1. Görüntüleme Kurulumu LOT kompakt ve hafif bir alana taşınabilir mimari 8 monte edilebilir ve alternatif kesit görüntüleme yeteneği ile bir optofluidic mikroskop gibi. 9 Bu raporda, ancak, statik tomografi doğru bir tezgah üst uygulanması için temel görüntüleme kurulum açıklayacağız örnekleri. <ol><li> Aydınlatma modülü: LOT, bu gibi ışık yayan-diyotları (LEDler) olarak kısmen koherent ışık kaynakları kullanılabilir. Deneysel esneklik için bir Xenon lamba (Cornerstone T260, Newport Corp) ile bir monokromatör kullanılır. Monokromatör örneğin 450-650 nm bir dalga boyunda merkezi etrafında ~ 1-10 nm spektral genişliğe sahip bir çıkış sağlamak üzere ayarlandı. Bu kısmen koherent çıkış daha sonra, sistem kısmen uyumlu ışık sunmak için bir çoklu fiber optik (Thorlabs AFS105/125Y) bağlanmıştır. Optik lif (a Thorlabs parçalarla üzerine monte edilmiştirPRM-1Z8 aydınlatma açısını değiştirmek için) Thorlabs TDC001 kontrolör tarafından tahrik. Motorlu aşaması, ışık kaynağı ile birlikte eklenmiş, belirli bir açıyla ışık kaynağının düzlem-kaymalar elde etmek için kullanılan iki boyutlu bir doğrusal XY aşama (Newport ILS50CC Newport MFA-PPD kontrolörleri ile tahrik) üzerine monte edilir. </li><li> Algılama: Lensfree optik tomografi dedektör dizi önemli ölçüde görüntü alma adımları veya veri işleme değiştirmeden uygulamaların gereksinimlerine göre seçilebilir bu tür ölçeklenebilir bir teknolojidir. Bu yazıda, 2.2 mikron piksel boyutunu (IDS Görüntüleme, UI-1485LE-M), 5 Mega piksel olan bir CMOS sensörü dizi istihdam. Dedektör aktif alanının doğrudan üstüne yerleştirildi örneklerinin geniş açılı alan görünümü (örneğin 24 mm 2) holografik görüntüleri kaydetmek için kullanılır. Bir çift eksenli tomografi kurulum uygulanmak üzere 7 ise, detektör gerektiğiLSO optik eksenine dik düzlemde örneği (araya dedektörü ile) döndürmek için bir yatay (optik tablosu ile ilgili) rotasyonu aşama (örn. Thorlabs RP-01) üzerine monte edilebilir. 7 </li></ol> 2. Numune Hazırlama Lensfree optik tomografi görüntü, hücre ve mikroorganizmalar gibi nesnelerin çeşitli olabilir ederken, bir C. üç boyutlu mikroskop yaparak temel ilkeleri gözler önüne serecektir elegans örneği. <ol><li> Bir neşter veya spatula kullanarak, C. içeren petri gelen agar küçük bir parça alır elegans kültürü. Her bir boyut boyunca birkaç milimetrelik bir kübik parçası nematodlar yüz ihtiva edecektir. </li><li> 1 ml deiyonize (Dİ) su ihtiva eden bir şişe içinde polipropilen agar, küçük parça yerleştirin. </li><li> 0.5-1 dakika ve solucanlar DI suya agar parçası çıktı taramamıza dek 10-15 dakika beklemek için yavaşça girdap. </li><li> Içingeçici, solucanlar hareketsiz 5-10 mM levamizol (Sigma-Aldrich) 1 ml ekleyin ve 10 dakika bekleyin. </li><li> Iki kapak fişleri arasında 5-10 şişe dibinden örnek ul ve sandviç Pipeti. Geçici immobilize kurtları (örneğin 50-100 solucanlar), çok sayıda içeren Bu örnek, verilerin alınması başlatmak için detektör üzerine yerleştirilebilir. </li></ol> 3. Veri Toplama Burada, LabView arayüzü geliştirilmiş, özel bir otomatik olarak tipik bir LOT deney için görüntü alma adımları özetlemektedir. <ol><li> 0 ° ışık kaynağının dikey konumunu karşılık -50 ° için dönme aşamasının başlangıç ​​açısını ayarlayın. </li><li> (0,0) XY ilk pozisyonunu ayarlayın, hangi ANA konumdur. </li><li> En iyi görüntü herhangi bir s olmadan mümkün olduğunca parlak olduğunu ve dinamik alanı gibi kullanmak için, dedektörün pozlama süresini ayarlayınaturated piksel. </li><li> Dönme aşamasında açısını değiştirmeden, 9 görüntüler yakalayabilir. Her bir görüntü için, her resim bir önceki oranla bir pikselin yaklaşık dörtte bir oranında kaydırır böyle bir 3x3 kare tablo içinde yeni bir konuma XY taşıyın. Bir 6μ6 ızgara örneğin, her açıda daha görüntüleri alma, nesne ve sinyal-gürültü oranı (SNR) türüne bağlı olarak çözünürlüğünü arttırmak olabilir. 