Questo lavoro è stato supportato da una RO1 dal NIH e recensioni Merito del Servizio di ricerca VA a C.-MT, e un NRSA individuale a CWL

Considerazioni generali di progettazione Se la lunghezza d&#39;onda di stimolazione foto è nel campo del visibile, come per gli esperimenti optogenetic, il layout del sistema sarà notevolmente più semplice per gli esperimenti di fotolisi UV. Basta acquistare un doppio modulo porta macchina fotografica che è disponibile presso tutte le aziende microscopio. L&#39;aereo DMD può essere posizionato sul piano dell&#39;immagine sia coniugato di una delle due porte della fotocamera. Ma nella fotolisi UV la luce che proviene dalla DMD devono essere portati attraverso l&#39;epi-illuminazione del percorso (fig. 3A), perché la lente del tubo che porta l&#39;imaging per la fotocamera non è corretto per lunghezza d&#39;onda UV in uno qualsiasi dei microscopi in commercio. L&#39;aberrazione sferica della lente tubo di imaging a 350 nm è abbastanza grave da impedire la possibilità di concentrare la luce in un punto stretto e di generare sufficiente risoluzione spaziale. Questo problema non si riscontra in microscopia confocale perché la lente del tubo viene rimosso quando un raggio laser collimato viene portato in lungo lo stesso asse ottico. Il problema di illuminazione UV può essere risolto da portare alla luce attraverso l&#39;epi-fluorescenza percorso perché le lenti del tubo ci sono tutti parzialmente corretto per l&#39;aberrazione sferica alla lunghezza d&#39;onda UV. Siamo in grado di espandere l&#39;ottica dei microscopi a qualsiasi grado i desideri rivista. La comunità scientifica è in generale interessato veloce in questo argomento e non ci sono molte fonti che insegna bene. Al fine di minimizzare le modifiche del microscopio ed a causa di vincoli di spazio stretto in epi-fluorescenza unità, il DMD è posto a un aereo di nuova creazione immagine coniugato creata da una lente relé (indicare la posizione dell&#39;unità DMD in relazione alle microscopio). Un UV commerciale corretto lente relè è stato acquistato (Ottica Specialità) (punto nel diagramma e sul microscopio). L&#39;illuminazione del DMD Dopo aver ricevuto il suo input binario uno microspecchio può passare da uno positivo e uno negativo 12 ° di inclinazione rispetto al piano del chip. L&#39;asse di rotazione si trova lungo la diagonale gli angoli di ogni specchio (45 ° rispetto ai lati del chip). L&#39;orientamento della rotazione dello specchio è designato da un triangolo color oro in un angolo sulla superficie anteriore del chip. L&#39;angolo di inclinazione dei microspecchi dettami l&#39;allineamento del campo e azithmus del fascio di luce in entrata. Il passo del raggio di illuminazione deve essere di 24 ° rispetto all&#39;asse perpendicolare del chip DMD e azithmus deve essere perpendicolare all&#39;asse di rotazione dello specchio. Allineamento del fascio preciso è fondamentale per il funzionamento efficiente. Nel sistema prototipo abbiamo progettato diverse regolazioni di inclinazione manuale che ci ha permesso di correggere le carenze di progettazione e lavorazione. Il risultato è un sistema che è molto più ingombrante di quanto sia necessario. Alternato, soluzione più compatta per portare alla luce è possibile (mostra di design alternativo se ritenuto utile). Per gli esperimenti fotolisi una sorgente laser è necessaria per generare l&#39;alta intensità del fascio di luce concentrata necessari per uncaging rapida. Per gli esperimenti optogenetic dove la luce ad alta intensità nettamente focalizzata non è richiesto, un non-coerente, sorgente di luce sarebbero adeguati. Nell&#39;esperimento fotolisi descritto qui abbiamo impiegato un quasi-continua pompato a diodi a stato solido (DPSS) frequenza triplicata NdVO4 laser (1 W, 355 nm). (Per una discussione dettagliata delle scelte delle fonti di luce per la fotolisi vedi riferimento 2). Un laser di potenza