = msdanalyzer (2, &#39;um&#39;, &#39;sec&#39;) dove um e sec sono le unità spazio-temporali fisici nel video. <ol><li> Importare le traiettorie delle particelle digitando: [tracce, md] = importTrackMateTracks (&#39;NomeFile.xml&#39;, &#39;clipZ&#39;,, &#39;Scalet&#39;) MA = ma.addAll (tracce); dove &#39;clipZ&#39; rimuove la dimensione z un video in 2D, e &#39;Scalet&#39;. </li><li> Tracciare le traiettorie Onto grafico utilizzando: ma.plotTracks; ma.labelPlotTracks; </li><li> Calcolare le DMS delle particelle mediante: ma = ma.computeMSD; ma.msd </li><li> Tracciare i DMS di ogni particella: figura ma.plotMSD </li><li> Combinare traiettorie delle particelle provenienti da altri video dello stesso categery (particelle ad esempio incorporato in wild-type microcolonie al giorno 3) ripetendo 3.5.1.1 a 3.5.1.4). </li><li> Tracciare la media insieme o la media su tutte le curve: ma.plotMeanMSD Cambiare la Y e asse x da lineare a scala logaritmica. In tempi di latenza lunghi, le curve diventano MSD rumorosa a causa di statistiche insufficienti a tempi più lunghi. Le curve MSD possono anche aumentare vertiginosamente a causa di errore dinamico. Queste regioni della curva può essere rimosso utilizzando lo strumento pennelloper selezionare la fine della curva e selezionando &#39;spazzolatura&#39;&gt; &#39;Rimuovi spettinati&#39; nel menu Strumenti. </li></ol></li><li> Scarica Ezyfit a <a href="http://www.fast.u-psud.fr/ezyfit/">http://www.fast.u-psud.fr/ezyfit/</a> e aggiungere Ezyfit a una cartella che appartiene al percorso Matlab (ad esempio C: Documents and Settings &lt;nome utente&gt; Documenti Matlab). Installare il menu Ezyfit digitando in Matlab lavoro efmenu installazione. Riavviare MATLAB con menù Ezyfit nella finestra di figura. Montare le curve MSD alla legge potere selezionando nel menu Ezyfit &#39;Mostra Fit&#39;&gt; &#39;Power&#39;&gt; &#39;a * x ^ n&#39; erano n è la stima esponente α diffusiva. NOTA: msdanalyzer richiede il Curve Fitting Toolbox in Matlab per il montaggio di curve MSD. Ezyfit è un&#39;alternativa e software gratuito in grado di eseguire curve fitting. </li><li> Cancella traiettorie delle particelle in corso e DMS per calcolare nuovi DMS particelle in altre posizioni o tempo: chiaro </li&gt; <li> Dopo il calcolo di varie curve medie MSD di particelle in media (controllo) e microcolonie e aree indifferenziate di vari ceppi, copiare e incollare le curve in un grafico per il confronto. Rigidità del campione e reticolazione aumento con valori più bassi MSD. Curve piatte hanno α inferiori e del campione è più elastica di curve più ripide con una maggiore α. </li><li> Selezionare la curva e andare in &quot;Strumenti&quot; a guardare le statistiche di dati, come ad esempio la mediana e la gamma. L&#39;MSD del tallone è proporzionale alla conformità creep, J (t) del materiale in cui il tallone è integrato secondo la relazione, <img alt="Equazione 1" src="/files/ftp_upload/53093/53093eq1.jpg" /> dove J = compliance creep, d = diametro delle particelle, k B = costante Boltzmann, T = temperatura e t = tempo. </li></ol></li></ol>

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The authors have nothing to disclose.

Microrheology è uno strumento utile per misure reologiche locali in sistemi eterogenei, come biofilm microbici. Si tratta di una tecnica non distruttiva, consentendo il monitoraggio in tempo reale dei cambiamenti reologici all&#39;interno dello stesso campione biologico su più punti di tempo. In questo protocollo,-particella monitoraggio microrheology è stato applicato alla Pel e mutanti esopolisaccaridi Psl per indagare come influenzano l&#39;elasticità ed efficace reticolazione della matrice biofilm. PSL favorisce...

