The financial support by DFG (German research foundation, project CZ 55/30-1) for parts of this work is gratefully acknowledged.

1. Montaggio del setup sperimentale <ol><li> Preparazione della parte prossimale <ol><li> Posizionare e fissare il laser che emette un fascio di luce collimato - o utilizzare un laser accoppiato in fibra ottica con collimazione alla sfaccettatura all&#39;uscita della fibra. </li><li> Mettere il divisore di fascio polarizzante (PBS) per dividere il raggio laser in riferimento e fascio oggetto. Ruotare l&#39;orientamento delle piastre semionda (HWP) ruotando il HWP nella sua rotazione montare finché l&#39;alimentazione del fascio di riferimento e fascio oggetto (sul lato distale) è circa la stessa. Controllare questo mettendo uno schermo in entrambi riferimento e fascio oggetto. Scegliere l&#39;orientamento della PBS in modo che la polarizzazione del fascio di riferimento si inserisce il modulatore spaziale di luce polarizzazione-sensitive (SLM). </li><li> Mettere un divisore di fascio (BS) nel fascio di riferimento per dividere il fascio di riferimento in due fasci. Posizionare i modulatori ottici (OM) tali che questi due fasci provenienti da BS1 possono passare OM1 e OM2, rispettivamente. &lt;/ Li&gt; <li> Unire i due fasci di passaggio OM1 e OM2 a BS2 impiegando due specchi. Regolare i separatori di fasci e specchi in modo che entrambi i fasci sono spazialmente separate. </li><li> allineare attentamente BS5 per assicurare che la direzione di incidenza di entrambi i fasci è perpendicolare al piano pixel del SLM, BS3 ignorando e BS4 all&#39;inizio. In un primo momento, non viene visualizzato sul SLM, cioè, agisce come uno specchio fino al termine della taratura (durante il passaggio 2). </li><li> Regolare la posizione e la distanza tra le due lenti (L) costituente un telescopio kepleriano per ottenere un&#39;immagine nitida del piano SLM sulla telecamera Complementary Metal-Oxide Semiconductor (CMOS). Osservare il corretto orientamento di L1 e L2 (facce piane di fronte all&#39;altra) per minimizzare le aberrazioni. </li></ol></li><li> Preparazione della parte distale <ol><li> Utilizzare BS7 per dividere il fascio oggetto in due fasci e combinarle a BS8 impiegando due specchi. Anche in questo caso, regolare i separatori di fasci e mirrors modo che entrambi i fasci sono spazialmente separate. </li><li> Deviare entrambe le travi utilizzando BS9 loro mirare all&#39;obiettivo del microscopio (OBJ). Fuoco OBJ2 sull&#39;estremità distale della fibra multimodale (MMF). Controllare la messa a fuoco osservando il riflesso indietro dal MMF impiegando L3 e un dispositivo di fotocamera ad accoppiamento di carica (CCD). </li></ol></li><li> Prossimale Collegamento e parte distale <ol><li> Collimare la luce dal fascio oggetto uscita dal MMF impiegando OBJ1. </li><li> Dividere il fascio oggetto utilizzando BS6, ignorare il