Name: label-free single molecule detection using microtoroid optical resonators
Uploaded: 2015-12-29T14:00:00.000+00:00
Duration: 8 min 53 s
Description: Watch this Scientific Journal Video about Label-free Single Molecule Detection Using Microtoroid Optical Resonators at JoVE.com

This research was supported in part by a National Research Service Award (T32GM07616) from the National Institute of General Medical Sciences.

1. Setup sperimentale e preparazione del campione <ol><li> Realizzare microtoroids utilizzando la litografia, incisione, e di fusione procedura descritta in precedenza 6. Realizzare microtoroids sulla cima di un wafer di silicio (chip) che tipicamente hanno un diametro maggiore di 80-100 micron, e un diametro minore di 2 micron. </li><li> Rilassatevi circa un metro di fibra ottica monomodale (125 rivestimento micron, 4,3 micron modo diametri campo) dalla bobina di fibra. </li><li> Al centro della parte srotolato della fibra ottica, spogliare un piccolo segmento (2,5 cm) del rivestimento polimerico attorno alla fibra ottica utilizzando spellafili. Nota: Questa è la porzione della fibra ottica che verrà utilizzato per evanescently luce coppia nel microtoroid. </li><li> Pulire la fibra spogliato con alcool isopropanolo e un panno senza strofinare. </li><li> Mantenere questa parte della fibra in posizione utilizzando un supporto di fibra in ganci magnetici. </li><li> Sottile la fibra spogliato a ~ 500 nm da Melting e tirando con una torcia idrogeno e due motori passo-passo che si muovono in direzioni opposte a 60 micron / min. Posizionare la fibra ottica all&#39;interno della porzione superiore della fiamma, che dovrebbe essere ~ altezza 10 mm. Arrestare tirando la fibra quando la trasmissione attraverso la fibra ferma fluttuante, che può essere controllato visivamente (guardando la luce che è dispersa lateralmente dal lampeggio fibra on e off) o collegando la fibra ad un fotodiodo che è attaccato ad un oscilloscopio . </li><li> Cleave una estremità della fibra ottica e inserirla in un adattatore fibra nuda. Inserire questa estremità della fibra all&#39;ingresso del fotoricevitore. </li><li> Fibra coppia l&#39;altra estremità della bobina fibra laser utilizzando un accoppiatore a fibre ottiche. </li><li> La fiche microtoroid sopra un portacampioni (acciaio inox, 37,8 mm x 6.4 mm x 3.2 mm) utilizzando resina epossidica o nastro biadesivo. </li><li> Montare il porta-campione in cima ad una fase di posizionamento che si compone di un 3 assi nano-posizionamento (nanocube) fase (vedere inventario) sulla cima di un micrometro a 3 assi. Eseguire tutti gli esperimenti su un tavolo ottico pneumaticamente isolata per ridurre al minimo le vibrazioni. </li><li> Posizione grossolana il chip campione utilizzando un micrometro a 3 assi. </li><li> Allineare il-microtoroid contenente chip di parallelamente alla fibra ottica utilizzando nano-posizionatore. Nota: allineare il microtoroid entro una distanza di una lunghezza d&#39;onda della luce di ingresso (~ 633 nm). Per la visualizzazione di questo processo utilizzare due colonne di imaging (tubo con lente obiettivo e telecamera, vedere inventario) posizionato sulla parte superiore e sul lato del chip. </li><li> Ottimizzazione della polarizzazione della luce laser diretta attraverso la fibra ottica utilizzando un controllore di polarizzazione in linea (vedere inventario) con una manopola per regolare la polarizzazione. Nota: polarizzazione ottimale si ottiene quando il dip misurata nella trasmissione della fibra ottica appare più stretto. Osserva questo tuffo su un oscilloscopio (vedere il punto 2.2 per maggiori dettagli). </li><li> Costruireuna camera del campione da epoxying vetrino coprioggetti sulla fase del campione con un vetrino per microscopio come distanziatore. Nota: Una recinzione in plexiglass che copre l&#39;intera installazione può essere utile per ridurre al minimo le correnti d&#39;aria. Una piccola apertura dovrebbe essere lasciato per consentire la soluzione da pompare in utilizzando tubi. </li><li> Equilibrare termicamente particelle sospensioni o soluzioni acquose singola molecola per ≥ 1 ora in un bagno d&#39;acqua RT (~ 500 ml). Nota: I campioni vengono diluiti alla concentrazione desiderata in provette da microcentrifuga utilizzando i buffer associati specificati dal costruttore, ad esempio, PBS o HEPES. Se non vengono rilevati eventi di legame, aumentare la concentrazione di sale del buffer. </li><li> Particella Vortex contenenti soluzioni (1 ml) per breve tempo per ~ 2 sec. </li><li> Iniettare soluzioni contenenti particelle nella camera campione a 1 ml / min usando una pompa a siringa da 1 ml. </li><li> Dopo la camera del campione ha riempito, spegnere la pompa a siringa. </li><li> Attendere 30 secondi prima di registrare i dati per ridurre al minimogli effetti delle vibrazioni meccaniche derivanti dal flusso di fluido che può influenzare la misura. </li></ol> Blocco 2. Frequenza <ol><li> Re-coppia toroide alla fibra ottica spostando il supporto del campione con il nanopositioner, perché l&#39;accoppiamento sarà disturbata a causa di iniezione di liquido. </li><li> Individuare la lunghezza d&#39;onda di risonanza del microtoroid attraverso la scansione laser ingresso computer controllato attraverso una varietà di lunghezze d&#39;onda. Eseguire questo passaggio inviando un segnale di forma d&#39;onda di tensione triangolare un elemento piezoelettrico all&#39;interno del controller laser che regola la lunghezza d&#39;onda del laser. Effettuare esperimenti usando la luce visibile (635 nm ± 2,5 nm) in quanto vi è un basso assorbimento di luce in acqua a questa lunghezza d&#39;onda. </li><li> Misurare la trasmissione della luce attraverso la fibra ottica collegando l&#39;uscita della fibra ottica in un fotoricevitore auto-equilibrata. Inserire l&#39;uscita del fotoricevitore in un oscilloscopio utilizzando un cavo BNC. Osservare on l&#39;oscilloscopio che alla lunghezza d&#39;onda di risonanza del microtoroid, trasmissione attraverso la fibra ottica gocce. </li><li> Attaccare l&#39;uscita del fotoricevitore all&#39;ingresso principale della frequenza bloccaggio regolatore a reazione (vedere inventario) tramite un cavo. </li><li> Eseguire il controller di feed back di blocco in modalità di blocco automatico con picco top di chiusura con una frequenza di dithering di 2 kHz e un&#39;ampiezza di lunghezza d&#39;onda di oscillazione del 19 fm. Empiricamente configurare le impostazioni proporzionale integrale derivati ​​nella finestra del software utilizzando Ziegler-Nichols regole di tuning 14. Nota: questi valori devono solo essere impostata una volta all&#39;inizio di tutti gli esperimenti. </li><li> Auto-bloccare la lunghezza d&#39;onda del laser alla lunghezza d&#39;onda di risonanza del microtoroid. Eseguire questo passaggio dopo aver riempito la camera del campione. Nota: Se lo spostamento della lunghezza d&#39;onda è troppo grande, quindi il regolatore di feedback perderà di blocco e passare automaticamente alla modalità di scansione al fine di individuare la posizione di risonanza lunghezza d&#39;onda. Questo occurs per lunghezza d&#39;onda si sposta più grande di circa un linewidth (almeno 600 fm per tutti i sistemi esaminati qui). </li><li> Registrare l&#39;uscita del regolatore a reazione a 20 kHz utilizzando una scheda di acquisizione dati a 24 bit. Esportare i dati come file di testo tramite software di acquisizione dati. </li></ol> 3. Elaborazione Dati e analisi <ol><li> Fourier trasformare i dati in MATLAB. </li><li> Passa-basso i dati utilizzando un filtro &quot;muro di mattoni&quot; con un cutoff a 1 kHz per rimuovere la frequenza di dithering imposto di 2 kHz (vedi codice supplementare sul file Schermata 1). </li><li> Notch computazionalmente filtrare i dati utilizzando una dimensione della finestra di 16 Hz. Nota: Questo viene fatto per rimuovere fonti note di rumore, in questo caso, 60 Hz rumore elettronico linea e le sue armoniche, nonché 100 Hz (proveniente dal driver laser) e sue armoniche (vedi codice supplementare file Schermata 1). </li><li> Fourier inversa trasformare i dati di nuovo nel dominio del tempo. </li><li> Filtro mediano i dati utilizzando una finestradimensioni di 1.001 campioni (vedi Codice supplementare sul file Schermata 2). </li><li> Individuare cambio di passo nella lunghezza d&#39;onda di risonanza utilizzando l&#39;algoritmo passo ricerca di Kerssemakers et al. 15. </li><li> Genera istogrammi di ampiezza di ciascuna fase di legame. </li><li> Calcola dimensione delle particelle con Eq. (1) (vedi Discussione). </li></ol>

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Lett. 28, 272-274 (2003).</li><li>Vollmer, F., Arnold, S., Keng, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+virus+detection+from+the+reactive+shift+of+a+whispering-gallery+mode.">Single virus detection from the reactive shift of a whispering-gallery mode.</a> Proc. Natl. Acad. Sci. U. S. A. 105, 20701-20704 (2008).</li><li>He, L., Ozdemir, S. K., Zhu, J., Kim, W., Yang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+single+viruses+and+nanoparticles+using+whispering+gallery+microlasers.">Detecting single viruses and nanoparticles using whispering gallery microlasers.</a> Nat Nano. 6, 428-432 (2011).</li><li>Dantham, V. 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The authors declare that they have no competing financial interests.

Come si lega una particella, la lunghezza d&#39;onda di risonanza (λ) del toroide aumenta. Se una particella non associa, la lunghezza d&#39;onda di risonanza diminuisce corrispondentemente (un evento step-down). Il diametro delle particelle (d) può essere determinata attraverso istogrammi di ampiezza di ogni passo di lunghezza d&#39;onda. L&#39;altezza di ogni gradino d&#39;onda varia a causa di variazioni dimensionali della particella legato e causa la posizione sulla microtoroid cui si lega la par...

Esperimenti rilevamento molecole singole sono utili per ridurre la quantità di analita usato in biosensori, per la diagnosi precoce della malattia, e per esaminare le proprietà fondamentali delle molecole 3. Tali esperimenti sono tipicamente eseguite utilizzando etichette, tuttavia, le etichette non sono sempre possibile ottenere per una particolare proteina, aumenta il costo, può perturbare gli eventi oggetto di studio, e può essere scomodo, particolarmente per tempo reale in loco esperimenti o point-of- Diagnostica di cura. Il gold standard attuale per biosensing label-free è risonanza plasmonica di superficie 4, ma i sistemi commerciali di risonanza plasmonica di superficie in genere hanno un tipico limite inferiore di rilevamento dell&#39;ordine di nM. Recentemente, risonatori ottici sono emersi come una tecnologia promettente per libero etichetta singola molecola biorilevazione 5. Risonatori ottica lavoro basato sul lungo termine (ns) confinamento della luce 6,7. La luce è evanescentlyaccoppiato in questi dispositivi tipicamente attraverso una fibra ottica. Quando la lunghezza d&#39;onda della luce che attraversa la fibra corrisponde alla lunghezza d&#39;onda di risonanza del risonatore, illuminare efficientemente accoppia il risonatore. Questa luce accoppiata riflette totalmente internamente la cavità del risonatore a generare un campo evanescente in prossimità della circonferenza del risonatore. Come particelle entrano campo evanescente e si legano al risuonatore, la lunghezza d&#39;onda di risonanza dei cambiamenti risonatore in proporzione al volume della particella 8. In termini di capacità di rilevamento, risonatori di microsfere sono stati in precedenza utilizzata per rilevare l&#39;influenza A solo particelle virali (100 nm) 9,10. Recentemente, plasmonically avanzata microsfere risonatori ottici sono stati utilizzati per rilevare siero albumina bovina singola molecole 11 e oligonucleotidi 8-mer 12, tuttavia questo approccio limita l&#39;area di cattura delle particelle di 0,3 micron 2 per device. Più grandi biosensori zona di cattura sono ideali per massimizzare la probabilità di rilevamento delle particelle. Le attuali tecnologie basate su soluzioni label-free biosensori con grandi (&gt; 100 micron 2) aree di acquisizione si sono limitati a rilevare particelle di polistirene ≥ 25 nm. Abbiamo sviluppato un sistema di biosensori label-free basato sulla tecnologia risonatore ottico noto come frequenza di blocco ottico Whispering Evanescent risonatore (FIORE) 13 (Figura 1), che è in grado di rilevare in tempo risolto di singole molecole in soluzione. FIORE utilizza la lunga durata fotone di risonatori ottici microtoroid combinati con frequenza di blocco di controllo di retroazione, il rilevamento equilibrato, e il filtraggio di calcolo per rilevare piccole particelle fino a singole molecole proteiche. L&#39;uso di aggancio in frequenza consente al sistema di monitorare sempre la risonanza spostamento del microtoroid come particelle si legano, senza la necessità di spazzare o la scansione della lunghezza d&#39;onda del laser sopragrandi intervalli. I principi di FLOWER possono essere utilizzati per migliorare le capacità di rilevazione delle altre tecniche, tra cui la valorizzazione plasmonica. In ciò che segue, vengono descritte le procedure per l&#39;esecuzione FIORE.

<table><tbody><tr><td>Tunable diode laser</td><td>Newport</td><td>TLB-6300</td><td></td></tr><tr><td>Laser controller</td><td>Newport</td><td>TLB-6300-LN</td><td></td></tr><tr><td>Frequency locking feedback controller</td><td>Toptica Photonics</td><td>Digilock 110</td><td></td></tr><tr><td>Auto-balanced photoreceiver</td><td>Newport</td><td>Model 2007</td><td></td></tr><tr><td>In-line polarization controller</td><td>General Photonics</td><td>PLC-003-S-90</td><td></td></tr><tr><td>24-bit data acquisition card</td><td>National Instruments</td><td>NI-PCI-4461</td><td></td></tr><tr><td>Recombinant human interleukin-2</td><td>Pierce Biotechnology</td><td>R201520</td><td></td></tr><tr><td>20 nm polystyrene beads</td><td>Thermo Scientific</td><td>3020A</td><td></td></tr><tr><td>NanoCube XYZ Piezo Stage</td><td>Physik Instrumente</td><td>P-611.3</td><td></td></tr><tr><td>Optical table</td><td>Newport</td><td>VH3660W-OPT</td><td></td></tr><tr><td>Objective lens for imaging column</td><td>Navitar Machine Vision</td><td>1-60228</td><td></td></tr><tr><td>Imaging column (adaptor tube)</td><td>Navitar Machine Vision</td><td>1-60228</td><td></td></tr><tr><td>High-Res CCD camera for imaging column</td><td>Edmund Industrial Optics</td><td>NT39244</td><td></td></tr></tbody></table>

label-free single molecule detection using microtoroid optical resonators

Rilevamento piccole concentrazioni di molecole fino al limite singola molecola ha un impatto su settori quali la diagnosi precoce della malattia, e studi fondamentali sul comportamento delle molecole. Tecniche di rilevazione di singola molecola comunemente utilizzano etichette come etichette fluorescenti o punti quantici, tuttavia, le etichette non sono sempre disponibili, aumentare i costi e la complessità, e possono perturbare gli eventi in fase di studio. Risonatori ottici sono emersi come un mezzo promettente per rilevare singole molecole senza l&#39;uso di etichette. Attualmente la più piccola particella rilevata da un sistema di risuonatore non plasmonically potenziato nudo ottica in soluzione è a 25 nm di polistirolo sfera 1. Abbiamo sviluppato una tecnica nota come frequenza di blocco ottico Whispering Evanescent risonatore (fiore) in grado di superare questo limite e ottenere la diagnosi singola molecola label-free in soluzione acquosa 2. La potenza del segnale scale con il volume delle particelle, il nostro lavoro rappresenta un&gt; 100x improvement nel rapporto segnale-rumore (SNR) rispetto allo stato attuale della tecnica. Qui le procedure dietro fiore sono presentati in uno sforzo per aumentare il suo utilizzo nel settore.

Eventi di legame delle particelle sono chiaramente visto come variazioni a gradino in lunghezza d&#39;onda di risonanza del microtoroid nel tempo (Figura 2A). Le altezze di questi passaggi sono indicati come un istogramma della figura 2B. Figure 2-4 mostrano tracce rappresentative del legame di esosomi (nanovesicles), 5 nm perline di silice, e single umane interleuchina-2 molecole, rispettivamente. Il fatto che gli eventi a gradino scala con granulometr...

Watch this Scientific Journal Video about Label-free Single Molecule Detection Using Microtoroid Optical Resonators at JoVE.com

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

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 ...

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9.2K Views.  California Institute of Technology. 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. 

Video: Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Fabrication of Silica Ultra High Quality Factor Microresonators

Whispering gallery resonant cavities confine light in circular orbits at their periphery.1-2 The photon storage lifetime in the cavity, quantified by the quality factor (Q) of the cavity, can be in excess of 500ns for cavities with Q factors above 100 million. As a result of their low material losses, silica microcavities have demonstrated some of the longest photon lifetimes to date1-2. Since a portion of the circulating light extends outside the resonator, these devices can also be used to probe the surroundings. This interaction has enabled numerous experiments in biology, such as single molecule biodetection and antibody-antigen kinetics, as well as discoveries in other fields, such as development of ultra-low-threshold microlasers, characterization of thin films, and cavity quantum electrodynamics studies.3-7
The two primary silica resonant cavity geometries are the microsphere and the microtoroid. Both devices rely on a carbon dioxide laser reflow step to achieve their ultra-high-Q factors (Q&gt;100 million).1-2,8-9 However, there are several notable differences between the two structures. Silica microspheres are free-standing, supported by a single optical fiber, whereas silica microtoroids can be fabricated on a silicon wafer in large arrays using a combination of lithography and etching steps. These differences influence which device is optimal for a given experiment.
Here, we present detailed fabrication protocols for both types of resonant cavities. While the fabrication of microsphere resonant cavities is fairly straightforward, the fabrication of microtoroid resonant cavities requires additional specialized equipment and facilities (cleanroom). Therefore, this additional requirement may also influence which device is selected for a given experiment.
Introduction
An optical resonator efficiently confines light at specific wavelengths, known as the resonant wavelengths of the device. 1-2 The common figure of merit for these optical resonators is the quality factor or Q. This term describes the photon lifetime (&tau;o) within the resonator, which is directly related to the resonator's optical losses. Therefore, an optical resonator with a high Q factor has low optical losses, long photon lifetimes, and very low photon decay rates (1/&tau;o). As a result of the long photon lifetimes, it is possible to build-up extremely large circulating optical field intensities in these devices. This very unique property has allowed these devices to be used as laser sources and integrated biosensors.10
A unique sub-class of resonators is the whispering gallery mode optical microcavity. In these devices, the light is confined in circular orbits at the periphery. Therefore, the field is not completely confined within the device, but evanesces into the environment. Whispering gallery mode optical cavities have demonstrated some of the highest quality factors of any optical resonant cavity to date.9,11 Therefore, these devices are used throughout science and engineering, including in fundamental physics studies and in telecommunications as well as in biodetection experiments. 3-7,12
Optical microcavities can be fabricated from a wide range of materials and in a wide variety of geometries. A few examples include silica and silicon microtoroids, silicon, silicon nitride, and silica microdisks, micropillars, and silica and polymer microrings.13-17 The range in quality factor (Q) varies as dramatically as the geometry. Although both geometry and high Q are important considerations in any field, in many applications, there is far greater leverage in boosting device performance through Q enhancement. Among the numerous options detailed previously, the silica microsphere and the silica microtoroid resonator have achieved some of the highest Q factors to date.1,9 Additionally, as a result of the extremely low optical loss of silica from the visible through the near-IR, both microspheres and microtoroids are able to maintain their Q factors over a wide range of testing wavelengths.18 Finally, because silica is inherently biocompatible, it is routinely used in biodetection experiments.
In addition to high material absorption, there are several other potential loss mechanisms, including surface roughness, radiation loss, and contamination loss.2 Through an optimization of the device size, it is possible to eliminate radiation losses, which arise from poor optical field confinement within the device. Similarly, by storing a device in an appropriately clean environment, contamination of the surface can be minimized. Therefore, in addition to material loss, surface scattering is the primary loss mechanism of concern.2,8
In silica devices, surface scattering is minimized by using a laser reflow technique, which melts the silica through surface tension induced reflow. While spherical optical resonators have been studied for many years, it is only with recent advances in fabrication technologies that researchers been able to fabricate high quality silica optical toroidal microresonators (Q&gt;100 million) on a silicon substrate, thus paving the way for integration with microfluidics.1
The present series of protocols details how to fabricate both silica microsphere and microtoroid resonant cavities. While silica microsphere resonant cavities are well-established, microtoroid resonant cavities were only recently invented.1 As many of the fundamental methods used to fabricate the microsphere are also used in the more complex microtoroid fabrication procedure, by including both in a single protocol it will enable researchers to more easily trouble-shoot their experiments.

We describe the use of a carbon dioxide laser reflow technique to fabricate silica resonant cavities, including free-standing microspheres and on-chip microtoroids. The reflow method removes surface imperfections, allowing long photon lifetimes within both devices. The resulting devices have ultra high quality factors, enabling applications ranging from telecommunications to biodetection.

Fabbricazione di Microrisonatori silice Ultra High Quality Factor

Whispering gallery resonant cavities confine light in circular orbits at their periphery.1-2 The photon storage lifetime in the cavity, quantified by the ...

Ricerca

Ingegneria

Optical Trapping of Nanoparticles

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping and manipulating small biological particles. Ashkin and co-workers first demonstrated optical tweezers using a single focused beam1. The single beam trap can be described accurately using the perturbative gradient force formulation in the case of small Rayleigh regime particles1. In the perturbative regime, the optical power required for trapping a particle scales as the inverse fourth power of the particle size. High optical powers can damage dielectric particles and cause heating. For instance, trapped latex spheres of 109 nm in diameter were destroyed by a 15 mW beam in 25 sec1, which has serious implications for biological matter2,3.
A self-induced back-action (SIBA) optical trapping was proposed to trap 50 nm polystyrene spheres in the non-perturbative regime4. In a non-perturbative regime, even a small particle with little permittivity contrast to the background can influence significantly the ambient electromagnetic field and induce a large optical force. As a particle enters an illuminated aperture, light transmission increases dramatically because of dielectric loading. If the particle attempts to leave the aperture, decreased transmission causes a change in momentum outwards from the hole and, by Newton's Third Law, results in a force on the particle inwards into the hole, trapping the particle. The light transmission can be monitored; hence, the trap can become a sensor. The SIBA trapping technique can be further improved by using a double-nanohole structure.
The double-nanohole structure has been shown to give a strong local field enhancement5,6. Between the two sharp tips of the double-nanohole, a small particle can cause a large change in optical transmission, thereby inducing a large optical force. As a result, smaller nanoparticles can be trapped, such as 12 nm silicate spheres7 and 3.4 nm hydrodynamic radius bovine serum albumin proteins8. In this work, the experimental configuration used for nanoparticle trapping is outlined. First, we detail the assembly of the trapping setup which is based on a Thorlabs Optical Tweezer Kit. Next, we explain the nanofabrication procedure of the double-nanohole in a metal film, the fabrication of the microfluidic chamber and the sample preparation. Finally, we detail the data acquisition procedure and provide typical results for trapping 20 nm polystyrene nanospheres.

The following setup approach details low power optical trapping of dielectric nanoparticles using a double-nanohole in metal film.

Trapping ottica di nanoparticelle

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping ...

Synthesis and Operation of Fluorescent-core Microcavities for Refractometric Sensing

This paper discusses fluorescent core microcavity-based sensors that can operate in a microfluidic analysis setup. These structures are based on the formation of a fluorescent quantum-dot (QD) coating on the channel surface of a conventional microcapillary. Silicon QDs are especially attractive for this application, owing in part to their negligible toxicity compared to the II-VI and II-VI compound QDs, which are legislatively controlled substances in many countries. While the ensemble emission spectrum is broad and featureless, an Si-QD film on the channel wall of a capillary features a set of sharp, narrow peaks in the fluorescence spectrum, corresponding to the electromagnetic resonances for light trapped within the film. The peak wavelength of these resonances is sensitive to the external medium, thus permitting the device to function as a refractometric sensor in which the QDs never come into physical contact with the analyte. The experimental methods associated with the fabrication of the fluorescent-core microcapillaries are discussed in detail, as well as the analysis methods. Finally, a comparison is made between these structures and the more widely investigated liquid-core optical ring resonators, in terms of microfluidic sensing capabilities.

Fluorescent-core microcavity sensors employ a high-index quantum-dot coating in the channel of silica microcapillaries. Changes in the refractive index of fluids pumped into the capillary channel cause shifts in the microcavity fluorescence spectrum that can be used to analyze the channel medium.

Sintesi e funzionamento del fluorescente-core microcavità per il Rilevamento rifrattometrico

This paper discusses  fluorescent core microcavity-based sensors that can operate in a microfluidic  analysis setup. These structures are based on the ...

Implementation of a Reference Interferometer for Nanodetection

A thermally and mechanically stabilized fiber interferometer suited for examining ultra-high quality factor microcavities is fashioned. After assessing its free spectral range (FSR), the module is put in parallel with a fiber taper-microcavity system and then calibrated through isolating and eliminating random shifts in the laser frequency (i.e. laser jitter noise). To realize the taper-microcavity junction and to maximize the optical power that is transferred to the resonator, a single-mode optical fiber waveguide is pulled. Solutions containing polystyrene nanobeads are then prepared and flown to the microcavity in order to demonstrate the system&#8217;s ability to sense binding to the surface of the microcavity. Data is post-processed via adaptive curve fitting, which allows for high-resolution measurements of the quality factor as well as the plotting of time-dependent parameters, such as resonant wavelength and split frequency shifts. By carefully inspecting steps in the time-domain response and shifting in the frequency-domain response, this instrument can quantify discrete binding events.

A reference interferometer technique, which is designed to remove undesirable laser jitter noise for nanodetection, is utilized for probing an ultra-high quality factor microcavity. Instructions for assembly, setup, and data acquisition are provided, alongside the measurement process for specifying the cavity quality factor.

Realizzazione di un interferometro di riferimento per Nanodetection

A thermally and mechanically stabilized fiber interferometer suited for examining ultra-high quality factor microcavities is fashioned. After assessing ...

Fabrication and Testing of Microfluidic Optomechanical Oscillators

Cavity optomechanics experiments that parametrically couple the phonon modes and photon modes have been investigated in various optical systems including microresonators. However, because of the increased acoustic radiative losses during direct liquid immersion of optomechanical devices, almost all published optomechanical experiments have been performed in solid phase. This paper discusses a recently introduced hollow microfluidic optomechanical resonator. Detailed methodology is provided to fabricate these ultra-high-Q microfluidic resonators, perform optomechanical testing, and measure radiation pressure-driven breathing mode and SBS-driven whispering gallery mode parametric vibrations. By confining liquids inside the capillary resonator, high mechanical- and optical- quality factors are simultaneously maintained.

Parametric optomechanical excitations have recently been experimentally demonstrated in microfluidic optomechanical resonators by means of optical radiation pressure and stimulated Brillouin scattering. This paper describes the fabrication of these microfluidic resonators along with methodologies for generating and verifying optomechanical oscillations.

Fabbricazione e la sperimentazione di microfluidici optomechanical Oscillatori

Cavity optomechanics experiments that parametrically couple the phonon modes and photon modes have been investigated in various optical systems including ...

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) ...

Bioingegneria

Fabrication and Operation of a Nano-Optical Conveyor Belt

The technique of using focused laser beams to trap and exert forces on small particles has enabled many pivotal discoveries in the nanoscale biological and physical sciences over the past few decades. The progress made in this field invites further study of even smaller systems and at a larger scale, with tools that could be distributed more easily and made more widely available. Unfortunately, the fundamental laws of diffraction limit the minimum size of the focal spot of a laser beam, which makes particles smaller than a half-wavelength in diameter hard to trap and generally prevents an operator from discriminating between particles which are closer together than one half-wavelength. This precludes the optical manipulation of many closely-spaced nanoparticles and&#160;limits the&#160;resolution of optical-mechanical systems. Furthermore, manipulation using focused beams requires beam-forming or steering optics, which can be very bulky and expensive. To address these limitations in the system scalability of conventional optical trapping our lab has devised an alternative technique which utilizes near-field optics to move particles across a chip. Instead of focusing laser beams in the far-field, the optical near field of plasmonic resonators produces the necessary local optical intensity enhancement to overcome the restrictions of diffraction and manipulate particles at higher resolution. Closely-spaced resonators produce strong optical traps which can be addressed to mediate the hand-off of particles from one to the next in a conveyor-belt-like fashion. Here, we describe how to design and produce a conveyor belt using a gold surface patterned with plasmonic C-shaped resonators and how to operate it with polarized laser light to achieve super-resolution nanoparticle manipulation and transport. The nano-optical conveyor belt chip can be produced using lithography techniques and easily packaged and distributed.

The scalability and resolution of conventional optical manipulation techniques are limited by diffraction. We circumvent the diffraction limit and describe a method of optically transporting nanoparticles across a chip using a gold surface patterned with a path of closely spaced C-shaped plasmonic resonators.

Fabbricazione e funzionamento di un nano-ottica Nastro trasportatore

The technique of using focused laser beams to trap and exert forces on small particles has enabled many pivotal discoveries in the nanoscale biological ...

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 ...

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.

Uso di pinzette ottiche doppie e microfluidica per studi a singola molecola

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the ...

Biochimica

Optical Biosensing

Optical biosensors utilize light to detect the binding of a target molecule. These sensors can utilize a label molecule, which produces a measurable signal such as fluorescence,&#160;or these sensors can be label-free&#160;and use the changes in optical properties, such as refractive index, to sense for the binding of the target molecule. This video introduces both label and label-free optical biosensors, demonstrates their use in the laboratory, and shows some applications of the technology.

Types of Optical Biosensing; Surface Plasmon Resonance SPR

Optical biosensors utilize light to detect the binding of a target molecule. These sensors can utilize a label molecule, which produces a measurable signal such as fluorescence, or these sensors can be label-free&#160;and use the changes in optical properties, such as refractive index, to sense for the binding of the target molecule. This video introduces both label and label-free optical biosensors, demonstrates their use in the laboratory, and shows some applications of the technology.

Biorilevamento ottico

Optical biosensors utilize light to detect the binding of a target molecule. These sensors can utilize a label molecule, which produces a measurable ...