La maggior parte delle cellule batteriche sono in grado di utilizzare sia (sessili) le modalità superficiali allegato di crescita 1 planctonici (senza vita) e. Nella modalità superficie divisoria di crescita, le cellule batteriche secernono e si racchiudono in grandi quantità di sostanze polimeriche extracellulari (EPS) per formare biofilm. L&#39;EPS consiste principalmente di proteine, DNA extracellulare esopolisaccaride, ed è essenziale per la formazione di biofilm 2. Serve come un&#39;impalcatura fisico da cui i batteri possono utilizzare per differenziare spazialmente e protegge i batteri da condizioni ambientali nocive e le risposte di accoglienza. Diversi componenti di EPS hanno ruoli distinti nella formazione di biofilm 3 ei cambiamenti nell&#39;espressione di componenti EPS può drammaticamente rimodellare strutture biofilm 4. Componenti EPS possono anche funzionare come molecole di segnalazione 5, e studi recenti hanno dimostrato alcuni componenti EPS interagiscono con le cellule microbiche per guidare le loro diff migrazione e biofilmerenziazione 6-8. La ricerca sulla EPS è notevolmente avanzato sulla base delle analisi morfologiche del biofilm prodotti da mutanti difettosi in un componente specifico dei EPS 9,10. Inoltre, l&#39;EPS è generalmente caratterizzato alla macro-scala (caratterizzazione bulk) 11. Morfologica analizza però può mancare dettaglio quantitativa e la caratterizzazione di massa, che restituisce i valori medi, perde dettaglio che esiste all&#39;interno della eterogeneità del biofilm. Vi è ora una tendenza crescente per avanzare al caratterizzazione in tempo reale delle proprietà meccaniche di EPS in microscala. Questo protocollo dimostra come particella-tracking microrheology è in grado di determinare gli effetti spazio-temporali delle componenti della matrice Pel e esopolisaccaridi Psl sulla viscoelasticità ed efficace reticolazione di Pseudomonas aeruginosa biofilm 4. Microrheology passiva è un rh semplice e poco costosoMetodo eology che offre il massimo throughput di campionamento microrheological spaziale di un materiale ad oggi 12,13. In microrheology passiva, sfere sonda vengono inseriti nel campione e il loro moto browniano, guidato da energie termiche (k B T) è seguita da microscopio video. Diverse le particelle possono essere monitorati contemporaneamente, e le coordinate dipendenti dal tempo delle particelle seguono un random walk convenzionale. Pertanto, in media, le particelle rimangono nella stessa posizione. Tuttavia, la deviazione standard degli spostamenti o spostamento quadratico medio (MSD) delle particelle, non è zero. Poiché scorrono fluidi viscosi, la particella MSD in un fluido viscoso cresce linearmente con il tempo progredisce. Al contrario, la reticolazione polimerica trovato in viscoelastica o sostanze elastiche aiutare loro di resistere flusso, e particelle diventa limitato nel loro spostamento, portando a plateau nella curva MSD (Figura 1A). Questa osservazione segue la relazione MSDαt <sup&gt; α, dove α è l&#39;esponente diffusiva che è rapporto tra contributi elastici e viscosi della sostanza correlata. Per le particelle si muovono in fluidi viscosi alfa = 1, a sostanze viscoelastiche 0 &lt;α &lt;1, e in sostanze elastiche α = 0. L&#39;MSD può essere utilizzato anche per calcolare l&#39;osservanza creep, che è la tendenza del materiale a deformarsi in modo permanente su tempo e stima quanto facilmente un diffonde materiali. La chimica dimensioni, la densità e la superficie della particella sono fondamentali per la corretta applicazione delle esperimento microrheological e sono scelti in relazione al sistema studiato (in questo caso i polimeri della matrice biofilm, vedere Figura 1B). In primo luogo, la particella misura la reologia della sostanza con strutture che sono molto più piccola della particella stessa. Se le strutture della sostanza sono di dimensioni simili alla particella, il moto del particolo è perturbata dalla forma e l&#39;orientamento delle singole strutture. Tuttavia, se le strutture circostanti particella sono molto più piccoli, questo effetto è piccola e media, presentando un ambiente omogeneo alla particella (Figura 1B). In secondo luogo, la densità della particella dovrebbe essere simile al mezzo (1,05 g ml -1 per mezzi acquosi) in modo che sia evitata la sedimentazione e forze inerziali sono trascurabili. La maggior parte delle particelle con grate di polistirene soddisfano i criteri di cui sopra. Idealmente, la particella non interagisce con i polimeri della matrice biofilm l&#39;interpretazione reologico MSD particella è valido solo se il movimento è casuale, guidata da energia termica e la collisione con strutture sostanza. Ciò può essere osservato controllando se la particella sonda tende a legarsi o rimbalzare la superficie di un biofilm pre-adulto. Tuttavia, nonostante la mancanza di attrazione per il biofilm, le particelle devono essere in grado di essere incorporati nella matrice.Inoltre, l&#39;eterogeneità fisiochimica del biofilm, particelle differenti che sono più adatti come sonde in diverse regioni del biofilm. Così, le particelle di diverse dimensioni e chimica di superficie dovrebbero essere applicati al biofilm. Come tale, la particella MSD è in grado di fornire utili informazioni su come i diversi componenti contribuiscono alla reologia e la diffusione del biofilm. Inoltre, l&#39;uso di diverse sonde permette di ricavare informazioni sul eterogeneità fisiochimica spaziale del biofilm. Questo metodo può essere utilizzato per testare l&#39;effetto trattamento antimicrobico sulle proprietà meccaniche del biofilm, oppure applicato a biofilm specie miste per studiare come le proprietà meccaniche del biofilm sono cambiati dall&#39;introduzione di un&#39;altra specie. Particle DMS può essere utile anche per la caratterizzazione di biofilm dispersione. Tali studi potrebbero essere utili per la nostra comprensione di biofilm, migliorando potenzialmente trattamenti biofilm unnd ingegnerizzazione di biofilm per le attività utili.

<table>
	<tbody>
		<tr>
			<td>Fluorspheres</td>
			<td>Invitrogen</td>
			<td>F-8821</td>
			<td>1.0 um red fluorescent (580/605) microspheres with carboxylate modification</td>
		</tr>
		<tr>
			<td>Zeiss Axio Imager M1</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Epifluorescent Microscope</td>
		</tr>
		<tr>
			<td>Masterflex L/S Digital Drive 07523-80</td>
			<td>Cole-Parmer</td>
			<td>EW-07523-80</td>
			<td>Peristaltic pump</td>
		</tr>
		<tr>
			<td>Flow Cell Chambers</td>
			<td colspan="3">Technical University of Denmark</td>
		</tr>
		<tr>
			<td>Bubble Trap</td>
			<td colspan="3">Technical University of Denmark</td>
		</tr>
		<tr>
			<td>Silicone Tubing</td>
			<td>Dow Corning</td>
			<td></td>
			<td>3 mm outer diameter, 1 mm inner diameter</td>
		</tr>
		<tr>
			<td>Clear polypropylene plastic connectors&#160;</td>
			<td>Cole Parmer</td>
			<td>06365-83</td>
			<td>1/16 in. (1.588 mm)</td>
		</tr>
		<tr>
			<td>Binder Clips</td>
			<td></td>
			<td></td>
			<td>To clamp tubing</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td colspan="2">Thermo Scientific&#8482; Nunc&#8482;</td>
			<td>50 x 24 mm</td>
		</tr>
		<tr>
			<td>Syringe 3 mL</td>
			<td>Terumo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>27G Needle</td>
			<td>Terumo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2L Storage/Media Bottles</td>
			<td>VWR&#174;&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trolley</td>
			<td></td>
			<td></td>
			<td>To hold biofilm setup</td>
		</tr>
	</tbody>
</table>

in situ mapping of the mechanical properties of biofilms by particle-tracking microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

Le proprietà viscoelastiche locali del biofilm nelle diverse regioni del biofilm, che comprendeva i vuoti (media sopra il biofilm), pianure (strato piatto indifferenziata di cellule) e microcolonie (vedi etichette in Figura 2A) sono stati studiati. Le variazioni temporali delle proprietà viscoelastiche del biofilm durante la maturazione da 3 a 5 giorni sono stati determinati. La MSD delle particelle nei vuoti è stato utilizzato come controllo e paragonabile al MSD di particelle in media pura. Al cont...