10 </li><li> (0,0) XY ilk pozisyonunu ayarlayın, hangi ANA konumdur. </li><li> +50 ° ulaşıncaya kadar, 2 ° artışlarla dönme aşamasında açısını artırın. Her bir artım sonra, her yeni açısı, 3-5 adımları tekrarlayın az yani. Açısal artışlarla alma süresi vs görüntüleme çözünürlük optimizasyonu bağlı olarak ince ya da kalın olabilir. </li><li> (A çift eksenli tomografi düzeni kullanılmak üzere ise isteğe bağlı adım) detektörü 90 ° monte edildiği yatay aşaması döndürür. Daha sonra,1-6 adımları tekrarlayın. </li></ol> 4. Veri Analizi Bölüm 3, aydınlatma 51 farklı açılardan her biri için 9 alt-piksel kaydırılır görüntüleri içeren kazanılır 459 görüntü kümesi, içinde 1-6 adımları izleyerek. Birincisi, 9 görüntülerin her set dijital bir yüksek çözünürlüklü (İK) Her açı için projeksiyon hologram elde etmek için piksel çözünürlüklü süper algoritmaları 10, kullanarak işlenir. Bu piksel çözünürlükte süper-lensfree görüntülerin mekansal çözünürlüğüne, daha ziyade kırınımı sınırından daha, piksel boyutu sınırlama üstesinden ifade ettiği belirtilmelidir. Piksel süper giderilmiş hologramlar sonra 51 dijital projeksiyon görüntüleri elde etmek için 7-10 sökülmelidir. 51 projeksiyon görüntüleri Bu set daha sonra TomoJ, bir plug-in ImageJ (açık kaynak kodlu bir görüntü analiz yazılımı) için. 11 Bu arka projeksiyon çalışması örnek bir üç boyutlu görüntü (tomogram) çıktılar. Kullanılarak geri yansıtılır olduğunu Burada uygulanan olmasa da,Referans tarif edildiği gibi, bir çift eksenli tomografi düzeni de yararlanılabilir. 7. Bu düzende, tomogram bir ikinci dizi örnek ile ilgili daha fazla bilgi sağlar mekansal ve eksenel çözünürlüğünü arttırır birinci dönme ekseni, ile ilgili olarak ortogonal bir eksen üzerinde dönen bir ışık kaynağı ile elde edilir. 5.. Temsilcisi Sonuçlar Lensfree optik tomografi geniş görüş alanı (FOV) Şekil 1&#39;de gösterilmiştir. Örnek dedektör-dizi doğrudan üstüne konur, nesnelerin holografik görüntüleri daha büyük aktif alanlar ile ortaya çıkan algılayıcı dizileri kullanılarak artırılabilir bir 24 FOV mm 2, üzerinde kaydedilebilir. Dedektör-dizinin piksel boyutu kaydedilen holografik görüntülerin çözünürlüğünü sınırlamasına rağmen, piksel süper-çözünürlük teknikleri bu sorunu hafifletmek. Şekil 2 boyunca piksel süper giderilmiş hologramlar gösteririle aydınlatma üç farklı açılar için, alt-mikrometre uzaysal çözünürlüğü ile rekonstrüksiyon görüntüleri (yani projeksiyon görüntüleri). Projeksiyon görüntüleri numunenin tomogram (yani üç boyutlu görüntü) hesaplamak için (örneğin filtre arka projeksiyon) tomografik görüntü oluşturma teknikleri ile kombine edilebilir. Şekil 3 solucanın ön aracılığıyla xy düzleminde üç dilim görüntüleri gösterir, burada faringeal tüp sadece ~ 5 mm dış çaplı bu kabaca silindirik yapıdaki beklenen z = 8 mikron aracılığıyla dilim görünür. Ayrıca, xz düzleminde kesitsel görüntü (Şekil 3 üst panelinde inset) açıkça solucanın sınırları ve farenks başarılı 3D görüntüleme gösteren faringeal tüp içinde (oklarla gösterilen) gösterir. <img src="/files/ftp_upload/4161/4161fig1.jpg" alt="Şekil 1" /> <strong&gt; Şekil 1 lensfree optik tomografi (LOT) dikey aydınlatma için kurulum kullanılarak kaydedildi tam açılı alan görünümü (24 mm 2) holografik görüntü gösterir. LOT geniş görüntüleme alanı sayesinde, pek çok solucan, aynı anda tek bir veri toplama adım ile görüntülenebilir. <img src="/files/ftp_upload/4161/4161fig2.jpg" alt="Şekil 2" /> Şekil 2 süper giderilmiş hologramlar (sol panel) ve dijital olarak yeniden projeksiyon görüntüleri (sağ panel) bir C için piksel gösterir üç farklı aydınlatma açılarda elegans solucan (geniş açılı alan görünümü kırpılmış). Her projeksiyon görüntü süzülmüş bir arka projeksiyon çalışması yoluyla 3D yapı hesaplama sağlayan farklı bir görüş açısı ile ilgili bilgileri içerir. <img src="/files/ftp_upload/4161/4161fig3.jpg" alt="Şekil 3" /> Şekil 3, C için hesaplanan tomogram gösterir elegans solucan of Şekil 2. (Üst sıra) z = 3 mikron altında olan tüm solucanın bir dilim görüntüsünü gösterir. Inset solucanın anterior bir kesitsel görüntüsünü gösterir. Inset Ölçeği bar 50 mikron olduğunu. (Alt satır) lensfree optik tomografi optik kesit alma yeteneğini gösteren, solucan ön üzerinden üç dilim görüntüleri gösterir. Rakam boyunca Oklar solucanın faringeal tüp üzerinde aynı noktaya işaret etmektedir. Bir mikroskop görüntünün (x40, 0.65-NA), aynı zamanda görsel karşılaştırma için verilmiştir.