relativamente alta è necessaria nel esperimenti descritti qui perché solo una piccola frazione della potenza laser viene effettivamente consegnato al campione quando si utilizza un sistema di DMD. La quantità di luce consegnato al campione è proporzionale al rapporto tra il numero di ON / OFF specchi all&#39;interno della regione illuminata dal fascio laser. Se una sorgente di luce laser è richiesto, è meglio per lanciare l&#39;uscita del laser in una fibra multimodale in modo che possa essere facilmente posizione e orientato lungo l&#39;asse corretto per l&#39;illuminazione della DMD. La trasmissione della luce attraverso la fibra ottica risolve un secondo problema, come eliminare l&#39;speckle pattern inerenti all&#39;illuminazione coerente. La fibra è avvolto attorno ad una barella fibra piezoelettrico (modello 915, Strumentazione canadese e Ricerca, Ltd) che oscilla a ~ 40 kHz. (Punto in tal senso nel rig) Il microscopico allungamento della fibra è sufficiente per spostare il speckle modello molte volte durante la durata millisecondi di ogni impulso stimolazione foto, così eliminando efficacemente l&#39;effetto di screziature. L&#39;uscita della fibra ottica è poi collimato con un obiettivo microscopio UV (Olympus DApo20UV). (Notare questo) Una calibrazione della risoluzione ottica del sistema è mostrato in fig. 3B. La risoluzione effettiva fisiologica misurata dalla ampiezza del flusso di corrente in funzione della posizione del punto di fotostimolazione è mostrato in fig. 3C. Attuali risposte fotostimolazione di diversa intensità sono illustrati nella fig. 3D. Funzionamento del sistema: Co-registrazione del pixel del CCD con gli specchi DMD Un in-house software è stato scritto che determina la corrispondenza dei singoli specchi DMD ai pixel specifiche della macchina fotografica di immagini CCD. L&#39;interfaccia utente grafica (GUI) all&#39;interno di questo software permette poi all&#39;utente di assegnare gli specchi DMD che corrisponde alla regione l&#39;immagine CCD tag con il mouse. Così, il luogo per la stimolazione foto può essere etichettato semplicemente spostando il cursore sopra l&#39;immagine visualizzata sullo schermo del computer e cliccando la regione tag di interesse. (Una immagine fluorescente di un dendritiche pergolato sarà visualizzato sullo schermo del computer. Student segnerà un certo numero di posizioni sopra l&#39;immagine sullo schermo del computer.) Programmazione dei modelli per la consegna della luce Il modello segnato sullo schermo del computer da parte dell&#39;operatore è memorizzato come una serie di immagini separate. La tempistica degli impulsi laser per ogni modello spaziale è poi programmato il software che si integra con il programma di acquisizione dati (pClamp). (Lo dimostrano). Coordinare gli impulsi laser per i modelli di DMD Il programma di acquisizione dati avvia e coordina i tempi dell&#39;elettronica DMD, il gating del laser, e l&#39;acquisizione della patch bloccato segnale elettrico dal neurone bersaglio. (Simulare questa sequenza sul computer) I dati sperimentali: Non lineare sommatoria tra distale punti di ramificazione dendritica Integrazione dendritiche con elettrodi è stata tradizionalmente eseguite variando l&#39;ampiezza di stimolazione in luoghi distinti, piuttosto che ampliare l&#39;area di stimolazione pur mantenendo una intensità costante. Le questioni relative alla saturazione dei elettrogenesi e il reclutamento di canali posizione dipendente può influenzare il risultato e la conclusione. Distribuito stimolazione dendritiche possono essere facilmente implementata utilizzando un sistema basato DMD (Fig. 4A). Intensità di input è stato variato aumentando il numero di macchie di fotostimolazione. Possiamo vedere che la sommatoria spaziale può essere non lineare attraverso punti di ramificazione sempre più sovra-lineare con ampiezze di input che arrivano sempre più sui due rami figlia. Il