Watch this Scientific Journal Video about In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology at JoVE.com

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

In Situ mappatura delle proprietà meccaniche del biofilm da-Particle tracciamento Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

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Research

JoVE Journal

Bioengineering

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

9.6K Views.  Nanyang Technological University. Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Video: In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Multiparametrica mappatura ottica del Langendorff-perfuso Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Ricerca

Bioingegneria

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

Mappatura ottico dei potenziali d&#39;azione e transitori Calcio nel Cuore mouse

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

Quantificare le proprietà meccaniche del glicocalice endoteliale con microscopia a forza atomica

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Misurare le proprietà meccaniche del Living cellule utilizzando microscopia a forza atomica

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in these structures are held together by physical interactions mediated by a network of extracellular polymeric substances. Bacterial biofilms impact many human activities and the understanding of their properties is crucial for a better control of their development &#8212; maintenance or eradication &#8212; depending on their adverse or beneficial outcome. This paper describes a novel methodology aiming to measure in situ the local physical properties of the biofilm that had been, until now, examined only from a macroscopic and homogeneous material perspective. The experiment described here&#160;involves introducing magnetic particles into a growing biofilm to seed local probes that can be remotely actuated without disturbing the structural properties of the biofilm. Dedicated magnetic tweezers were developed to exert a defined force on each particle embedded in the biofilm. The setup is mounted on the stage of a microscope to enable the&#160;recording of time-lapse images of the particle-pulling period. The particle trajectories are then extracted from the pulling sequence and the local viscoelastic parameters are derived from each particle displacement curve, thereby providing the 3D-spatial distribution of the parameters. Gaining insights into the biofilm mechanical profile is essential from an engineer&#39;s point of view for biofilm control purposes but also from a fundamental perspective to clarify the relationship between the architectural properties and the specific biology of these structures.

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

This paper shows an original methodology based on the remote actuation of magnetic particles seeded in a bacterial biofilm and the development of dedicated magnetic tweezers to measure in situ the local mechanical properties of the complex living material built by micro-organisms at interfaces.

Azionamento a distanza magnetica micrometrici Sonde per<em&gt; In situ</em&gt; Mappatura 3D di biofilm batterici Proprietà fisiche

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

Investigare la separazione tridimensionale del flusso indotta da un modello di polipo piega vocale

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

Misurazione longitudinale di matrice extracellulare rigidità in 3D Tumore modelli utilizzando-particelle inseguimento Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Un<em&gt; In Vitro</em&gt; Organ Culture Modello del disco intervertebrale Murine

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the flexural behavior of these LBBSs is important for understanding the origins of their remarkable mechanical functions.
We describe a protocol for performing three-point bending tests using a custom-built mechanical testing device that can measure forces ranging from 10-5 to 101 N and displacements ranging from 10-7 to 10-2 m. The primary advantage of this mechanical testing device is that the force and displacement capacities can be easily adjusted for different LBBSs. The device&#39;s operating principle is similar to that of an atomic force microscope. Namely, force is applied to the LBBS by a load point that is attached to the end of a cantilever. The load point displacement is measured by a fiber optic displacement sensor and converted into a force using the measured cantilever stiffness. The device&#39;s force range can be adjusted by using cantilevers of different stiffnesses.
The device&#39;s capabilities are demonstrated by performing three-point bending tests on the skeletal elements of the marine sponge Euplectella aspergillum. The skeletal elements&#8212;known as spicules&#8212;are silica fibers that are approximately 50 &#181;m in diameter. We describe the procedures for calibrating the mechanical testing device, mounting the spicules on a three-point bending fixture with a &#8776;1.3 mm span, and performing a bending test. The force applied to the spicule and its deflection at the location of the applied force are measured.

We present a protocol for performing three-point bending tests on sub-millimeter scale fibers using a custom-built mechanical testing device. The device can measure forces ranging from 20 &#181;N up to 10 N and can therefore accommodate a variety of fiber sizes.

Un sistema di prova millimetro scala flessionale per misurare le proprietà meccaniche di spicole di spugna marina

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Un protocollo per il monitoraggio in tempo reale 3D singola particella

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...