<ol>
	<li>Schmitt, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+(OCT):+a+review,+J.+Sel.+Top.">Optical coherence tomography (OCT): a review, J. Sel. Top.</a> Quant. Elect. 5, 1205-1215 (1999).</li><li>Keller, P. J., Schmidt, A. D., Wittbrodt, J., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+zebrafish+early+embryonic+development+by+scanned+light+sheet+microscopy.">Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy.</a> Science. 322, 1065-1069 (2008).</li><li>Sharpe, J., Ahlgren, U., Perry, P., Hill, B., Ross, A., Hecksher-S&#248;rensen, J., Baldock, R., Davidson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Projection+Tomography+as+a+Tool+for+3D+Microscopy+and+Gene+Expression+Studies.">Optical Projection Tomography as a Tool for 3D Microscopy and Gene Expression Studies.</a> Science. 296, 541-545 (2002).</li><li>Sung, Y., Choi, W., Fang-Yen, C., Badizadegan, K., Dasari, R. R., Feld, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+diffraction+tomography+for+high+resolution+live+cell+imaging.">Optical diffraction tomography for high resolution live cell imaging.</a> Opt. Exp. 17, 266-277 (2009).</li><li>Debailleul, M., Simon, B., Georges, V., Haeberle, O., Lauer, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Holographic+microscopy+and+diffractive+microtomography+of+transparent+samples.">Holographic microscopy and diffractive microtomography of transparent samples.</a> Meas. Sci. Technol. 19, 074009 (2008).</li><li>Charri&#232;re, F., Pavillon, N., Colomb, T., Depeursinge, C., Heger, T. J., Mitchell, E. A. D., Marquet, P., Rappaz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+specimen+tomography+by+digital+holographic+microscopy:+Morphometry+of+testate+amoeba.">Living specimen tomography by digital holographic microscopy: Morphometry of testate amoeba.</a> Opt. Exp. 14, 7005-7013 (2006).</li><li>Isikman, S. O., Bishara, W., Mavandadi, S., Yu, S. W., Feng, S., Lau, R., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lens-free+optical+tomographic+microscope+with+a+large+imaging+volume+on+a+chip.">Lens-free optical tomographic microscope with a large imaging volume on a chip.</a> Proc. Nat. Acad. Sci. 108, 7296-7301 (2011).</li><li>Isikman, S. O., Bishara, W., Sikora, U., Yaglidere, O., Yeah, J., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field-portable+Lensfree+Tomographic+Microscope.">Field-portable Lensfree Tomographic Microscope.</a> Lab Chip. 11, 2222-2230 (2011).</li><li>Isikman, S. O., Bishara, W., Zhu, H., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optofluidic+tomography+on+a+chip.">Optofluidic tomography on a chip.</a> App. Phys. Lett. 98, 161109 (2011).</li><li>Bishara, W., Su, T. W., Coskun, A., Ozcan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lensfree+on-chip+microscopy+over+a+wide+field-of-view+using+pixel+super-resolution.">Lensfree on-chip microscopy over a wide field-of-view using pixel super-resolution.</a> Opt. Exp. 18, 11181-11191 (2010).</li><li>Messaoudi, C., Boudier, T., Sorzano, C. O. S., Marco, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TomoJ:+tomography+software+for+three-dimensional+reconstruction+in+transmission+electron+microscopy.">TomoJ: tomography software for three-dimensional reconstruction in transmission electron microscopy.</a> BMC Bioinformatics. 8, 288 (2007).</li><li>Heng, X., Erickson, D., Baugh, L. R., Yaqoob, Z., Sternberg, P. W., Psaltis, D., Yang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optofluidic+Microscopy:+A+Method+for+Implementing+High+Resolution+Optical+Microscope+On+A+Chip.">Optofluidic Microscopy: A Method for Implementing High Resolution Optical Microscope On A Chip.</a> Lab on a Chip. 6, 1274-1276 (2006).</li></ol>

