Biz Biyoloji Mühendisliği ve University of Missouri Christopher S. Bond Life Sciences Merkezi Bölümü desteğiyle kabul etmiş sayılırsınız. Biz Missouri Yaşam Bilimleri Araştırma Kurulu&#39;nun 09-1034 ve NIH R21CA139186-0 hibe desteği kabul etmiş sayılırsınız. Ayrıca, mali destek için, Missouri, Missouri Moleküler Sitoloji Core Üniversitesi ve University of Missouri College of Engineering Üniversitesi Yaşam Bilimleri Araştırma Fırsat Lisans Programı teşekkür ederiz.

1. Kan numune hazırlama <ol><li> Evre IV Kanser hastanın tam kan yaklaşık 10 ml, 5 ml iki vacutainers içine çizin. </li><li> On dakika boyunca dakikada 3.000 devrimler (rpm) tüpleri santrifüjleyin. </li><li> Kan numunesi üç farklı katmanları ayırmak, buffy coat denilen eritrosit, orta tabaka, alt tabaka oluşur, lökositler ve melanoma hücreleri içeren ve üst tabaka, plazma ve trombosit içerir. Buffy coat rahatsız etmeden, mümkün olduğu kadar plazma gibi çıkarın. </li><li> Yeri 250, iki adet 1 ml Wintrobe tüpleri Histopaque 1077 mcL. </li><li> 9 &quot;Pasteur Pipet kullanarak, &lt;500 mcL toplamak için emin olun, kalan plazma, tüm buffy coat, ve hatta en üst tabakası eritrositlerin ayıklayın. </li><li> Çözüm Histopaque katmanı tepesine yerleştirin ve 15 ml konik tüpler 3000 rpm&#39;de 10 dakika süreyle Wintrobe tüpler santrifüj. </li><li> Kan yenidenayrı, ama bu sefer eritrositler buffy coat kolay çıkarılması için izin Histopaque ayrılmış olacak. 9 &quot;Pasteur Pipet tüm buffy coat almak ve 2 ml Eppendorf tüp yerleştirmek için kullanın. </li><li> % 2 v / v Tween 20 ile 1.5 mL PBS buffy coat askıya alınması. </li></ol> 2. Akış Odası İnşaat <ol><li> Akrilik bir silindir, 20 mm dış çap, 17 mm iç çapı, 8 mm ve uzun boylu alın. Birbirlerine 90 derecelik açıyla yan silindir ile üç adet 5 mm çapında delikler delin. Polimer boru üç adet (1.6 mm iç çapı 5 mm dış çapı) Kes ve delinmiş deliklerin içine koyun. </li><li> Su geçirmez bir mühür yapmak için akrilik halkasının alt kısmında Parafilm gerin. Parafilm akrilik halka ile sığ bir kase formları ve bir akış odası oluşturacak olan akrilamid çözüm yapacak. </li><li> Karşısında boru parçaları ile 1.6 mm çaplı tel askıyasadece tel ring ortasında görünür olduğundan emin birbirlerine. Üçüncü parça, 1.6 mm çapında tel ve konumu tüp asma tel 1 mm ile boru doldurun. Bu teller tüp girmesini önlemek akrilamid. Akrilamid katılaşır sonra, teller, silindirik bir akış yolu ve bir yere bırakarak, bir fiber optik konumuna çıkartılacaktır akış yolu üzerine odaklanır. </li><li> [20 g akrilamid (Sigma Aldrich), 0.7 g bisacrylamide (Sigma Aldrich), 100 ml distile su] küçük bir beher 5 ml hazırlanan akrilamid ekle. 0.02 g amonyum persülfat (Sigma) ölçün ve beher ekleyin. 3 dakika boyunca bir karıştırıcı ile karıştırın. Beher 20 mcL tetramethylethylenediamene (Fisher Scientific) ekleyin. Hızla üst dolgu, akrilik halka içine karışımı dökün. Birkaç saniye sonra, çözüm, jel, bir akış odasında kalıp yapım olacak. Dikkatlice akrilamid jel rahatsız değil emin olun, teller çekin. Akış chamber artık kullanıma hazırdır. </li></ol> 3. Foto akustik flowmetre set-up <ol><li> Akustik çift ultrason jel kullanılarak doğrudan akış yolu üzerinde bir akış odasına polivinil florür piezoelektrik akustik transdüser (Panametrics). </li><li> Süngü Neill-Concelman (BNC) kablo kullanarak, bir RITEC Geniş Bant Alıcı-640A girişi (çıkışı, yüksek geçiren filtresi: 1 MHz Giriş Empedansı: 50 Ohm, amplifikasyon: 35 dB alçak geçiren filtre) transdüser bağlayın. Filtre çıkışı osiloskop (Tektronix TDS 2024B) bağlayın. Osiloskop tetiklemek için bir fotodiyot (Thorlabs) transdüser yakın kurmak ve bir BNC kablosu kullanarak osiloskop takılı. </li><li> Bir t-konnektör (Küçük Parça) akış odasının bir kola takılır. İki şırınga pompaları (Braintree Bilimsel) konektör diğer dallara bağlanır. Diğer bölüm 1 hazırlanan hücre süspansiyonu içeren bir şırınga, hava içerir. Akış oranları ayarlayın.100 mcL / dakika. </li><li> Akış odasının küçük tüp