Nous reconnaissons l&#39;appui du Département de Génie Biologique et le Christopher S. Bond Life Sciences Center de l&#39;Université du Missouri. Nous reconnaissons le soutien octroi du Missouri Life Sciences Research Board 09-1034 et les NIH R21CA139186-0. Nous remercions également les sciences de la vie Programme de premier cycle Research Opportunity à l&#39;Université du Missouri, de l&#39;Université du Missouri moléculaire Cytologie Core et l&#39;Université du Missouri College of Engineering de soutien financier.

1. Préparation de l&#39;échantillon de sang <ol><li> Dessinez environ 10 ml de sang entier d&#39;un patient du cancer de stade IV en deux 5 ml vacutainers. </li><li> Centrifuger les tubes pendant dix minutes à 3000 tours par minute (rpm). </li><li> L&#39;échantillon de sang sera séparé en trois couches différentes: la couche inférieure comprend les érythrocytes, la couche intermédiaire, appelée aussi la couche leucocytaire, contient les leucocytes et les cellules de mélanome, et la couche supérieure contient le plasma et les plaquettes. Retirez le plasma autant que possible sans perturber la couche leuco-plaquettaire. </li><li> Placer 250 pi d&#39;Histopaque 1077 dans deux tubes 1 ml Wintrobe. </li><li> Avec une pipette 9 &quot;Pasteur, extraire le plasma restant, la couche leuco-ensemble, et même la couche supérieure des globules rouges, en veillant à collecter &lt;500 ul. </li><li> Placer la solution au sommet de la couche d&#39;Histopaque et centrifuger les tubes Wintrobe dans 15 ml tubes coniques de 10 minutes à 3000 rpm. </li><li> Le sang sera de nouveauséparés, mais cette fois les érythrocytes seront séparés par le Histopaque, permettant l&#39;extraction facile de la couche leuco-plaquettaire. Utilisez un 9 &quot;pipette Pasteur pour obtenir la couche leuco-ensemble et le placer dans un tube Eppendorf de 2 ml. </li><li> Suspendre la couche leucocytaire dans 1,5 ml de PBS avec 2% v / v de Tween 20. </li></ol> 2. Construction Chambre de circulation <ol><li> Obtenir un cylindre acrylique, 20 mm de diamètre extérieur, 17 mm de diamètre intérieur, et de 8 mm de haut. Percez trois trous 5 mm de diamètre dans le côté du cylindre à angles de 90 degrés les uns des autres. Couper trois morceaux de tube polymère (1,6 mm de diamètre interne, 5 mm de diamètre extérieur), et les placer dans les trous percés. </li><li> Étirez Parafilm à travers le fond de l&#39;anneau de l&#39;acrylique pour faire un joint étanche à l&#39;eau. Le parafilm forme un bol peu profond avec l&#39;anneau acrylique et tiendra la solution d&#39;acrylamide qui forment une chambre de flux. </li><li> Suspendre un fil de 1,6 mm de diamètre à travers les morceaux de tubes qui sont en face de lal&#39;autre, en s&#39;assurant que le fil seul est visible à travers le milieu du ring. Remplissez le troisième morceau de tube avec un fil de 1,6 mm de diamètre et la position du tube 1 mm du fil de suspension. Ces fils de prévenir l&#39;acrylamide de pénétrer dans le tuyau. Après l&#39;acrylamide se solidifie, le fils sera extraite, laissant une voie d&#39;écoulement cylindrique et un endroit pour placer une fibre optique se concentre sur le chemin d&#39;écoulement. </li><li> Ajouter 5 ml d&#39;acrylamide préparés [20 g d&#39;acrylamide (Sigma Aldrich), 0,7 g de bisacrylamide (Sigma Aldrich), 100 ml d&#39;eau distillée] pour un petit bécher. Mesurer 0,02 g de persulfate d&#39;ammonium (Sigma) et l&#39;ajouter au bécher. Mélanger avec une barre de remuer pendant 3 minutes. Ajouter 20 uL tetramethylethylenediamene (Fisher Scientific) dans le bécher. Verser rapidement le mélange dans l&#39;anneau acrylique, le remplissage vers le haut. Après quelques secondes, la solution sera de gel, fabriquer un moule chambre d&#39;écoulement. Retirez délicatement les fils hors, en veillant à ne pas perturber le gel d&#39;acrylamide. La Cham des fluxbre est maintenant prêt à utiliser. </li></ol> 3. Débitmétrie photoacoustique mise en place <ol><li> Acoustiquement quelques un transducteur piézoélectrique acoustiques fluorure de polyvinylidène (Panametrics) pour une chambre d&#39;écoulement à l&#39;aide de gel échographique directement au-dessus du chemin d&#39;écoulement. </li><li> L&#39;utilisation d&#39;un Bayonet Neill-Concelman (BNC) par câble, connecter le capteur à l&#39;entrée d&#39;un récepteur large bande RITEC-640A (filtre passe-bas: out, filtre passe-haut: 1 MHz, imput impédance de 50 Ohms, d&#39;amplification: 35 dB). Connectez la sortie du filtre à l&#39;oscilloscope (Tektronix TDS 2024B). Pour déclencher l&#39;oscilloscope, une photodiode (Thorlabs) est établi à proximité du capteur et est branché sur l&#39;oscilloscope en utilisant un câble BNC. </li><li> Un connecteur en T (petites pièces) est attaché à un bras de la chambre d&#39;écoulement. Deux pompes à seringues (Braintree scientifique) sont connectés aux autres branches du connecteur. Une seringue contient de l&#39;air, tandis que l&#39;autre contient la suspension cellulaire préparée dans la partie 1. Régler les débits à100 ul / minute. </li><li> Extraire le tube de la chambre de petite couler jusqu&#39;à une petite poche d&#39;air de formes, et d&#39;insérer une fibre de 1 mm de diamètre coeur optique ayant une ouverture numérique 0,37 dans cet emplacement. Remplissez la poche avec de l&#39;eau afin de réduire les signaux d&#39;interface et d&#39;augmenter la quantité de lumière laser appliquée à la voie d&#39;écoulement. En raison de l&#39;ouverture numérique de la fibre optique et à courte distance de la trajectoire d&#39;écoulement, le laser ne irradient une limace de l&#39;échantillon à un moment donné, faire en sorte que l&#39;échantillon directement en face du laser est le même échantillon de créer des signaux photoacoustique. L&#39;énergie du laser devrait être d&#39;environ 28 mJ / cm 2 avec une taille de spot de 7 mm 2. </li><li> Tournez les deux pompes seringue sur. Lorsque les deux fluides non miscibles atteindre la jonction en T, ils seront divisées en limaces homogène et des flux passés du transducteur. </li><li> Irradier les échantillons qui coule avec un laser pulsé nanoseconde pour générer des ondes photoacoustique dans les cellules de mélanome. Comme la mélanine est un broadbet optiques d&#39;absorption, toute longueur d&#39;onde laser visible peut être utilisé. </li><li> Si un signal apparaît sur l&#39;oscilloscope quand un slug est au sommet de la sonde, la limace contient un mélanome, et devraient être suivis par le système jusqu&#39;à tomber dans un flacon de collecte. </li></ol> 4. Immunocytochimie <ol><li> Prenez les limaces capturées à un Cytospin 4 Centrifugeuse (Thermo Scientific) afin de les fixer à des lames de verre. Placer les lames de verre en Cytofunnels (Shandon EZ Cytofunnel simple avec Filtre brun carte Thermo Scientific) et les charger dans la Cytospin 4 Centrifugeuse. Mettez 100 uL de 1% de sérumalbumine bovine (Sigma) dans du PBS dans chaque entonnoir et puis centrifuger à 600 rpm pendant 3 minutes afin d&#39;aider les cellules capturées coller à la lame de verre. </li><li> Après centrifugation, ajouter les échantillons de cellules à l&#39;Cytofunnels puis centrifuger à 600 rpm pendant 3 minutes. Vaporiser ensuite les lames avec un fixateur à base d&#39;éthanol. </li><li> Laver les lames dans du PBS pendant 10 minutes deux fois. </li><li&gt; Pour bloquer les liaisons non spécifiques, incuber chaque diapositive avec un mélange de 10 ul de sérum de chèvre (Sigma) et 90 ul de PBS pendant 1 heure à température ambiante. </li><li> Après l&#39;incubation, décanter les diapositives et incuber avec la solution d&#39;anticorps primaire pendant 1 heure dans une chambre humide à température ambiante. Chaque diapositive doit contenir 1% MART-1 anticorps élevé chez le lapin, 1% de CD-45 anticorps élevé chez la souris, du sérum de chèvre 10%, et 89% de PBS. </li><li> Après incubation, laver les lames dans du PBS pendant 10 minutes trois fois. </li><li> Les lames sont ensuite incubés dans une solution d&#39;anticorps secondaire, qui a fluorophore lié à l&#39;anticorps. Le mélange contient 0,05% de chèvre anticorps anti-lapin, 0,05% de chèvre anticorps anti-souris, du sérum de chèvre de 0,1%, et 99% de PBS. Les lames sont incubées dans l&#39;obscurité pendant 1 heure à température ambiante. </li><li> Après incubation, décanter les diapositives laver dans du PBS pendant 5 minutes trois fois. </li><li> Pour les taches pour un noyau, incuber les cellules avec 0,1 pg / ml de DAPI (Invitrogen) pour1 minute. Puis transvaser les diapositives et les laver dans du PBS. </li><li> Monter une lamelle sur l&#39;échantillon de cellules au moyen de 20 uL d&#39;un média à base de résine de fixation par lame. Sceau de la lamelle avec du vernis à ongles transparent pour préserver la diapositive. Maintenant, les diapositives sont prêts à être imagées avec un microscope à fluorescence. </li></ol> 5. Les résultats représentatifs: <img src="/files/ftp_upload/3559/3559fig1.jpg" alt="Figure 1"/> Figure 1. Les formes d&#39;onde photoacoustique à partir de globules blancs et cellules de mélanome sont montrés ici. Les globules blancs, n&#39;ayant pas de pigmentation inhérents, ne produisent pas de vagues photoacoustique et manifeste une ligne fixe de bruit électronique (à gauche). Les cellules du mélanome pigmentées produit une onde robustes photoacoustique (à droite). <img src="/files/ftp_upload/3559/3559fig2.jpg" alt="Figure 2"/> Figure 2. Après coloration immunocytochimique, les cellules de mélanome en culture montrent des signaux DAPI dans blue tout MART1 est surligné en vert. <img src="/files/ftp_upload/3559/3559fig3.jpg" alt="Figure 3"/> Figure 3. Superposition des images pour DAPI et MART1, comme dans la Figure 2, avec CD45 comme un indicateur de leucocytes, cette figure montre les cellules de mélanome parmi plusieurs globules blancs.

