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

1. Deneysel Kurulum ve Numune Hazırlama <ol><li> , Litografi kullanarak gravür ve daha önce 6 açıklanan prosedür erime microtoroids Üretiyor. Tipik olarak 80-100 um büyük bir çapa ve 2 um küçük bir çapa sahip olan, bir silikon göbek (çip) üzerinde microtoroids üretmektedirler. </li><li> Kabaca kendi fiber makaradan tek modlu fiber optik (125 mikron kaplama, 4.3 mikron mod alan çapı) bir metre gevşeyin. </li><li> Fiber optiğin çözülür bölümünün ortasında, tel şeritler kullanılarak optik elyafın yaklaşık polimer kaplama gibi küçük bir bölümünü (2.5 cm) şerit. Not: Bu microtoroid içine evanescently birkaç ışığa kullanılacak fiber optiğin kısmıdır. </li><li> Izopropanol alkol ve ücretsiz silin bir tüy bırakmayan soyulmuş lif temizleyin. </li><li> Tutucu mıknatıs yapılan bir lif tutucu kullanılarak yerinde lif bu bölümünü tutun. </li><li> Meltin tarafından ~ 500 nm İnce elimden lifg ve hidrojen meşale ve 60 mm / dak zıt yönlerde hareket eden iki step motorlar kullanılarak çekme. ~ 10 mm boyunda olmalıdır alev üst kısmı, iç fiber optik yerleştirin. Fiber boyunca transmisyon (kapalı fiber yanıp yanal olarak dağılmış ve ışık izleme) veya bir osiloskop bağlanmış bir foto diyot için elyafın bağlanması yoluyla görsel olarak izlenebilir olan dalgalı durduğunda lif çekme durdurma . </li><li> Fiber optik bir ucunu yarmak ve çıplak fiber adaptöre takın. Foto alıcı girişine içine lif bu ucunu yerleştirin. </li><li> Fiber çift fiber optik bağlayıcı kullanılarak lazer fiber makaranın diğer ucu. </li><li> Epoksi veya çift taraflı bant kullanarak bir numune tutucu üstüne microtoroid çip (paslanmaz çelik, 37.8 mm x 6,4 mm x 3,2 mm) yerleştirin. </li><li> (3 eksenli nano konumlandırma içeren bir konumlandırma kademesinin üzerine numune tutucuyu monte n3 eksenli mikrometre üstünde anocube) aşama (ekipman listesine bakınız). Titreşimleri en aza indirmek için bir pnömatik izole optik masa üzerindeki tüm deneyler. </li><li> Kaba pozisyon 3 eksenli mikrometre kullanarak örnek çip. </li><li> Nano-pozisyoner kullanarak optik fiber microtoroid içeren çip paralel hizaya getirin. Not: Giriş ışığı (~ 633 nm) bir dalga boyunun bir mesafede microtoroid hizalayın. Bu sürecin görselleştirme için iki görüntüleme sütunları kullanın üstünde ve çip tarafında konumlandırılmış (objektif lens ve kamera ile boru, ekipman listesine bakınız). </li><li> Bir in-line polarizasyon kontrolörü kullanarak optik fiber üzerinden yönlendirilen lazer ışığının polarizasyonu Optimize kutuplaşmayı ayarlamak için bir düğme ile (ekipman listesine bakınız). Not: fiber optik iletimi ölçülen dip dar göründüğünde Optimum polarizasyon elde edilir. Bir osiloskop (Daha fazla bilgi için adım 2.2) Bu dalış gözlemleyin. </li><li> Inşa etmekbir aralama olarak cam mikroskop lamı kullanılarak örnek sahne üzerinde bir cam lamel epoxying bir örnek odası. Not: Tüm kurulum kapsayan pleksiglas muhafaza hava akımları en aza indirmek için yararlı olabilir. Küçük bir açıklık boru kullanılarak pompalanan edilecek çözelti sağlamak için bırakılmalıdır. </li><li> Isı, bir RT su banyosunda (~ 500 mi) içinde ≥ 1 saat boyunca tanecik süspansiyonlar ya da tek-molekül sulu çözeltileri dengeye getirin. Not: Örnekler üreticiler, örneğin PBS veya HEPES belirtilen ilgili tamponlar kullanılarak mikrosantrifüj tüpleri içinde istenen konsantrasyona seyreltilir. Hiçbir bağlayıcı olay tespit edilirse, tampon tuz konsantrasyonu artırır. </li><li> ~ 2 sn çözümler (1 ml) kısaca içeren Vortex parçacık. </li><li> 1 ml numune bölmesine partikül içeren çözeltiler enjekte / 1 ml şırınga pompası kullanılarak Min. </li><li> Numune odası doldurdu sonra, şırınga pompası kapatın. </li><li> Kayıt verileri en aza indirmek için önce 30 saniye bekleyinölçümünü etkileyebilecek sıvı akışı kaynaklanan mekanik titreşimler etkileri. </li></ol> 2. Frekans Kilitleme <ol><li> Bağlama sıvısının enjeksiyonu nedeniyle rahatsız olacak çünkü, nanopositioner numune tutucu hareket ettirerek fiber optik için toroid-çift Re. </li><li> Dalga boyu çeşitli yoluyla bilgisayar kontrollü giriş lazer tarama microtoroid rezonans dalga boyu bulun. Lazerin dalga boyunu düzenleyen lazer denetleyicisi içinde piezoelektrik eleman bir üçgen dalga gerilim sinyali göndererek bu adımı gerçekleştirin. Bu dalga boyunda suyun ışık düşük emme olarak orada görünür ışığı (2.5 nm ± 635 nm) kullanarak deneyler yapın. </li><li> Oto-dengeli foto alıcı içine fiber optik çıkışını takarak fiber optik aracılığıyla ışık geçirgenliği ölçün. BNC kablosu kullanarak bir osiloskop içine foto alıcı çıkışını takın. O gözlemleyinn osiloskop microtoroid rezonans dalga boyunda optik fiber üzerinden iletim düşer. </li><li> Bir kablo ile geribildirim denetleyicisi (ekipman listesine bakınız) kilitleme frekansı ana girişine foto alıcı çıkışını takın. </li><li> 2 kHz titreşim frekansı ve 19 fm dalga boyu titreşim genliği ile kilitleme üstü tepe kullanarak kilitlenme modunda frekans kilitleme geribildirim denetleyicisi çalıştırın. Ampirik Ziegler-Nichols ayar kurallarını 14 kullanarak yazılım penceresindeki oransal-integral-türev ayarlarını. Not: Bu değerler tüm deneyler başında bir kez ayarlanması yeterlidir. </li><li> Microtoroid rezonans dalga boyuna lazerin dalga boyu otomatik kilitleyin. Numune odasını doldurduktan sonra bu adımı gerçekleştirin. Not: dalgaboyu kayması çok büyükse, o zaman geri denetleyici kilidi kaybedecek ve otomatik rezonans dalgaboyu yerini bulmak için tarama moduna. Bu ocDalga boyu Curs yaklaşık bir çizgi genişliği (araştırılan tüm sistemler için en az 600 fm) daha büyük kaydırır. </li><li> 24 bit veri toplama kartı kullanarak 20 kHz geribildirim denetleyicisi çıkışını kaydedin. Veri toplama yazılımı ile bir metin dosyası olarak veri verir. </li></ol> 3. Veri İşleme ve Analiz <ol><li> Fourier MATLAB&#39;da verileri dönüştürmek. </li><li> Düşük 2 kHz dayatılan taklidinin frekansını çıkarmak için 1 kHz bir kesim ile bir &quot;tuğla duvar&quot; filtre kullanarak veri geçmek (Yan Kod Screenshot 1 Dosya bakınız). </li><li> Hesaplama çentik 16 Hz pencere boyutunu kullanarak verileri süzmek. Not: Bu, bu durumda, gürültü bilinen kaynaklarını ortadan kaldırmak için yapılır, 60 Hz, elektronik hat gürültüsü ve harmonikleri, (lazer sürücüsünden gelen) yanı sıra, 100 Hz ve harmonik (Yan Kod Screenshot 1 Dosya bakınız). </li><li> Ters Fourier zaman alanına geri verileri dönüştürmek. </li><li> Medyan filtre penceresini kullanarak veri1001 örneklerin boyutu (Yan Kodu Screenshot 2 Dosya bakınız). </li><li> Kerssemakers ve ark., 15 adım bulma algoritması kullanılarak rezonans dalga adım değişiklikleri bulun. </li><li> Her bağlama aşamasında genliği histogramları oluşturun. </li><li> Denklem kullanarak parçacık boyutunu hesaplayın. (1) (Tartışma). </li></ol>

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The authors declare that they have no competing financial interests.

Bir parçacık bağlanan olarak, toroid artar rezonans dalgaboyu (λ). Bir parçacık keser ise, rezonans dalga boyu buna (a step-down olayı) azalır. Parçacık çapı (d), her dalga boyunda adım amplitüdü histogramları yoluyla belirlenebilir. Her dalga boyu basamağın yüksekliği bağlı parçacık büyüklüğü varyasyonları nedeniyle partikül bağlayan microtoroid üzerinde konumu nedeniyle değişir. Parçacıklar elektrik alan (E 0, max) maksimum olduğu mic...

Tek moleküllü saptama deneyleri Hastalığın erken teşhisi için biyosensörler kullanılan analit miktarının azaltılması için faydalıdır, ve moleküllerin 3 temel özelliklerini incelemek için. Bu tür deneyler, tipik olarak etiketler, bununla birlikte, etiketler her olay üzerinde çalışılan yapabilirliğini bozmakta ve bu, maliyet artışı, belirli bir protein için elde etmek mümkün değildir, ve özellikle de bünyesinde deneyleri veya nokta-of, gerçek süre, rahatsız edici olabilir kullanılarak gerçekleştirilir bakım teşhis. Etiket içermeyen biosensörleme şu anki altın standart Ancak, ticari yüzey plazmon rezonans sistemi tipik haliyle nM mertebesinde tespit tipik bir alt sınırı, yüzey plazmon rezonans 4&#39;tür. Son zamanlarda, optik rezonatörler etiket içermeyen tek bir molekül biodetection 5 için umut verici bir teknoloji olarak ortaya çıkmıştır. Uzun vadeli (ns) dayalı optik rezonatörler çalışma ışığı 6,7 hapsi. Işık evanescently olduğunutipik bir fiber optik aracılığıyla bu cihazların içine birleştiğinde. Fiber geçiyor ışığın dalga boyu rezonatör rezonans dalga boyu eşleştiği zaman, verimli rezonatör çiftler yanar. Bu birleştirilmiş ışık tamamen dahili rezonatörün çevresinin yakınında genliği azalan bir alan üreten rezonatörün en boşluk içinde yansıtır. Parçacıklar rezonatör, parçacığın 8 hacmi ile orantılı olarak rezonatör değişiklikleri rezonans dalgaboyu kaybolan alan ve bağlayıcılığı girerken. Algılama yeteneği açısından mikrosfer rezonatörler daha önce tek bir influenza A virüs partiküllerini (100 nm), 9,10 tespit etmek için kullanılmıştır. Son zamanlarda, plasmonically geliştirilmiş mikroküre optik rezonatörler Ancak bu yaklaşım de yüzde 0.3 um ila 2 tane çekim alanı sınırlar molekülleri 11 ve 8-mer oligonükleotitlerin 12 albumin, tek sığır serumu tespit etmek için kullanılmaktadıryardımcısı. Büyük çekim alanı biyosensörler parçacık algılama şansını maksimize etmek için idealdir. Büyük (&gt; 100 mikron 2) yakalama alanları ile Güncel çözüm tabanlı etiket içermeyen biyoalgı teknolojileri polistiren tanecikleri ≥ 25 nm tespit sınırlı kalmıştır. Bu çözelti içinde tek moleküllerin zamana bağımlı algılama kapasitesine sahip optik Whispering Evanescent Resonator (çiçek) 13 (Şekil 1) kilitleme Frekans olarak bilinen optik rezonatör teknolojisine dayalı bir etiket içermeyen biyo-algılayıcı sistemi geliştirmiştir. ÇİÇEK frekans kilitleme geri besleme kontrolü, dengeli algılama ve tek bir protein molekülleri aşağı küçük partikülleri tespit etmek için hesaplamalı filtreleme ile birlikte microtoroid optik rezonatörlerin uzun foton ömrünü kullanır. Frekans kilitleme kullanımı süpürüp veya lazer dalga boyu üzerinden tarama gerek kalmadan, parçacıklar bağlamak gibi sistem daima microtoroid kayması rezonans izlemenize olanak sağlarBüyük aralığındadır. Çiçeğin ilkeleri plasmonik geliştirilmesi de dahil olmak üzere diğer tekniklerin algılama yeteneklerini geliştirmek için kullanılabilir. Aşağıda, ÇİÇEK gerçekleştirmek için prosedürler açıklanmaktadır.

<table border="0" cellpadding="0" cellspacing="0" width="396">
	<tbody>
		<tr height="20" style="height:15.0pt">
			<td class="xl63" height="20" style="height:15.0pt;width:99pt" width="132">Tunable diode laser</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">TLB-6300</td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl63" height="20" style="height:15.0pt;width:99pt" width="132">Laser controller</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">TLB-6300-LN</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Frequency locking feedback controller</td>
			<td class="xl63" style="width:99pt" width="132">Toptica Photonics</td>
			<td class="xl63" style="width:99pt" width="132">Digilock 110</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Auto-balanced photoreceiver</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">Model 2007</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">In-line polarization controller</td>
			<td class="xl63" style="width:99pt" width="132">General Photonics</td>
			<td class="xl63" style="width:99pt" width="132">PLC-003-S-90</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">24-bit data acquisition card</td>
			<td class="xl63" style="width:99pt" width="132">National Instruments</td>
			<td class="xl63" style="width:99pt" width="132">NI-PCI-4461</td>
		</tr>
		<tr height="60" style="height:45.0pt">
			<td class="xl63" height="60" style="height:45.0pt;width:99pt" width="132">Recombinant human interleukin-2</td>
			<td class="xl63" style="width:99pt" width="132">Pierce Biotechnology</td>
			<td class="xl63" style="width:99pt" width="132">R201520</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">20 nm polystyrene beads</td>
			<td class="xl63" style="width:99pt" width="132">Thermo Scientific</td>
			<td class="xl63" style="width:99pt" width="132">3020A</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">NanoCube XYZ Piezo Stage</td>
			<td class="xl63" style="width:99pt" width="132">Physik Instrumente</td>
			<td class="xl63" style="width:99pt" width="132">P-611.3</td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl63" height="20" style="height:15.0pt;width:99pt" width="132">Optical table</td>
			<td class="xl63" style="width:99pt" width="132">Newport</td>
			<td class="xl63" style="width:99pt" width="132">VH3660W-OPT</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Objective lens for imaging column</td>
			<td class="xl63" style="width:99pt" width="132">Navitar Machine Vision</td>
			<td class="xl64">1-60228</td>
		</tr>
		<tr height="40" style="height:30.0pt">
			<td class="xl63" height="40" style="height:30.0pt;width:99pt" width="132">Imaging column (adaptor tube)</td>
			<td class="xl63" style="width:99pt" width="132">Navitar Machine Vision</td>
			<td class="xl65">1-60228</td>
		</tr>
		<tr height="60" style="height:45.0pt">
			<td class="xl63" height="60" style="height:45.0pt;width:99pt" width="132">High-Res CCD camera for imaging column</td>
			<td class="xl63" style="width:99pt" width="132">Edmund Industrial Optics</td>
			<td class="xl64">NT39244</td>
		</tr>
	</tbody>
</table>

label-free single molecule detection using microtoroid optical resonators

Aşağı tek bir molekül sınırına moleküllerinin küçük konsantrasyonlarını algılama gibi Hastalığın erken teşhisi ve moleküllerin davranışına temel çalışmaları gibi alanlarda bir etkiye sahiptir. Tek molekül algılama teknikleri yaygın, ancak etiket maliyeti ve karmaşıklığı artırmak, her zaman mevcut değildir ve olaylar çalışılan perturb böyle floresan etiket veya kuantum noktaları gibi etiketler kullanır. Optik rezonatörler ve etiket kullanılmaksızın tek moleküllerin tespit için umut verici bir araç olarak ortaya çıkmıştır. Şu anda çözelti içinde olmayan plasmonically geliştirilmiş çıplak optik rezonatör sistemi tarafından tespit edilen en küçük parçacık 25 nm polistiren küre 1&#39;dir. Biz Optik Whispering Evanescent Resonator sulu çözelti 2 etiket içermeyen tek bir molekül algılama bu sınırı aşmak ve elde edebilirsiniz (ÇİÇEK) Kilitleme Frekans olarak bilinen bir teknik geliştirdiler. Sinyal gücü partikül hacmi ile ölçekler gibi, bizim çalışma&gt; 100x improveme temsilnt sanatın mevcut durumu üzerinde gürültü oranı (SNR) sinyalde. İşte ÇİÇEK arkasında prosedürleri alanındaki kullanımını artırmak amacıyla sunulmuştur.

Olayları bağlayıcı Parçacık açıkça zaman (Şekil 2A) üzerinde microtoroid rezonans dalga adım gibi değişiklikler olarak görülmektedir. Bu adımların yükseklikleri. Şekil 2B&#39;de, bir histogram olarak gösterilen 2-4, sırasıyla Örnek eksozom bağlanma izleri (nanovesicles), 5 mil silika boncuklar ve tek bir insan interlökin-2 moleküllerinin Şekil edilir. Adım gibi olaylar parçacık boyutu ile ölçek gerçeği tekniği doğru ...

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

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

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

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 ...

label-free-single-molecule-detection-using-microtoroid-optical

Research

JoVE Journal

Bioengineering

9.1K Görüntüleme.  California Institute of Technology. We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented. 

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

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

Manyetik cımbız kullanarak Multiplexed Tek molekül Kuvvetleri Proteoliz Ölçümleri

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

Biyomühendislik

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

Hücre Fusion Tespit Cre-ETOX P Rekombinasyon Yaklaşım In Vivo</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Raman Spektroskopisi kullanarak Hücreler ve Ekstrasellüler Matriks Temassız, Etiket ücretsiz izleme

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

Optik Algılama<em&gt; E. coli</emMezopor Silikon Biyosensörlerin tarafından&gt; Bakteri

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

G-protein bağlı reseptörler ile Potasyum kanallarının modülasyonunu Algılama Label içermeyen optik Biyosensör kullanımı

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

Optik cımbız ile Tek RNA Moleküllerin Nanomanipulation

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Etiket-serbest DNA Hibridizasyon Analiz için mikroakışkan tabanlı Elektrokimyasal Biochip

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Tek Hücre Salgıları uzay-zamansal Görüntüleme için bir etiket içermeyen Tekniği

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

Planar on Tek Molekül Floresans Mikroskopi bilayers Desteklenen

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

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

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

Damlacık Mikroakiskan için renkli Floresan Algılama Optik Elyaf kullanma

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