hava formlarının küçük bir cep kadar ayıklayın ve bu yuvaya 0.37 sayısal diyafram ile 1 mm çap çekirdekli fiber optik yerleştirin. Arabirim sinyallerini azaltmak ve akış yolu uygulanan lazer ışığı miktarı artırmak için su ile cebe doldurun. Lazer, fiber optik ve akış yolu kısa mesafe sayısal diyafram nedeniyle, doğrudan lazer önünde örnek foto akustik sinyalleri oluşturarak aynı örnek olmasını sağlamak, herhangi bir zamanda örnek bir sümüklü böcek ışın tedavisi olacaktır. Lazer enerjisi, bir spot büyüklüğü ile 7 mm 2 yaklaşık 28 mJ / cm 2 olmalıdır . </li><li> Iki şırınga pompaları açın. Iki karışmayan sıvılar t-kavşak yerine varınca homojen salyangozlar ve transdüser geçmiş akışı içine bölümlenmiş olacaktır. </li><li> Nanosaniyelik melanom hücrelerinde foto akustik dalgaları oluşturmak için lazer darbeli akan örnek ışın tedavisi. Melanin bir broadb olduğu gibive optik emici, görünür bir lazer dalga boyu kullanılabilir. </li><li> Bir sümüklü böcek transdüser tepesine bir sinyal osiloskop belirirse, sümüklü böcek melanom içeren ve bir koleksiyon şişenin içine bırakarak kadar sistem üzerinden takip edilmelidir. </li></ol> 4. İmmünositokimya <ol><li> Cam slaytlar bunları düzeltmek amacıyla yakalanan salyangozlar sitospin 4 Santrifüj (Thermo Scientific) ele alalım. Place cam Cytofunnels slaytlar (Kahverengi Filtre Kart Thermo Scientific Shandon EZ Tek Cytofunnel) ve sitospin 4 Santrifüj içine yerleştirin. Her huni içine PBS içinde% 1 (Sigma) Bovine Serum Albumin 100 mcL koyun ve daha sonra yakalanan hücrelerin cam slaytlar sopa yardımcı olmak için 3 dakika boyunca 600 rpm&#39;de santrifüj. </li><li> Santrifüj sonra, hücre örnekleri Cytofunnels ekleyin ve daha sonra 3 dakika boyunca 600 rpm&#39;de santrifüj. Sonra bir etanol bazlı fiksatif slaytlar spreyi. </li><li> PBS içinde slaytları iki kez 10 dakika boyunca yıkayın. </li><li&gt;, nonspesifik bağlanma blok Keçi Serumu 10 mcL (Sigma) ve oda sıcaklığında 1 saat 90 mcL PBS karışımı ile her slayt kuluçkaya yatmaktadır. </li><li> Inkübasyondan sonra, birincil antikor çözeltisi ile nemlendirilmiş bir odasında oda sıcaklığında 1 saat inkübe slaytlar ve süzün. Her slayt% 1 tavşan büyüdü SMART-1 antikor fare gündeme CD-45 antikor,% 1,% 10 Keçi serumu ve% 89 PBS içermelidir. </li><li> Inkübasyondan sonra, slaytları PBS içinde 10 dakika üç kez yıkayın. </li><li> Daha sonra slaytlar fluorofor antikorlara bağlı ikincil bir antikor çözüm, inkübe edilir. Karışımı anti-tavşan antikoru% 0.05 keçi,% 0.05 &#39;i keçi anti-fare antikor,% 0.1&#39; keçi serumu ve% 99 PBS içerir. Slaytlar, oda sıcaklığında 1 saat boyunca karanlıkta inkübe edilir. </li><li> Inkübasyondan sonra, slaytlar, 5 dakikada üç kez PBS içinde yıkayın süzün. </li><li> Bir çekirdek için leke DAPI 0.1 mikrogram / mL (Invitrogen) hücreleri inkübasyon1 dakika. Sonra süzün ve slaytları PBS içinde yıkayın. </li><li> 20 mcL kullanarak slayt başına bir reçine bazlı montaj medya hücre örneği üzerinde lamel monte edin. Slayt korumak için şeffaf tırnak cilası ile lamel Seal. Şimdi slaytlar floresan mikroskop ile görüntülü olmaya hazırız. </li></ol> 5. Temsilcisi Sonuçlar: <img src="/files/ftp_upload/3559/3559fig1.jpg" alt="Şekil 1"/> Şekil 1 beyaz kan hücreleri ve melanom hücrelerinin foto akustik dalga şekilleri burada gösterilmiştir. Doğasında herhangi bir pigmentasyon olan beyaz kan hücreleri, elektronik gürültü düz bir çizgi (sol) olarak, hiçbir foto akustik dalgaları ve tezahür üretmek. Pigmentli melanom hücrelerinin sağlam bir foto akustik dalga (sağda) üretir. <img src="/files/ftp_upload/3559/3559fig2.jpg" alt="Şekil 2"/> Şekil 2. immünositokimyasal boyama sonra, kültürlü melanom hücrelerinin bl DAPI sinyalleri gösteriyorue ise MART1 yeşil vurgulanır. <img src="/files/ftp_upload/3559/3559fig3.jpg" alt="Şekil 3"/> Şekil 3. CD45 lökositlerin bir göstergesi olarak, Şekil 2&#39;de, DAPI ve MART1 görüntüleri kaplanacağı, bu rakam, bazı beyaz kan hücreleri arasında melanom hücreleri gösterir .

<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. 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John A. Viator Viator Technologies Inc, insan sağlığının iyileştirilmesi için foto akustik teknolojileri ticarileştirmek için kurulmuş bir şirket kurucusu ve başkanı.

Nedenle kapalı zaman eğrileri algılama nadir tümör hücreleri izole zor bir problem nedeniyle çok az sayıda klinik uygulamaları ile hala yoğun bir araştırma alanı. CTC algılama, RT-PCR, mikroakışkan hücre yakalama, immunomagnetic yakalama ve diğer yöntemlerle 8-12 dahil pek çok diğer teknikler değerlendirilmektedir . Ücretsiz etiket ve küçük, ışık emici parçacıkları yakalamak için hızlı ve doğru bir yol sağlar Ancak, CMCs gösterir foto akustik algılama söz veriyorum. Protokol, beyaz kan hücreleri izole etmek için açıklanan son derece verimli, ama kırmızı kan hücrelerinin az sayıda örnek var. Bu rouge kirlenmesine neden olan hücreleri de lazer ışığı emer, ancak büyük ölçüde azalmış bir düzeyde. Kırmızı kan hücreleri, bizim melanom algılama sistemi ile karışmaz sağlamak için, sistem üzerinden beş sağlıklı hasta numunelerinin getirilmiştir ve hiçbiri, kırmızı kan hücrelerinin yeterince düşük konsantrasyonlarda mevcut olduğunu gösteren bir sinyal verdidikkate alınmayacaktır. Konsantrasyon çalışmaları bir dizi kültür melanoma hücreleri kullanarak ve foto akustik akış sistemi hassas aşağı 1 hücre / mcL (yayınlanmamış veri) olduğu kanıtlanmıştır yapılmıştır. Bu çalışmalar, devam eden ve yakında kanser hasta numuneleri kullanılarak çalışmalarda yer alacak. Bu foto akustik tekniği de eksojen kromoforlar ekleyerek meme ve prostat kanseri gibi olmayan pigmentli nedenle kapalı zaman eğrileri, kullanmak için değerlendirilmektedir. Biz kanser cells13 altın nanopartiküller kullanarak ön çalışma gerçekleştirdik. Bu testler nanopartiküller foto akustik dalgaları üretmek için gerekli optik soğurma sağlayabileceğini göstermiştir. Kalan teknik zorluk gibi parçacıklar kanser hücrelerini seçici eklemek.

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </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

Dolaşımdaki tümör hücreleri (nedenle kapalı zaman eğrileri), makroskopik tümör ayrılmış ve kan ve lenf sistemleri tohum ikincil tümörler 1,2,3 yoluyla yayılan bu hücrelerdir . Nedenle kapalı zaman eğrileri metastatik hastalığı ve kan örnekleri algılama göstergeleri kanser teşhis ve hastanın tedaviye yanıtı izlemek için kullanılıyor olabilir. Nedenle kapalı zaman eğrileri nadir olduğundan, bir tümör ileri evre kanser hastalarında, normal kan hücreleri milyarlarca arasında hücre, bunların tespiti ve sayımı ile ilgili oluşan zor bir iştir. Foto akustik oluşturmak için en melanom hücrelerinde pigment varlığı istismar veya lazer, ultrasonik dalgalar dolaşan melanom hücrelerinin (CMCs) 4,5 tespiti için özel bir akış sitometresi indüklenen . Bu süreç, santrifüj kullanılarak ve beyaz kan hücre tabakasından elde tam kan örneği ayıran gerektirir. Varsa tam kan, CMCs beyaz kan hücreleri ile benzer yoğunluğu nedeniyle ayrı olacaktır. Bu hücreler yeniden süspansefosfat tamponlu salin (PBS) ve debimetre içine tanıtıldı. Kan hücre süspansiyonu sürekli bir akış yerine, daha fazla çalışma için bu hücreleri yakalamak için iki faz akış kaynaklı. İki fazlı akış, bir mikroakışkan sistemde iki karışmayan sıvılar sıvı 6,7 salyangozlar bir alternatif kavşak ve form buluşuyor. PBS, sırayla, lazer ışığı ile ışınlanmış beyaz kan hücreleri ve hava form mikrolitrelik salyangozlar askıya aldı. Sıvı faz bir yüzey yanı sıra düzgün sümüklü böcek oluşumunu sağlar ve kullanıcı iki faz akış oranları değiştirerek farklı büyüklükteki salyangozlar oluşturabilirsiniz. Beyaz kan hücreleri ile hava ve PBS salyangozlar salyangozlar ışık emiciler içeren ve dolayısıyla, foto akustik dalgaları üretmek değil. Ancak, salyangozlar, hatta tek bir CMCs içeren beyaz kan hücrelerinin lazer ışığı emer ve yüksek frekanslı akustik dalgalar üretir. Foto akustik dalgaları oluşturan bu salyangozlar verific birikir ve Sitokimyasal boyama için toplananCMCs, ation.

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

Biz lazer kaynaklı ultrason kullanılarak dolaşımdaki melanom hücrelerinin metastatik hastalığın erken bir göstergesi olarak algılamak için bir akış sitometresinin geliştirdik.

Sirkülasyon Melanom Hücreler foto akustik flowmetre kullanarak Algılama ve İzolasyonu

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 Görüntüleme.  University of Missouri. Biz lazer kaynaklı ultrason kullanılarak dolaşımdaki melanom hücrelerinin metastatik hastalığın erken bir göstergesi olarak algılamak için bir akış sitometresinin geliştirdik.

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.

Üç boyutlu optik çözünürlük foto akustik Mikroskopi

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

Biyomühendislik

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.

Hasta Kan Örneklerinden Canlı Dolaşımdaki Tümör Hücreleri Rapid İzolasyonu

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.

Elektrokimyasal Sensörler kullanarak Bakteriyel Algılama ve Tanıma

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.

Entegre Photoacoustic Oftalmoskopi ve Spektral-domain optik koherens tomografi

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.

Foto akustik Sistografi

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.

Kas Yenilenmesinin Fare Modellerinde İndüklenmiş Pluripotent Kök Hücre türevi Mezoangioblast benzeri Miyojenik Progenitörlerin Nakli

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.

Microtoroid Optik Rezonatörler Kullanarak Etiket serbest Tek Molekül Algılama

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.

Altın nanorods İçeren Hücresel Araçların Hazırlama ve foto akustik Analizi

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.

MicroRNA Niceleme DNA bağlayıcı Boya Kimya ve Damlacık Dijital PCR kullanarak Sirkülasyon

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.

Için değiştirilebilir Akustik ve Optik Çözünürlük Fotoakustik Mikroskopi<emIn vivo</em&gt; Küçük Hayvan Kan Vaskülatür Görüntüleme

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