<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 est le fondateur et président de Technologies Viator Inc, une société formée pour commercialiser des technologies photoacoustique pour l&#39;amélioration de la santé humaine.

Détecter les CTC est encore un domaine de recherche intense avec très peu d&#39;applications cliniques en raison de l&#39;épineux problème d&#39;isoler les cellules tumorales rares. Beaucoup d&#39;autres techniques sont en cours d&#39;évaluation pour la détection de la CCT, y compris la RT-PCR, la capture cellulaire microfluidique, la capture immunomagnétique, et d&#39;autres méthodes 8-12. Cependant, la détection photoacoustique de spectacles CMC promets que c&#39;est une étiquette libre et fournit un moyen rapide et précis pour capturer les petits, légers particules absorbant. Le protocole décrit à isoler les globules blancs est très efficace, mais un faible nombre de globules rouges dans l&#39;échantillon. Ces cellules Rouge contaminent également absorber la lumière laser, mais à un niveau considérablement diminué. Pour assurer les globules rouges ne pas interférer avec notre système de détection du mélanome, cinq échantillons de patients sains ont été introduits dans le système et aucun a donné un signal, indiquant que les globules rouges sont présents à de faibles concentrations suffisantes pourêtre négligé. Une série d&#39;études de concentration ont été réalisées en utilisant des cellules de mélanome en culture et le système de flux photoacoustique a prouvé sensibles à 1 cellule / uL (données non publiées). Ces études sont en cours et comprendra bientôt des essais en utilisant des échantillons patient atteint de cancer. Cette technique photoacoustique est également en cours d&#39;évaluation pour une utilisation chez les non-pigmentées CTC, comme du sein et cancer de la prostate en attachant des chromophores exogènes. Nous avons effectué des travaux préliminaires en utilisant des nanoparticules d&#39;or sur le cancer cells13. Ces tests ont montré que les nanoparticules peuvent fournir l&#39;absorption optique nécessaire pour générer des ondes photoacoustique. Le défi reste technique consiste à fixer ces particules aux cellules cancéreuses de manière sélective.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires </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

Des cellules tumorales circulantes (CTC) sont les cellules qui sont séparés d&#39;une tumeur macroscopique et la propagation à travers les systèmes sanguin et lymphatique des tumeurs secondaires de graines 1,2,3. CTC sont des indicateurs de la maladie métastatique et leur détection dans les échantillons sanguins peuvent être utilisés pour diagnostiquer le cancer et surveiller la réponse du patient au traitement. Depuis CTC sont rares, comprenant environ une cellule tumorale parmi des milliards de cellules sanguines normales chez les patients cancéreux avancés, leur détection et leur énumération est une tâche difficile. Nous exploitons la présence du pigment dans les cellules de mélanomes les plus à générer photoacoustique, ou induite par laser ondes ultrasonores dans un cytomètre de flux personnalisées pour la détection des cellules de mélanome en circulation (CMC) 4,5. Ce processus implique la séparation d&#39;un échantillon de sang total par centrifugation et l&#39;obtention de la couche de globules blancs. Si elle est présente dans le sang total, les CMC seront séparées avec les globules blancs à cause de la densité similaire. Ces cellules sont remises en suspension danstampon phosphate salin (PBS) et introduite dans le débitmètre. Plutôt que d&#39;un flux continu de la suspension de cellules sanguines, nous avons provoqué deux flux de phase afin de capturer ces cellules pour une étude plus approfondie. Dans deux flux de phase, deux liquides non miscibles dans un système microfluidique répondre à un carrefour et la forme en alternance les limaces de 6,7 liquide. PBS a suspendu les globules blancs et les limaces de l&#39;air sous forme microlitre qui sont séquentiellement irradiés avec une lumière laser. L&#39;ajout d&#39;un tensioactif à la phase liquide permet la formation limaces uniforme et l&#39;utilisateur peut créer différentes limaces de taille en modifiant les débits des deux phases. Les limaces de l&#39;air et les limaces de PBS avec les globules blancs ne contiennent pas d&#39;absorbeurs de lumière et donc, ne produisent pas de vagues photoacoustique. Toutefois, les limaces de globules blancs qui contiennent même des CMC seule absorber la lumière laser et de produire des ondes acoustiques de haute fréquence. Ces limaces qui génèrent des ondes photoacoustique sont séquestrés et collectées pour la coloration cytochimique pour verifiction de la 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

Nous avons développé un cytomètre en flux en utilisant des ultrasons pour détecter induite par laser circulant cellules de mélanome comme un indicateur précoce de la maladie métastatique.

Détection et isolement des cellules du mélanome de circulation à l&#39;aide Débitmétrie photoacoustique

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 vues.  University of Missouri. Nous avons développé un cytomètre en flux en utilisant des ultrasons pour détecter induite par laser circulant cellules de mélanome comme un indicateur précoce de la maladie métastatique.

Vidéo: 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.

Tridimensionnelle optique résolution microscopie photoacoustique

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

Recherche

Bio-ingénierie

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.

Isolement rapide des cellules tumorales circulantes viables partir d&#39;échantillons sanguins des patients

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.

Bacterial Detection &amp; identification au moyen de capteurs électrochimiques

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.

Intégré ophtalmoscopie photoacoustique et Spectral-domaine tomographie par cohérence optique

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.

Cystographie Photoacoustique

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.

Transplantation de progéniteurs myoangioblastes dérivés de cellules souches pluripotentes induites dans des modèles murins de régénération musculaire

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.

Simple détection de molécule libre Label-Utilisation Microtoroid optiques résonateurs

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.

Préparation et Photoacoustique Analyse des véhicules cellulaires contenant de l&#39;or 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.

Circulant microARN Quantification Utilisation DNA-binding Dye Chemistry and Droplet PCR numérique

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.

Microscopie photoacoustique à résolution acoustique et optique à commutation commutable pour<em&gt; In vivo</em&gt; Imagerie vasculaire de sang de petit animal

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