polarizzatore lineare (LP) in un primo momento. Combinare entrambi i fasci di oggetto con entrambi i fasci di riferimento a BS3 e BS4 impiegano uno specchio. Regolare i divisori di fascio e specchi in modo che ciascuna coppia di riferimento e la sovrapposizione fascio oggetto allo SLM, intersecante con un piccolo angolo (meno di 1 °). </li><li> Assicurarsi che la potenza di riferimento e la trave oggetto sono all&#39;incirca uguali ruotando l&#39;orientamento del HWP, secondo la fase 1.1.2. </li><li> Controllare l&#39;interferenza pattern (fuori asse ologramma) alla telecamera CMOS e regolare l&#39;angolo di intersezione di conseguenza. Aumentare l&#39;angolo, fino a quando la distanza interferenza frangia è uguale o meno le dimensioni di due pixel della fotocamera CMOS. </li><li> Regolare l&#39;orientamento del LP per abbinare la polarizzazione del fascio di riferimento dell&#39;oggetto e per ottenere un contrasto massimo della figura di interferenza immagine della telecamera CMOS, in modo che l&#39;immagine della telecamera mostra frange distinti. </li></ol></li></ol> 2. Calibrazione del sistema <ol><li> Calibrazione della relazione pixel tra SLM e CMOS <ol><li> Illuminare tutta SLM utilizzando solo uno dei fasci di riferimento e bloccare l&#39;altro riferimento e oggetti travi. </li><li> Acquisire un&#39;immagine SLM con la fotocamera CMOS. </li><li> Ottenere le coordinate dell&#39;angolo superiore sinistro della SLM immagine della telecamera CMOS, ad esempio, utilizzando software di grafica e il cursore del mouse sul PC. Utilizzare queste coordinate pixel come il punto di origineper quanto riguarda l&#39;SLM. </li><li> Rimuovere tutti i blocchi del fascio. </li></ol></li><li> Calibrazione dei percorsi di segnale <ol><li> Bloccare entrambi riferimento fascio 2 e l&#39;oggetto del fascio 2. </li><li> Acquisire un&#39;immagine dell&#39;ologramma con la fotocamera CMOS. Valutare la fase nell&#39;ologramma registrati utilizzando il metodo dello spettro angolare 19. Calcolare la fase invertita nella regione corrispondente trave 1. </li><li> Rimuovere i blocchi ex fascio e ora bloccare sia di riferimento trave 1 e oggetto trave 1. </li><li> Acquisire un&#39;immagine dell&#39;ologramma con la fotocamera CMOS. Misurare la fase nell&#39;ologramma registrato con nuovo metodo spettro angolare. Calcolare la fase invertita al corrispondente regione del fascio 2. </li><li> Rimuovere tutti i blocchi del fascio. </li></ol></li></ol> 3. trasmettere i segnali <ol><li> Bloccare il fascio oggetto. </li><li> Unire le immagini di fase invertite calcolati ai corrispondenti regioni del fascio 1 e 2 insieme e visualizzare ilintera immagine sullo SLM, tipicamente utilizzando la porta grafica computerizzata. </li><li> Avviare la modulazione dei segnali di ingresso 1 e 2 attivando OM1 e OM2. </li><li> Osservare i segnali di uscita 1 e 2 sulla fotocamera CCD. </li></ol>

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The authors have nothing to disclose.

Il montaggio del setup sperimentale (passaggio 1 nel protocollo) richiede un allineamento accurato dei componenti ottici rispetto all&#39;altro. L&#39;aspetto più importante è l&#39;incidenza rettangolare delle travi di riferimento e la SLM, al fine di garantire un elevato PBR. Al fine di migliorare la configurazione a più di due segnali trasmessi, separatori di fasci aggiuntivi potrebbero essere utilizzati. In alternativa, una implementazione a base di fibre sarebbe più compatta e robus...

La trasmissione di più segnali di luce attraverso una fibra multimodale (MMF) è evidente nelle comunicazioni di ingegneria 1 e 2 biofotonica. In Ingegneria delle telecomunicazioni, spazio-division multiplexing (SDM) si crede di essere una soluzione praticabile al fine di migliorare la capacità di trasmissione delle fibre ottiche per le future applicazioni di trasferimento dati che beneficiano di un maggiore utilizzo dello spazio limitato, rispetto a più fibre monomodali 3. In biofotonica, campioni biologici sono manipolati trasmettendo la luce attraverso un endoscopio MMF 4. Ad esempio, il controllo ottico indipendente dei singoli neuroni utilizzando endoscopi MMF è di interesse per optogenetics per studiare reti neuronali nel cervello 5. Tuttavia, la luce proiettata sulla sfaccettatura di ingresso MMF è soggetto a distorsione dovuta alla modalità di miscelazione e dispersione durante la propagazione al output aspetto del MMF. Come risultato, la propagazione della luce viene alterata, che rende difficile la trasmissione del segnale. Wavefront metodi di formatura 6, 7 sono applicati in dispersione media utilizzando modulatori spaziali di luce (SLM) e consentire la compensazione per la distorsione a causa di dispersione durante la propagazione della luce 8. Ci sono approcci iterativi che ottimizzano l&#39;uscita utilizzando un feedback ottico 9. Questi approcci sono piuttosto tempo a causa della necessità per numerose iterazioni e l&#39;alto grado di libertà, corrispondente ad un gran numero di elementi modulatori. Un altro approccio è quello di determinare completamente i distorsione ai MMF descritto dal suo matrice di trasmissione 10. Se il numero di modalità da trasmettere è grande, questo sarà tempo pure. In contrasto, digitale coniugazione di fase ottica (DOPC) è consideratoveloce e vantaggioso qui, dal momento che solo pochi punti focali devono essere generata nel sfaccettatura uscita del MMF. Approcci coniugazione di fase sono stati dimostrati per focalizzare o immagini attraverso tessuto biologico 12, 13, 14. Finora, DOPC stato impiegato per un singolo segnale volta solo 15, 16, ed è stato applicato per la trasmissione della luce attraverso un MMF 17. Un approccio DOPC per molteplici segnali indipendenti non è stato realizzato. Abbiamo sviluppato un metodo DOPC migliorata fornendo la trasmissione indipendente di più segnali luminosi utilizzando wavefront individuale sagomatura per ciascun segnale impiegando un singolo di sola fase SLM 18. A questo scopo, il SLM è segmentata in regioni, uno per ogni segnale da trasmettere. L&#39;apparato sperimentale proposto è mostrata in figura 1, Dove una calibrazione viene eseguita in a) prima della trasmissione effettiva accade in b). <img alt="Figura 1" src="/files/ftp_upload/55407/55407fig1.jpg" /> Figura 1: configurazione sperimentale. BS = divisore di fascio, CCD = dispositivo ad accoppiamento di carica, OM = modulatore ottico, CMOS = Complementary Metal-Oxide Semiconductor, HWP = metà piatto dell&#39;onda, L = lente, LP = polarizzatore lineare, MMF = fibra multimodale, obiettivo OBJ = microscopio, PBS = polarizzazione divisore di fascio, SLM = modulatore spaziale di luce (fase unica) - solo travi rilevanti per (a) la calibrazione e (b) la trasmissione sono raffigurati <a href="http://ecsource.jove.com/files/ftp_upload/55407/55407fig1large.jpg" target="_blank">cliccate qui per vedere una versione più grande di questa figura.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>spatial light modulator</td>