sovra-linearità è in gran parte mediata recettori NMDA e non alla tensione di canali gated (Fig. 5) Rappresentante Risultati <img src="/files/ftp_upload/2003/2003fig1.jpg" alt="Figura 1" /> Figura 1. Funzionamento dei microspecchi nella DMD. (Vista laterale) forze elettrostatiche dirette a singoli specchi causa allo specchio per essere inclinato in una delle due possibili orientazioni 12 ° rispetto al piano orizzontale. In posizione ON un fascio incidente è diretto lungo l&#39;asse ottico perpendicolare del chip. In posizione OFF il fascio incidente è diretto fuori asse 48 °. Luce che colpisce il divario sottile tra gli specchi è diretto 24 ° fuori asse. (Vista dall&#39;alto), l&#39;inclinazione è orientata a 45 ° rispetto ai lati degli specchi e di chip. <img src="/files/ftp_upload/2003/2003fig2.jpg" alt="Figura 2" /> Figura 2. Layout di base del microscopio a fluorescenza moderna. Il cubo filtro è posizionato nel &quot;spazio infinito&quot; tra l&#39;obiettivo e la lente del tubo. C&#39;è un percorso di immagini che conduce alla fotocamera e vi è un percorso di illuminazione che porta la luce al campione. La lente del tubo per il percorso di questi due leggeri sono tipicamente differenti nel design e nella lunghezza focale. L&#39;illuminazione, ma non l&#39;immagine, lente del tubo è progettato per il funzionamento nello spettro UV. <img src="/files/ftp_upload/2003/2003fig3.jpg" alt="Figura 3" /> Figura 3. Layout per UV sistema basato DMD. Se l&#39;illuminazione è nello spettro UV, allora deve essere portato attraverso la lente del tubo che è stato corretto per UV. La lente del tubo nel percorso di imaging non è progettato per UV. Un sistema di relè è necessario per creare un facile accesso piano dell&#39;immagine coniugato. I diversi piani di coniugare immagine nel microscopio è caratterizzata da linee tratteggiate rosse. Modelli luminosi in un unico piano dell&#39;immagine vengono proiettate su piani coniugato altri. Durante l&#39;imaging lo schema del piano di campionamento è proiettata sul sensore / fotocamera. Per l&#39;illuminazione del modello generato dalla DMD è proiettata sul campione. <img src="/files/ftp_upload/2003/2003fig4.jpg" alt="Figura 4" /> Figura 4. Layout per la luce visibile sistema di proiezione DMD. Se la luce illuminazione è nel campo del visibile, è possibile portare il modello DMD luce generata attraverso il percorso della luce di imaging. Questo può essere facilmente implementato con commerciAl doppia fotocamera allegati porta e lo specchio dicroico appropriato. <img src="/files/ftp_upload/2003/2003fig5.jpg" alt="Figura 5" /> Figura 5. Risoluzione laterale del sistema. (A) Risoluzione ottica sul target fluorescente è ~ 2 micron. (B) La risoluzione effettiva, misurata dalla fotolisi di glutammato ingabbiato in una dendrite è ~ 5 micron. La distanza è aumentata a causa di una combinazione di diffusione di glutammato e la larghezza finita della dendrite. (C) Fotolisi in grado di simulare la cinetica di eventi sinaptici. La famiglia di tensione bloccato attuali risposte è dovuto alle variazioni di energia luminosa consegnato al dendrite. <img src="/files/ftp_upload/2003/2003fig6.jpg" alt="Figura 6" /> Figura 6. Sommatoria non lineare attraverso punti di ramificazione dendritica. (A) gli ingressi distribuiti sono applicati a due rami dendritici separatamente. Le loro risposte rispettivi tensione sono riportati di seguito. (B) Gli stimoli vengono poi somministrati contemporaneamente. La risposta misurata (traccia rossa) è diversa dalla somma aritmetica delle due risposte individuali (grigio traccia). <img src="/files/ftp_upload/2003/2003fig7.jpg" alt="Figura 7" /> Sommatoria figura 7. Sopralineare è recettore NMDA dipendente. (A) APV, un antagonista selettivo del recettore NMDA blocca la sommatoria sopralineare. (B) Il rapporto intensità dello stimolo e la sua sensibilità di antagonista del recettore NMDA è tracciata.

<ol>
	<li>Scanziani, M., Hausser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiology+in+the+age+of+light.">Electrophysiology in the age of light.</a> Nature. 461, 930-939 (2009).</li><li>Tang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photolysis+of+caged+neurotransmitters:+Theory+and+procedures+for+light+delivery.">Photolysis of caged neurotransmitters: Theory and procedures for light delivery.</a> Curr. Prot. Neurosci. , 6.21.1-6.21.12 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+Technology+Feature,+Cell+imaging:+Light+activated.">Nature Technology Feature, Cell imaging: Light activated.</a> Nature. 456, 826-827 (2008).</li><li>Lutz, C., Otis, T. S., DeSars, V., Charpak, S., DeGregorio, D. A., Emiliani, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Holographic+photolysis+of+caged+neurotransmitters.">Holographic photolysis of caged neurotransmitters.</a> Nature Methods. 5, 821-827 (2008).</li><li>Hornbeck, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Digital Light Processing and MEMs: An overview. , .</li></ol>

Nessun conflitto di interessi dichiarati.

Il vantaggio di DMD approccio basato fotostimolazione è più evidente nelle situazioni in cui il target occupa un&#39;area relativamente grande. Se il bersaglio di interesse è molto piccolo, come un paio di spine dendritiche, sistemi di scansione sequenziale confocale e 2-fotoni possono essere l&#39;approccio migliore. Un punto debole significativo dell&#39;approccio DMD è il suo uso inefficiente della luce disponibile. La maggior parte della luce disponibile è necessariamente rivolta al specchi OFF e non utilizzati...

Erratum: Patterned Photostimulation with Digital Micromirror Devices to Investigate Dendritic Integration Across Branch Points

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<td>Modern upright fluorescent microscope</td>
<td>&nbsp;</td>
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<td>CCD camera and image acquisition software</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Computer and data acquisition/interface system</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>DLP Discovery Developer Kit </td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>ALP3 USB interface</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>S2 + Optics w/LED</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Dual camera port unit</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>355nm frequency tripled NdVO4 laser (~1 W)</td>
<td>&nbsp;</td>
<td>DPSS Laser Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Laser shutter Model LS6</td>
<td>&nbsp;</td>
<td>Uniblitz</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Multimode optical fiber and fiber stretcher Model# 915</td>
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<td>Canadian Instrument and Research, Ltd </td>
<td>&nbsp;</td>
<td>100 um core multimode fiber</td>
</tr>
<tr>
<td>Multimode Fiber launcher </td>
<td>&nbsp;</td>
<td>Oz Optics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Signal generator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>up to 50 kHz</td>
</tr>
<tr>
<td>Beam collimator</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>DApo20UV340</td>
<td>&nbsp;</td>
<tr>
<td>UV relay lens</td>
<td>&nbsp;</td>
<td>Special Optics</td>
<td>#: 54-25-60-355</td>
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patterned photostimulation with digital micromirror devices to investigate dendritic integration across branch points

Light is a versatile and precise means to control neuronal excitability. The recent introduction of light sensitive effectors such as channel-rhodopsin and caged neurotransmitters have led to interests in developing better means to control patterns of light in space and time that are useful for experimental neuroscience. One conventional strategy, employed in confocal and 2-photon microscopy, is to focus light to a diffraction limited spot and then scan that single spot sequentially over the region of interest. This approach becomes problematic if large areas have to be stimulated within a brief time window, a problem more applicable to photostimulation than for imaging. An alternate strategy is to project the complete spatial pattern on the target with the aid of a digital micromirror device (DMD). The DMD approach is appealing because the hardware components are relatively inexpensive and is supported by commercial interests. Because such a system is not available for upright microscopes, we will discuss the critical issues in the construction and operations of such a DMD system. Even though we will be primarily describing the construction of the system for UV photolysis, the modifications for building the much simpler visible light system for optogenetic experiments will also be provided. The UV photolysis system was used to carryout experiments to study a fundamental question in neuroscience, how are spatially distributed inputs integrated across distal dendritic branch points. The results suggest that integration can be non-linear across branch points and the supralinearity is largely mediated by NMDA receptors.

Watch this Scientific Journal Video about Patterned Photostimulation with Digital Micromirror Devices to Investigate Dendritic Integration Across Branch Points at JoVE.com

Patterned Photostimulation with Digital Micromirror Devices to Investigate Dendritic Integration Across Branch Points

Dispositivi digitali micromirror (DMD) in grado di generare modelli complessi nel tempo e nello spazio con cui controllare l&#39;eccitabilità neuronale. Questioni rilevanti per la progettazione, costruzione e funzionamento dei sistemi di DMD sono discussi. Tale sistema ha permesso la dimostrazione di non-lineare integrazione tra distale punti di ramificazione dendritica.

Fotostimolazione modellato con i dispositivi digitali Micromirror per Indagare integrazione dendritiche Attraverso punti di ramificazione

Light is a versatile and precise means to control neuronal excitability. The recent introduction of light sensitive effectors such as channel-rhodopsin ...

patterned-photostimulation-with-digital-micromirror-devices-to

Research

JoVE Journal

Bioengineering

15.6K Views.  University of Maryland School of Medicine. Dispositivi digitali micromirror (DMD) in grado di generare modelli complessi nel tempo e nello spazio con cui controllare l'eccitabilità neuronale. Questioni rilevanti per la progettazione, costruzione e funzionamento dei sistemi di DMD sono discussi. Tale sistema ha permesso la dimostrazione di non-lineare integrazione tra distale punti di ramificazione dendritica.

Video: Patterned Photostimulation with Digital Micromirror Devices to Investigate Dendritic Integration Across Branch Points

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Progettazione di un bioreattore per la pressione ciclica Ex Vivo Studio delle valvole cardiache aortica

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Ricerca

Bioingegneria

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

Modelli e metodi per valutare il trasporto di Drug Delivery Systems attraverso le barriere cellulari

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment2.
Coagulation Factor VIII (FVIII)3 is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A&#160;-&#160;a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting4. Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete5. Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa6,7.
In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.&#160;The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.

We present a combination of Cryo-electron microscopy, lipid nanotechnology, and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory&#160;to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.

Elicoidale Organization of Blood fattore VIII della coagulazione su Lipid nanotubi

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Planar gradiente di diffusione del sistema per Indagare Chemiotassi in un 3D collagene Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

Realizzazione di un funzionalizzato magnetico batterica Nanocellulose con nanoparticelle di Ossido di Ferro

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Fabricating Cotton Analytical Devices

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and provide information for follow-up treatment. Here, we demonstrate the development of a cotton-based urinalysis (i.e., nitrite, total protein, and urobilinogen assays) analytical device that employs a lateral flow-based format, and is inexpensive, easily fabricated, rapid, and can be used to conduct multiple tests without cross-contamination worries. Cotton is composed of cellulose fibers with natural absorptive properties that can be leveraged for flow-based analysis. The simple but elegant fabrication process of our cotton-based analytical device is described in this study. The arrangement of the cotton structure and test pad takes advantage of the hydrophobicity and absorptive strength of each material. Because of these physical characteristics, colorimetric results can persistently adhere to the test pad. This device enables physicians to receive clinical information in a timely manner and shows great potential as a tool for early intervention.

To investigate simple fabrication approaches for multiple assay needs, we created a fluid-absorbing channel system made of cotton material. This device was used to establish a multiple detection platform, and solve contamination issues that commonly affect lateral flow-based biomedical devices, for clinical urinalysis of nitrite, total protein, and urobilinogen.

Fabbricazione di dispositivi analitici cotone

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

Utilizzando adesivo Patterning per costruire dispositivi microfluidici di carta 3D

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

Uno Sperimentale e Finite Element protocollo per indagare il trasporto di soluti neutri e carichi attraverso cartilagine articolare

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

Creazione di tessuto cardiaco che mostra l&#39;integrazione meccanica degli spheroidi usando la biopittura 3D

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable microfluidic devices. Here, a protocol for the fabrication of plastic microfluidic devices is demonstrated, where soft lithographic techniques are used to embed transparent Calcium Fluoride (CaF2) view-ports in connection with observation chamber(s). The method is based on a replica casting approach, where a polydimethylsiloxane (PDMS) mold is produced through standard lithographic procedures and then used as the template to produce a plastic device. The plastic device features ultraviolet/visible/infrared (UV/Vis/IR) -transparent windows made of CaF2 to allow for direct observation with visible and IR light. The advantages of the proposed method include: a reduced need for accessing a clean room micro-fabrication facility, multiple view-ports, an easy and versatile connection to an external pumping system through the plastic body, flexibility of the design, e.g., open/closed channels configuration, and the possibility to add sophisticated features such as nanoporous membranes.

A protocol for the fabrication of plastic microfluidic devices with transparent view-ports for visible and infrared light imaging is described.

Procedura litografica morbido per la produzione di dispositivi microfluidici plastica con vista-porte trasparente alla luce visibile e infrarossa

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable ...