Noi riconosciamo il sostegno del Dipartimento di Ingegneria Biologica e la Christopher S. James Bond Life Sciences Center presso la University of Missouri. Riconosciamo sovvenzioni da Missouri Life Sciences Research Board 09-1034 e NIH R21CA139186-0. Ringraziamo anche la Vita di laurea Scienze programma Opportunità di ricerca presso l&#39;Università del Missouri, l&#39;Università di Core Missouri Citologia molecolare, e l&#39;Università del Missouri College of Engineering per il sostegno finanziario.

1. Preparazione del campione di sangue <ol><li> Prelevare circa 10 ml di sangue intero da un paziente Stadio Cancro IV in due vacutainer 5 ml. </li><li> Centrifugare le provette per dieci minuti a 3.000 giri al minuto (rpm). </li><li> Il campione di sangue viene separato in tre diversi strati: lo strato inferiore comprende il eritrociti, lo strato centrale, detto anche buffy coat, contiene leucociti e cellule di melanoma, e lo strato superiore contiene plasma e piastrine. Rimuovere il plasma più possibile senza disturbare il buffy coat. </li><li> Posto 250 microlitri di HISTOPAQUE 1077 in due tubi Wintrobe 1 ml. </li><li> Utilizzando un 9 &quot;pipetta Pasteur, estrarre il plasma residuo, il cappotto tutto buffy, e anche lo strato superiore degli eritrociti, avendo cura di raccogliere &lt;500 microlitri. </li><li> Posizionare la soluzione sopra lo strato HISTOPAQUE e centrifugare i tubi Wintrobe in provette da 15 ml conica per 10 minuti a 3000 rpm. </li><li> Il sangue sarà di nuovoseparati, ma questa volta il eritrociti saranno separati dal HISTOPAQUE, consentendo una facile estrazione del buffy coat. Utilizzare un 9 &quot;pipetta Pasteur per ottenere il cappotto intero buffy e metterla in una provetta 2 ml Eppendorf. </li><li> Sospendere il buffy coat in 1,5 ml di PBS con il 2% v / v Tween 20. </li></ol> 2. Camera di flusso Costruzione <ol><li> Ottenere un cilindro in acrilico, 20 mm di diametro esterno, 17 mm di diametro interno, e 8 mm di altezza. Praticare tre fori di 5 mm di diametro attraverso il lato del cilindro ad angoli di 90 gradi l&#39;uno dall&#39;altro. Tagliate tre pezzi di tubo di polimeri (1,6 mm di diametro interno, 5 mm di diametro esterno), e metterli nei fori. </li><li> Stretch Parafilm nella parte inferiore dell&#39;anello acrilico per fare una perfetta tenuta d&#39;acqua. Il parafilm forma una ciotola con l&#39;anello in acrilico e terrà la soluzione di acrilammide che si forma una camera di flusso. </li><li> Sospendere un filo di 1,6 mm di diametro attraverso i pezzi di tubo che sono di fronte al&#39;altro, facendo in modo che il filo da solo è visibile attraverso il centro del ring. Riempire il terzo pezzo di tubo con un filo di 1,6 millimetri di diametro e la posizione del tubo di 1 millimetro di distanza dal filo sospeso. Questi fili impedisce di acrilammide di entrare nel tubo. Dopo l&#39;acrilamide si solidifica, i cavi saranno estratti, lasciando un sentiero cilindrico flusso e un posto per posizionare una fibra ottica si concentra sul percorso del flusso. </li><li> Aggiungere 5 mL di acrilamide preparati [20 g di acrilammide (Sigma Aldrich), 0,7 g bisacrylamide (Sigma Aldrich), 100 ml di acqua distillata] in un recipiente di piccole dimensioni. Misurare 0,02 g di persolfato di ammonio (Sigma) e aggiungerlo al bicchiere. Mescolare con ancoretta per 3 minuti. Aggiungere 20 l tetramethylethylenediamene (Fisher Scientific) al bicchiere. Velocemente versare il composto sul ring acrilico, riempiendo verso l&#39;alto. Dopo pochi secondi, la soluzione in gel, facendo uno stampo camera di flusso. Estrarre delicatamente i fili fuori, facendo attenzione a non disturbare il gel di acrilammide. Il flusso di ChamBER è ora pronto per l&#39;uso. </li></ol> 3. Flussimetria fotoacustico set-up <ol><li> Acusticamente paio di fluoruro di polivinile trasduttore acustico piezoelettrico (Panametrics) per una camera di flusso usando gel per ultrasuoni direttamente sopra il percorso del flusso. </li><li> Utilizzando un Bayonet Neill-Concelman (BNC), cavo, collegare il trasduttore con l&#39;ingresso di un ricevitore a banda larga RITEC-640A (filtro passa-basso: fuori, filtro passa-alto: 1 MHz, Impedenza d&#39;ingresso: 50 Ohm, amplificazione: 35 dB). Collegare l&#39;uscita del filtro per l&#39;oscilloscopio (Tektronix TDS 2024B). Per attivare l&#39;oscilloscopio, un fotodiodo (Thorlabs) è istituito presso il trasduttore ed è collegato a dell&#39;oscilloscopio utilizzando un cavo BNC. </li><li> Un connettore a T (minuteria) è collegato a un braccio della camera di flusso. Due pompe siringa (Braintree scientifico) sono collegati ad altri rami del connettore. Una siringa contiene aria, mentre l&#39;altro conterrà la sospensione cellulare preparata nella parte 1. Impostare la velocità di flusso di100 l / minuto. </li><li> Estrarre il tubo piccolo della camera di flusso fino a quando una piccola tasca di forme d&#39;aria, e inserire un diametro di 1 millimetro in fibra ottica core con un apertura numerica 0,37 in quello slot. Riempire la tasca con acqua per ridurre segnali di interfaccia e di aumentare la quantità di luce laser applicata al percorso del flusso. A causa della apertura numerica della fibra ottica e breve distanza dal percorso del flusso, il laser solo irradiare uno slug del campione in qualsiasi momento, assicurando che il campione direttamente di fronte al laser è lo stesso campione di creare segnali fotoacustico. L&#39;energia laser dovrebbe essere circa 28 mJ / cm 2 con uno spot di 7 mm 2. </li><li> Accendere entrambe le pompe siringa su. Quando i due fluidi immiscibili raggiungere la t-giunzione saranno suddivisi in omogenei lumache e il flusso di oltre il trasduttore. </li><li> Irradiare il campione scorre un nanosecondo a impulsi laser per generare onde fotoacustico nelle cellule di melanoma. Come melanina è un broadbe ottiche assorbitore, ogni lunghezza d&#39;onda laser visibile può essere utilizzato. </li><li> Se un segnale appare sul oscilloscopio quando una lumaca è in cima al trasduttore, la lumaca contiene melanoma, e dovrebbero essere monitorati attraverso il sistema fino a cadere in una fiala di raccolta. </li></ol> 4. Immunocitochimica <ol><li> Prendere le lumache acquisiti in un Centrifuga 4 Cytospin (Thermo Scientific), al fine di correggerli per vetrini. Collocare i vetrini in vetro Cytofunnels (Shandon Cytofunnel EZ singola con Brown filtro carta Thermo Scientific) e li carico in centrifuga 4 Cytospin. Mettere 100 ml di 1% di albumina di siero bovino (Sigma) in PBS in ciascun imbuto, quindi si centrifuga a 600 giri al minuto per 3 minuti per aiutare le cellule catturate bastone alle diapositive di vetro. </li><li> Dopo la centrifugazione, aggiungere i campioni delle cellule al Cytofunnels, quindi si centrifuga a 600 giri al minuto per 3 minuti. Quindi spruzzare le diapositive con un fissativo a base di etanolo. </li><li> Lavare i vetrini in PBS per 10 minuti due volte. </li><li&gt; Per bloccare legame aspecifico, incubare ogni diapositiva con una miscela di 10 ml di siero di capra (Sigma) e 90 ml di PBS per 1 ora a temperatura ambiente. </li><li> Dopo l&#39;incubazione, far decantare le diapositive e incubare con la soluzione di anticorpo primario per 1 ora in una camera umidificata a temperatura ambiente. Ogni diapositiva deve contenere l&#39;1% MART-1 anticorpi cresciuto a coniglio, 1% CD-45 anticorpi cresciuto nel topo, nel siero di capra 10%, 89% e PBS. </li><li> Dopo l&#39;incubazione, lavare i vetrini in PBS per 10 minuti tre volte. </li><li> I vetrini vengono poi incubate in una soluzione di anticorpo secondario, che ha fluoroforo legate agli anticorpi. La miscela contiene 0,05% di capra anti-coniglio di anticorpi, 0,05% di capra anti-topo, siero di capra 0,1%, e il 99% PBS. Le diapositive vengono incubate al buio per 1 ora a temperatura ambiente. </li><li> Dopo l&#39;incubazione, decantare le diapositive lavare in PBS per 5 minuti tre volte. </li><li> A macchia di un nucleo, incubare le cellule con 0,1 mg / ml di DAPI (Invitrogen) per1 minuto. Poi decantare le diapositive e lavare in PBS. </li><li> Montare un vetrino sul campione cellulare utilizzando 20 l di un supporto di montaggio a base di resina per vetrino. Sigillare il coprioggetto con smalto trasparente per preservare la diapositiva. Ora le diapositive sono pronti per essere ripreso con un microscopio a fluorescenza. </li></ol> 5. Rappresentante dei risultati: <img src="/files/ftp_upload/3559/3559fig1.jpg" alt="Figura 1"/> Figura 1. Le forme d&#39;onda fotoacustico da globuli bianchi e cellule di melanoma sono mostrati qui. I globuli bianchi, non avendo pigmentazione intrinseca, non producono onde fotoacustico e si manifestano come una linea piatta di rumore elettronico (a sinistra). Le cellule di melanoma pigmentato produce un&#39;onda robusto fotoacustico (a destra). <img src="/files/ftp_upload/3559/3559fig2.jpg" alt="Figura 2"/> Figura 2. Dopo la colorazione immunocitochimica, cellule di melanoma in coltura mostrano segnali DAPI in blue mentre MART1 è evidenziato in verde. <img src="/files/ftp_upload/3559/3559fig3.jpg" alt="Figura 3"/> Figura 3. Sovrapposizione delle immagini per DAPI e MART1, come in Figura 2, con CD45 come un indicatore di leucociti, questa figura mostra cellule di melanoma tra le diverse cellule bianche del sangue.

<ol>
	<li>Pantel, K., Brakenhoff, R., Brandt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+clinical+relevance+and+specific+biological+properties+of+disseminating+tumour+cells.">Detection clinical relevance and specific biological properties of disseminating tumour cells.</a> Nature Reviews Cancer. 8, 329-340 (2008).</li><li>Pantel, K., Brakenhoff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+metastatic+cascade.">Dissecting the metastatic cascade.</a> Nature Reviews Cancer. 4, 448-456 (2004).</li><li>Yu, M., Stott, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells:+approaches+to+isolation+and+characterization.">Circulating tumor cells: approaches to isolation and characterization.</a> The Journal of Cell Biology. 192, 373-373 (2011).</li><li>Weight, R. M., Dale, P. S., Viator, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+circulating+melanoma+cells+in+human+blood+using+photoacoustic+flowmetry.">Detection of circulating melanoma cells in human blood using photoacoustic flowmetry.</a> , 106-109 (1985).</li><li>Weight, R. M., Dale, P. S., Lisle, A., Caldwell, C., Viator, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+detection+of+metastatic+melanoma+cells+in+the+human+circulatory+system.">Photoacoustic detection of metastatic melanoma cells in the human circulatory system.</a> Opt. Lett. 31, 2998-3000 (2006).</li><li>Gupta, A., Kumar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+geometry+on+droplet+formation+in+the+squeezing+regime+in+a+microfluidic+T-junction.">Effect of geometry on droplet formation in the squeezing regime in a microfluidic T-junction.</a> Microfluidics and Nanofluidics. 8, 799-812 (2010).</li><li>Mocellin, S., Hoon, D., Ambrosi, A., Nitti, D., Rossi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prognostic+value+of+circulating+tumor+cells+in+patients+with+melanoma:+a+systematic+review+and+meta-analysis.">The prognostic value of circulating tumor cells in patients with melanoma: a systematic review and meta-analysis.</a> Clinical Cancer Research. 12, 4605-4605 (2006).</li><li>Cristofanilli, M., Budd, G., Ellis, M., Stopeck, A., Matera, J., Miller, M., Reuben, J., Doyle, G., Allard, W., Terstappen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells,+disease+progression,+and+survival+in+metastatic+breast+cancer.">Circulating tumor cells, disease progression, and survival in metastatic breast cancer.</a> New England Journal of Medicine. 351, 781-781 (2004).</li><li>Cristofanilli, M., Hayes, D., Budd, G., Ellis, M., Stopeck, A., Reuben, J., Doyle, G., Matera, J., Allard, W., Miller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells:+a+novel+prognostic+factor+for+newly+diagnosed+metastatic+breast+cancer.">Circulating tumor cells: a novel prognostic factor for newly diagnosed metastatic breast cancer.</a> Journal of Clinical Oncology. 23, 1420-1420 (2005).</li><li>Ghossein, R., Scher, H., Gerald, W., Kelly, W., Curley, T., Amsterdam, A., Zhang, Z., Rosai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+circulating+tumor+cells+in+patients+with+localized+and+metastatic+prostatic+carcinoma:+clinical+implications.">Detection of circulating tumor cells in patients with localized and metastatic prostatic carcinoma: clinical implications.</a> Journal of Clinical Oncology. 13, 1195-1195 (1995).</li><li>Loberg, R., Fridman, Y., Pienta, B., Keller, E., McCauley, L., Taichman, R., Pienta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+isolation+of+circulating+tumor+cells+in+urologic+cancers:+a+review.">Detection and isolation of circulating tumor cells in urologic cancers: a review.</a> Neoplasia. 6, 302-302 (2004).</li><li>Viator, J. A., Gupta, S. K., Goldschmidt, B. S., Bhattacharyya, K., Raghuraman, R., Shukla, R., Dale, P. S., Boote, E., Katti, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+gold+nanoparticle+enhanced+prostate+cancer+cells+using+photoacoustic+flowmetry+with+optical+reflectance.">Detection of gold nanoparticle enhanced prostate cancer cells using photoacoustic flowmetry with optical reflectance.</a> J. Biomed. Nanotech. 6, 187-191 (2010).</li></ol>

John A. Viator è fondatore e presidente di Viator Technologies Inc, una società costituita per commercializzare le tecnologie fotoacustico per il miglioramento della salute umana.

Rilevare CTC è ancora un campo di intensa ricerca con applicazioni cliniche pochissime a causa del difficile problema di isolare le cellule tumorali rare. Molte altre tecniche sono in corso di valutazione per la rilevazione CTC, tra RT-PCR, la cattura delle cellule microfluidica, la cattura immunomagnetica, e altri metodi 8-12. Tuttavia, l&#39;individuazione fotoacustico di spettacoli CMC promessa in quanto è l&#39;etichetta gratuita e fornisce un mezzo rapido e preciso per catturare piccole particelle che assorbono la luce. Il protocollo descritto per isolare le cellule bianche del sangue è altamente efficiente, ma un numero basso di globuli rossi presenti nel campione. Queste cellule rouge contaminando anche assorbire la luce laser, ma ad un livello notevolmente diminuito. Per garantire i globuli rossi non interferiscono con il nostro sistema di rilevazione melanoma, cinque campioni di pazienti sani sono stati introdotti attraverso il sistema e nessuno ha dato un segnale, indicando che i globuli rossi sono presenti a basse concentrazioni abbastanzaessere ignorato. Una serie di studi di concentramento sono stati condotti utilizzando cellule di melanoma in coltura e del sistema a flusso fotoacustico si è dimostrato sensibile a 1 cellula / mL (dati non pubblicati). Questi studi sono in corso e presto includere prove usando campioni di pazienti di cancro. Questa tecnica fotoacustico è anche in corso di valutazione per l&#39;uso in non pigmentate CTC, come al seno e alla prostata allegando cromofori esogeni. Abbiamo effettuato un lavoro preliminare utilizzando nanoparticelle d&#39;oro sul cancro cells13. Questi test hanno dimostrato che le nanoparticelle in grado di fornire il necessario assorbimento ottico per generare onde fotoacustico. La sfida restante tecnico è quella di allegare tali particelle selettivamente alle cellule tumorali.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> ABCD1234 </td><td></td></tr></tbody></table>

detection and isolation of circulating melanoma cells using photoacoustic flowmetry

Cellule tumorali circolanti (CTC) sono quelle cellule che hanno separato da un tumore macroscopico e diffuso attraverso i sistemi di sangue e la linfa ai tumori secondari seme 1,2,3. CTC sono indicatori della malattia metastatica e la loro rilevazione in campioni di sangue possono essere utilizzati per diagnosticare il cancro e monitorare la risposta del paziente alla terapia. Dal momento che CTC sono rare, composto da circa una cellula tumorale tra miliardi di cellule del sangue normali in pazienti con cancro avanzato, la loro individuazione e l&#39;enumerazione è un compito difficile. Sfruttiamo la presenza del pigmento nella maggior parte delle cellule di melanoma per generare fotoacustica, o laser indotta onde ultrasoniche in un citofluorimetro personalizzati per la rilevazione di cellule di melanoma circolanti (CMC) 4,5. Questo processo comporta la separazione di un campione di sangue intero mediante centrifugazione e di ottenere il bianco strato di cellule del sangue. Se presente nel sangue intero, CMC sarà separato con i globuli bianchi a causa della densità simile. Queste cellule sono risospese intampone fosfato (PBS) ed introdotti nel misuratore di portata. Piuttosto che un flusso continuo della sospensione delle cellule del sangue, ci ha indotto due fasi del flusso al fine di catturare queste cellule per ulteriori studi. In due fasi del flusso, due liquidi immiscibili in un sistema di microfluidica si incontrano ad un bivio e la forma si alternano spezzoni di liquido 6,7. PBS sospesi i globuli bianchi e l&#39;aria lumache microlitro forma che sono in sequenza irradiati con luce laser. L&#39;aggiunta di un tensioattivo alla fase liquida permette di formazione uniforme lumaca e l&#39;utente può creare diversi proiettili di dimensioni modificando le portate delle due fasi. Lumache di aria e lumache di PBS con i globuli bianchi non contengono assorbitori di luce e, di conseguenza, non producono onde fotoacustico. Tuttavia, lumache di globuli bianchi che contengono anche CMC solo assorbono la luce laser e di produrre onde acustiche ad alta frequenza. Queste lumache che generano onde fotoacustico vengono sequestrati e raccolti per la colorazione citochimica per verificzione di CMC.

Watch this Scientific Journal Video about Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry at JoVE.com

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Abbiamo sviluppato un citofluorimetro mediante ultrasuoni laser indotto a rilevare in circolo cellule di melanoma come un indicatore precoce della malattia metastatica.

Rilevazione e isolamento delle cellule del melanoma circolanti con flussimetria fotoacustico

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

detection-isolation-circulating-melanoma-cells-using-photoacoustic

Research

JoVE Journal

Bioengineering

15.7K Views.  University of Missouri. Abbiamo sviluppato un citofluorimetro mediante ultrasuoni laser indotto a rilevare in circolo cellule di melanoma come un indicatore precoce della malattia metastatica.

Video: Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

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 ...

Ricerca

Bioingegneria

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

Isolamento rapido di cellule tumorali circolanti da campioni di sangue del paziente

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Rilevamento e identificazione batterica Utilizzando Sensori elettrochimici

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

Integrato oftalmoscopia fotoacustico e Spectral-dominio Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

Photoacoustic Cystography

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The risk of bladder cancers in children due to radiation exposure is more significant than many other cancers. Here we demonstrate the feasibility of nonionizing and noninvasive photoacoustic (PA) imaging of urinary bladders, referred to as photoacoustic cystography (PAC), using near-infrared (NIR) optical absorbents (i.e. methylene blue, plasmonic gold nanostructures, or single walled carbon nanotubes) as an optical-turbid tracer. We have successfully imaged a rat bladder filled with the optical absorbing agents using a dark-field confocal PAC system. After transurethral injection of the contrast agents, the rat's bladders were photoacoustically visualized by achieving significant PA signal enhancement. The accumulation was validated by spectroscopic PA imaging. Further, by using only a laser pulse energy of less than 1 mJ/cm2 (1/20 of the safety limit), our current imaging system could map the methylene-blue-filled-rat-bladder at the depth of beyond 1 cm in biological tissues in vivo. Both in vivo and ex vivo PA imaging results validate that the contrast agents were naturally excreted via urination. Thus, there is no concern regarding long-term toxic agent accumulation, which will facilitate clinical translation.

Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.

Cistografia fotoacustico

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Trapianto di progenitori miogenici pluripotenti derivati da cellule staminali indotte simili a mesoangioblasti nei modelli di topo di rigenerazione muscolare

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

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 ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Preparazione e fotoacustico Analisi dei veicoli cellulari contenenti oro Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

Di circolazione MicroRNA Quantificazione Utilizzando DNA-binding Dye Chimica e di gocce Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

Microscopio fotoacustico acustico e ottico<em&gt; In Vivo</em&gt; Piccolo animale Blood Imaging di Vasculature

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...