			<td>Holoeye</td>
			<td>PLUTO&#8208;VIS&#8208;016</td>
			<td></td>
		</tr>
		<tr>
			<td>CMOS camera</td>
			<td>Mikrotron</td>
			<td>MC4082</td>
			<td></td>
		</tr>
		<tr>
			<td>diode&#8208;pumped solid state laser</td>
			<td>Laser Quantum</td>
			<td>torus 532</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>IDS</td>
			<td>U3&#8208;3482LE&#8208;M</td>
			<td>CMOS camera; suitable as well</td>
		</tr>
		<tr>
			<td>lens 1</td>
			<td>Qioptiq</td>
			<td>G063204000</td>
			<td></td>
		</tr>
		<tr>
			<td>lens 2</td>
			<td>Qioptiq</td>
			<td>G063203000</td>
			<td></td>
		</tr>
		<tr>
			<td>lens 3</td>
			<td>Thorlabs</td>
			<td>AC508&#8208;180&#8208;A&#8208;ML</td>
			<td></td>
		</tr>
		<tr>
			<td>multimode fiber</td>
			<td>Thorlabs</td>
			<td>M14L02</td>
			<td></td>
		</tr>
		<tr>
			<td>beam splitters</td>
			<td>Thorlabs</td>
			<td>BS013</td>
			<td>9x</td>
		</tr>
		<tr>
			<td>polarizing beam splitters</td>
			<td>Thorlabs</td>
			<td>PBS251</td>
			<td></td>
		</tr>
		<tr>
			<td>mirrors</td>
			<td>Thorlabs</td>
			<td>PF10&#8208;03&#8208;P01</td>
			<td>5x</td>
		</tr>
		<tr>
			<td>microscope objectives</td>
			<td>Thorlabs</td>
			<td>RMS20X</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>half wave plates</td>
			<td>Thorlabs</td>
			<td>WPH10M&#8208;532</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>linear polarizer</td>
			<td>Thorlabs</td>
			<td>LPVISB050&#8208;MP2</td>
			<td></td>
		</tr>
		<tr>
			<td>optical modulators</td>
			<td>Thorlabs</td>
			<td>MC2000B&#8208;EC</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>linear and rotation stage for CMOS camera</td>
			<td>Thorlabs</td>
			<td>XYR1/M</td>
			<td></td>
		</tr>
		<tr>
			<td>fiber connector</td>
			<td>Thorlabs</td>
			<td>S120&#8208;SMA</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>reducing ring for microscope objectives</td>
			<td>Qioptiq</td>
			<td>G061621000</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>xy adjustment for objective adapters</td>
			<td>Qioptiq</td>
			<td>G061025000</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>z translation mount for fiber adapter</td>
			<td>Thorlabs</td>
			<td>SM1Z</td>
			<td>2x</td>
		</tr>
		<tr>
			<td>rods for fiber alignment to objectives</td>
			<td>Qioptiq</td>
			<td>G061210000</td>
			<td>8x</td>
		</tr>
		<tr>
			<td>mounts for lenses 1 and 2 plus two phantom mounts</td>
			<td>Qioptiq</td>
			<td>G061047000</td>
			<td>4x</td>
		</tr>
		<tr>
			<td>rail carriers for objective and lens mounts</td>
			<td>Qioptiq</td>
			<td>G061372000</td>
			<td>6x</td>
		</tr>
		<tr>
			<td>rail for rail carriers</td>
			<td>Qioptiq</td>
			<td>G061359000</td>
			<td>2x</td>
		</tr>
		<tr>
			<td colspan="2">adapter for CCD camera to 1 post</td>
			<td></td>
			<td>in-house</td>
		</tr>
		<tr>
			<td>adapter for laser to 4 posts</td>
			<td></td>
			<td></td>
			<td>in-house</td>
		</tr>
		<tr>
			<td>mount for lens 3</td>
			<td>Thorlabs</td>
			<td>LMR2/M</td>
			<td></td>
		</tr>
		<tr>
			<td>mounts for half wave plates</td>
			<td>Thorlabs</td>
			<td>RSP1D/M</td>
			<td>2</td>
		</tr>
		<tr>
			<td>mounts for mirrors</td>
			<td>Thorlabs</td>
			<td>KM100</td>
			<td>5x</td>
		</tr>
		<tr>
			<td>mount for linear polarizer</td>
			<td>Thorlabs</td>
			<td>RSP05/M</td>
			<td></td>
		</tr>
		<tr>
			<td>mounts for beam splitters and SLM</td>
			<td>Thorlabs</td>
			<td>KM100PM/M</td>
			<td>11x</td>
		</tr>
		<tr>
			<td>clamping arms for beam splitters and SLM</td>
			<td>Thorlabs</td>
			<td>PM4/M</td>
			<td>11x</td>
		</tr>
		<tr>
			<td>posts for mounts, rail carriers and adapters</td>
			<td>Thorlabs</td>
			<td>TR75/M</td>
			<td>29x</td>
		</tr>
		<tr>
			<td>holders for posts</td>
			<td>Thorlabs</td>
			<td>PH50/M</td>
			<td>29x</td>
		</tr>
		<tr>
			<td>pedestals for holders</td>
			<td>Thorlabs</td>
			<td>BE1/M</td>
			<td>29x</td>
		</tr>
		<tr>
			<td>clamping forks for pedestals</td>
			<td>Thorlabs</td>
			<td>CF125</td>
			<td>29x</td>
		</tr>
	</tbody>
</table>

transmission of multiple signals through an optical fiber using wavefront shaping

La trasmissione di più segnali ottici indipendenti attraverso una fibra multimodale viene eseguita mediante formatura fronte d&#39;onda al fine di compensare la distorsione della luce durante la propagazione all&#39;interno della fibra. La nostra metodologia si basa su digitale coniugazione di fase ottica utilizzando solo un singolo modulatore spaziale di luce, in cui il fronte d&#39;onda ottica è modulata individualmente in diverse regioni del modulatore, una regione al segnale luminoso. approcci coniugazione di fase ottica digitali sono considerati più veloce di altri wavefront modellatura approcci, dove (per esempio) viene eseguita una determinazione completa del comportamento di propagazione dell&#39;onda della fibra. Al contrario, il metodo presentato è tempo efficiente poiché richiede soltanto una calibrazione per ogni segnale luminoso. Il metodo proposto è potenzialmente appropriato per spaziale multiplexing divisione in Ingegneria delle telecomunicazioni. Ulteriori campi di applicazione sono la consegna luce endoscopica in biofotonica, in particolare in optogenetics, dove le cellule singole in tessuto biologico devono essere selettivamente illuminati con alta risoluzione spaziale e temporale.

<html> Segnali di uscita Tipici a lato distale della fibra 2 m di lunghezza sono descritte nella Figura 2. Si noti che il punto focale desiderata (picco) è accompagnato da un speckle indesiderato (background), che è dovuto all&#39;imperfezione della DOPC come questione di principio. Il corrispondente rapporto picco-a-fondo (PBR) è pari a 53 (solo il segnale 1 è &#39;on&#39;), 36 (solo segnale 2 è &#39;on&#39;) e 20 (entrambi i segnali 1 e 2 sono &#39;on&#39;) qui, ri...

Watch this Scientific Journal Video about Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping at JoVE.com

Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping

We demonstrate the transmission of multiple independent signals through a multimode fiber using wavefront shaping employing a single spatial light modulator. By modulating the wavefront for each signal individually, spatially separated foci are transmitted. Potential applications are multiplexed data transfer in communications engineering and endoscopic light delivery in biophotonics.

La trasmissione di segnali multipli attraverso una fibra ottica Utilizzando Wavefront Shaping

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the ...

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9.8K Views.  TU Dresden. We demonstrate the transmission of multiple independent signals through a multimode fiber using wavefront shaping employing a single spatial light modulator. By modulating the wavefront for each signal individually, spatially separated foci are transmitted. Potential applications are multiplexed data transfer in communications engineering and endoscopic light delivery in biophotonics.

Video: Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Tridimensionale con risoluzione ottica Microscopia fotoacustico

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

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Bioingegneria

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13 Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2 Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

Collegamento sonde biologiche ai biosensori ottici che utilizza silice agenti di accoppiamento silanici

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from regulation of cell migration and proliferation, to secretion and even differentiation. The signals underlying each of these complex processes arise from the molecular interactions of extracellular matrix receptors on the surface of the cell. Integrins are the prototypic receptors and provide a mechanical link between extracellular matrix and the cytoskeleton, as well as initiating some of the adhesion-dependent signaling cascades. However, it is becoming increasingly apparent that additional transmembrane receptors function alongside the integrins to regulate both the integrin itself and signals downstream. The most elegant of these examples is the transmembrane proteoglycan, syndecan-4, which cooperates with &alpha;5&beta;1-integrin during adhesion to fibronectin. In vivo models demonstrate the importance of syndecan-4 signaling, as syndecan-4-knockout mice exhibit healing retardation due to inefficient fibroblast migration1,2. In wild-type animals, migration of fibroblasts toward a wound is triggered by the appearance of fibronectin that leaks from damaged capillaries and is deposited by macrophages in injured tissue. Therefore there is great interest in discovering strategies that enhance fibronectin-dependent signaling and could accelerate repair processes.
The integrin-mediated and syndecan-4-mediated components of fibronectin-dependent signaling can be separated by stimulating cells with recombinant fibronectin fragments. Although integrin engagement is essential for cell adhesion, certain fibronectin-dependent signals are regulated by syndecan-4. Syndecan-4 activates the Rac1 protrusive signal3, causes integrin redistribution1, triggers recruitment of cytoskeletal molecules, such as vinculin, to focal adhesions4, and thereby induces directional migration3. We have looked for alternative strategies for activating such signals and found that low-intensity pulsed ultrasound (LIPUS) can mimic the effects of syndecan-4 engagement5. In this protocol we describe the method by which 30 mW/cm2, 1.5 MHz ultrasound, pulsed at 1 kHz (Fig. 1) can be applied to fibroblasts in culture (Fig. 2) to induce Rac1 activation and focal adhesion formation. Ultrasound stimulation is applied for a maximum of 20 minutes, as this combination of parameters has been found to be most efficacious for acceleration of clinical fracture repair6. The method uses recombinant fibronectin fragments to engage &alpha;5&beta;1-integrin, without engagement of syndecan-4, and requires inhibition of protein synthesis by cycloheximide to block deposition of additional matrix by the fibroblasts., The positive effect of ultrasound on repair mechanisms is well documented7,8, and by understanding the molecular effect of ultrasound in culture we should be able to refine the therapeutic technique to improve clinical outcomes.

This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.

L&#39;induzione di segnali di adesione-dipendenti utilizzando ultrasuoni a bassa intensità

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

In sintesi in vitro di Native, Spaziatura fibrosa lungo e segmentale collagene spaziatura lungo

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Singola molecola di rilevamento senza etichetta di testo usando Microtoroid ottici Risonatori

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Multicolore fluorescenza Detection per Droplet microfluidica tramite fibre ottiche

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

Determinazione della Zeta Potenziale tramite nanoparticelle Traslocazione Le velocità attraverso una Tunable Nanopore: usando le particelle di DNA modificato come esempio

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor analytes against the background of human serum are crucial.
Nanoimprinting lithography (NIL) was used to fabricate, at a low cost, sensing areas as large as 1.5 mm x 1.5 mm. The sensing surface was made of high-fidelity arrays of nanoholes, each with an area of about 140 nm2. The great reproducibility of NIL made it possible to employ a one-chip, one-measurement strategy on 12 individually manufactured surfaces, with minimal chip-to-chip variation. These nanoimprinted localized surface plasmon resonance (LSPR) chips were extensively tested on their ability to reliably measure a bioanalyte at concentrations varying from 2.5 to 75 ng/mL amidst the background of a complex biofluid-in this case, human serum. The high fidelity of NIL enables the generation of large sensing areas, which in turn eliminates the need for a microscope, as this biosensor can be easily interfaced with a commonly available laboratory light source. These biosensors can detect cardiac troponin in serum with a high sensitivity, at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. They also show low chip-to-chip variance (due to the high quality of the fabrication process). The results are commensurable with widely used enzyme-linked immunosorbent assay (ELISA)-based assays, but the technique retains the advantages of an LSPR-based sensing platform (i.e., amenability to miniaturization and multiplexing, making it more feasible for POC applications).

This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff &#8764;10-400 pg/mL, depending on the assay).

Utilizzando la trasmissione ottica straordinaria per quantificare i biomarcatori cardiaci nel siero umano

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

Targeting Neuronal Fiber Tracts for Deep Brain Stimulation Therapy Using Interactive, Patient-Specific Models

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for movement disorders and is being applied to a growing number of disorders. Computational modeling has been successfully used to predict the clinical effects of DBS; however, there is a need for novel modeling techniques to keep pace with the growing complexity of DBS devices. These models also need to generate predictions quickly and accurately. The goal of this project is to develop an image processing pipeline to incorporate structural magnetic resonance imaging (MRI) and diffusion weighted imaging (DWI) into an interactive, patient specific model to simulate the effects of DBS. A virtual DBS lead can be placed inside of the patient model, along with active contacts and stimulation settings, where changes in lead position or orientation generate a new finite element mesh and solution of the bioelectric field problem in near real-time, a timespan of approximately 10 seconds. This system also enables the simulation of multiple leads in close proximity to allow for current steering by varying anodes and cathodes on different leads. The techniques presented in this paper reduce the burden of generating and using computational models while providing meaningful feedback about the effects of electrode position, electrode design, and stimulation configurations to researchers or clinicians who may not be modeling experts.

The goal of this project is to develop an interactive, patient-specific modeling pipeline to simulate the effects of deep brain stimulation in near real-time and provide meaningful feedback as to how these devices influence neural activity in the brain.

Tratti della fibra di un neurone di targeting per la terapia di stimolazione cerebrale profonda utilizzando modelli interattivi, specifico per ogni paziente

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

La trasmissione di segnali multipli attraverso una fibra ottica Utilizzando Wavefront Shaping

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