Çıkar çatışması ilan etti.

Bu lensfree on-chip holografik mikroskop eşsiz geometri piksel süper-çözünürlük ve tomografik görüntüleme sağlamak için kritik etkinleştiricidir olduğunu vurgulamak önemlidir. Kaydedilen görüntülerin 12 yaklaşımlar tutarsız temas görüntüleme gibi projeksiyon görüntüleri olarak kabul edilmez, ama projeksiyon hologramlar, iletilen ışık kırınım bu dedektör impinges kadar dijital holografik yeniden düzeltilebilir beri. Bu nedenle, örnek ile sensör mesafe varyasyonlar ...

<table border="1"><tbody><tr><td> Isim </td><td> Compay </td><td> # Kataloğa </td><td> Yorum </td></tr><tr><td> Lineer XY aşamaları </td><td> Newport Corp </td><td> MFA-PP </td><td> Minyatür Doğrusal Sahne </td></tr><tr><td> Motorlu dönme aşamasında </td><td> Thorlabs </td><td> PRM1Z8 </td><td> Motorlu Hassas Rotasyon Dağı </td></tr><tr><td> Multimode fiber optik </td><td> Thorlabs </td><td> AFS105/125Y </td><td> Multimode Fiber </td></tr><tr><td> Işık kaynağı </td><td> Newport Corp </td><td> 6255 </td><td> Ozon serbest Xenon Lamba </td></tr><tr><td> Monokromatör </td><td> Newport Corp </td><td> 74100 </td><td> Cornerstone 260 1/4 m Monokromatör </td></tr><tr><td> CMOS sensör dizisi </td><td> Aptina A.Ş. </td><td> MT9P031STC </td><td> 5 Megapiksel CMOS Sensör </td></tr><tr><td> C. elegans örneği </td><td> Carolina Biosupply </td><td> 173500 </td><td> Yabani tip C. elegans </td></tr><tr><td> Levamizol </td><td> Sigma Aldrich </td><td> L9756-5G </td><td> Tetramisole hidroklorür </td></tr></tbody></table>

lensfree on-chip tomographic microscopy employing multi-angle illumination and pixel super-resolution

Farklı boyut ölçekleri nesneler ile ilgili üç boyutlu (3D) yapısal bilgi sağlamak gibi tomografik görüntüleme tıpta yaygın olarak kullanılan bir araç olmuştur. Mikrometre ve milimetrik ölçeklerde, optik mikroskopi yöntemleri görünür ışık iyonlaştırıcı olmayan doğaya artan kullanımı sayesinde bulmak ve zengin bir aydınlatma kaynaklarının seti (örneğin lazer ve ışık yayan diyotlar-gibi) ve tespit elemanları (örneğin kullanılabilirliğini geniş format CCD ve CMOS algılayıcı-dizileri). Yakın zamanda geliştirilen optik tomografik mikroskopi yöntemleri arasında, tek bir optik koherens tomografi, optik kırınım tomografi, optik projeksiyon tomografi ve ışık sayfalık mikroskobu içerebilir. 1-6 Bu platformlar hücreler, mikroorganizmalar ve C gibi model hayvan kesit görüntüleme sağlamak elegans, Zebra balığı ve fare embriyoları. Mevcut 3D optik kameralar genellikle inci sınırlayıcı, nispeten hantal ve karmaşık mimarileri varE gelişmiş laboratuarları bu ekipman durumu ve laboratuvar-on-a-chip platformları ve mikroakışkan çipleri ile entegrasyonu engelleyen. Alternatif tomografik mikroskop sağlamak için, son 7 LOT atar lensler ve hantal optik bileşenlerin kullanımı. Yüksek verimlilik, kompakt ve uygun maliyetli optik tomografi yöntemi olarak lensfree optik tomografi (LOT) geliştirilmiş ve bunun yerine çok açılı güveniyor aydınlatma ve geniş bir görüntüleme hacmi üzerinde mikro-objelerin derinlik çözülmesi görüntü elde etmek için dijital hesaplama. LOT için x &lt;1 mikron x &lt;3 15-100 mm 3 geniş bir görüntüleme hacmi üzerinden sırasıyla x, y ve z boyutlarda um, olabilir ve özellikle faydalı &lt;1 mikron mekansal çözünürlükte görüntü Biyolojik örnek olabilir lab-on-a-chip platformlar.

Watch this Scientific Journal Video about Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution at JoVE.com

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Lensfree optik tomografi &lt;1 um arasındaki konumsal bir çözünürlüğe sahip bir üç-boyutlu mikroskopi tekniktir x &lt;1 um x 15-100 mm arasında geniş bir görüntüleme-hacmi içinde, sırasıyla, x, y ve z, boyutları, içinde &lt;3 um,<sup&gt; 3</sup&gt;, Hangi lab-on-a-chip platformları ile entegrasyon için özellikle yararlı olabilir.

Çok açılı Aydınlatma ve Pixel Super çözünürlüklü istihdam Lensfree On-chip Tomografik Mikroskopi

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

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Bioengineering

11.4K Görüntüleme.  University of California, Los Angeles. Lensfree optik tomografi 

Video: Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

Biyomoleküler Algılama istihdam İnterferometrik Yansıtma Görüntüleme Sensörü (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Biyomühendislik

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration1,2 and efficient separation and extraction based on ionic mobility.3 For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation4-8 and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood.9-12
ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.13). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.
We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.

Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.

İyonların ayrılması ve Nükleik Asit Saflaştırılması için on-chip Isotachophoresis

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

On-Chip Endotel İnflamatuar fenotipleme

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells.

This article provides an in depth guide for the assembly and operation of a structured illumination microscope operating with total internal reflection fluorescence illumination (TIRF-SIM) to image dynamic biological processes with optical super-resolution in multiple colors.

Çoklu Renkler Yüksek hızında Yapısal Aydınlatma TIRF Mikroskopi A Guide

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

Kuru zar elektrokimyasal mikrosıvısal Biyoalgılayıcı fotorezist tabanlı Platform: Cihaz imalat, On-chip tahlil hazırlama ve sistemin çalışması

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

İmalat ve Organ-on-chip sistemin doğrudan Transendothelial elektrik direncini ölçmek için entegre elektrotlar ile doğrulama

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab and industrial scale. However, due to its toxicity and flammability, there is a desire to avoid methanol while maintaining the high productivity of P. pastoris. Small scale bioreactor cultivations (0.5-5 L working volume) are commonly used to evaluate a strain and its protein production characteristics since microscale cultivation in deep well plates can be hardly controlled or relies on expensive equipment. Furthermore, traditional protocols for the cultivation and induction of P. pastoris were established for constitutive expression or methanol induction and so far, no reliable protocols were described to screen P. pastoris expression strains with derepressible promoters in (controlled and monitored) parallel cultivations. To simplify such initial cultivations to characterize and compare new protein production strains, we established a simple shake flask cultivation system for methanol free expression that simulates bioreactor conditions including a constant slow glycerol feed and online monitoring, thereby coming closer to the real conditions in bioreactors compared to mostly applied small scale batch cultivations. To drive recombinant protein expression in P. pastoris, the carbon source repressed promoters PDC and PDF were applied. Polymer discs with embedded carbon source, releasing a constant amount of glycerol, assured a feed rate delivering the necessary energy for maintaining the promoters active while keeping the biomass generation low.

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

This method for the first time describes reliable experimental procedures for efficient inducible methanol-free recombinant protein production in Pichia pastoris (Komagataella phaffii), employing the carbon source regulated promoters PDCand PDF in small scale experiments while cultivation parameters can be monitored online, and a constant glycerol feed is applied.

Metanol bağımsız ifade tarafından de-baskı teknolojilerinden yararlanan Pichia Pastoris

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab ...

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single CRISPR RNA (crRNA) array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S.&#160;cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently colored carotenoid producing yeast strains that were constructed.

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

The CRISPR/Cas12a system in combination with a single crRNA array enables efficient multiplex editing of the S.&#160;cerevisiae genome at multiple loci simultaneously. This is demonstrated by constructing carotenoid producing yeast strains which are subsequently used to create yeast pixel art.

CRISPR/Cas12a Multiplex Genome düzenleme Saccharomyces cerevisiae ve Maya Pixel Art oluşturulması

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

Açık Kaynak Mikroplaka Uyumlu Aydınlatma Panelleri Uygulamaları

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...

Building a Simple and Versatile Illumination System for Optogenetic Experiments

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical control allows for an unprecedented ability to dissect function and holds the potential for enabling novel genetic therapies. However, optogenetic experiments require adequate light sources with spatial, temporal, or intensity control, often a bottleneck for researchers. Here we detail how to build a low-cost and versatile LED illumination system that is easily customizable for different available optogenetic tools. This system is configurable for manual or computer control with adjustable LED intensity. We provide an illustrated step-by-step guide for building the circuit, making it computer-controlled, and constructing the LEDs. To facilitate the assembly of this device, we also discuss some basic soldering techniques and explain the circuitry used to control the LEDs. Using our open-source user interface, users can automate precise timing and pulsing of light on a personal computer (PC) or an inexpensive tablet. This automation makes the system useful for experiments that use LEDs to control genes, signaling pathways, and other cellular activities that span large time scales. For this protocol, no prior expertise in electronics is required to build all the parts needed or to use the illumination system to perform optogenetic experiments.

This protocol describes how to perform optogenetic experiments for controlling gene expression with red and far-red light using PhyB and PIF3. Included are step-by-step instructions for building a simple and flexible illumination system, which enables the control of gene expression or other optogenetics with a computer.

Optogenetik Deneyler için Basit ve Çok Yönlü Bir Aydınlatma Sistemi Oluşturma

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical ...