<html> Burada LUCAS [1-3] dahil olan deneysel prosedürleri tartışacağız. LUCAS kavramı kanıtı göstermek için biz bütün bir kan örneği görüntüleme işlemi anlatacağım. A. Görüntüleme Set-up LUCAS Görüntüleme platformu, özellikle kaynak sınırlı ayarları için bir maliyet-etkin ve kompakt bir alternatif, mevcut bakım noktası sitometri ve tıbbi tanı araçları sağlamak için önemli avantajlar sergiliyor. LUCAS hücrelerin görüntü tespit yerine, her bir hücrenin arka plan ışığı ile dağınık ışık girişim tarafından oluşturulan hücrelerin yerine dijital hologramlar yakalar. Aydınlatma kısmi mekansal tutarlılığı dikkatli kontrol holografik kayıt etkinleştirmek için kritik öneme sahiptir. 1. Dijital sensör dizisi LUCAS platformu optoelektronik sensör, dijital olarak tek tek hücre hologramlar kaydetmek için bir dizi kullanır. Kullanılabilir: veya tamamlayıcı metal oksit yarı iletken çip (MT9P031, Micron CMOS, Örnek Model): Bu amaçla, çift cihazlar (KAI-11002, Kodak KAF-39000, Örnek Modeller CCD) ücret. Sırasıyla, aktif bir 10 cm2 FOV, 18 cm2, ve 24.4 mm2 Kodak piksel boyutlarında çift cihazları, KAI-11002, KAF-39000 ücret ve Micron CMOS görüntü sensörleri, 9 mm, 6.8 mm ve 2.2 mm. [1-2]. 2. Işık kaynağı Diğer çoğu mikroskopik görüntüleme yöntemlerinin aksine, LUCAS, bu nedenle bir lazer ve hatta basit bir ışık yayan diyot (LED) aydınlatma için kullanılan gerektirmez. Dalga boyu ayarlanabilir aydınlatma sağlamak için, biz de liflerin paket (77.564, Newport) ve iğne deliği oluşan standart bir not erimiş silika elyaf ile birlikte bir Xenon Lamba (Cornerstone T260, Newport Corp) ile monokromatör yararlanabilirler ~~ 100 mikron çapında ~ sensör yüzeyi üzerinde 5-10 cm bulunmaktadır. Bu ayarlanabilir dalga boyu aydınlatma yapılandırma hücrelerin holografik imzaları ayarlanabilir esnek bir platform sağlar ve melez dijital imzalar sinyal gürültü oranı daha iyi karakterizasyonu doğruluk ve özgüllük için geliştirmek için sentezlenmiş olabilir. [3] B. Numune Hazırlama ve Görüntüleme Aşağıda açıklandığı gibi heterojen bir çözüm kullanarak görüntüleme-çip tabanlı LUCAS kavramı bir kanıtı sunulacaktır. Benzer bir protokol, çeşitli hücre tipleri [1-3] için uygulanabilir. 1. Tam kan sulandırma ve heterojen bir çözümün hazırlanması <ol><li> Sürecine başlamak için, bütün bir kan örneği ve çeşitli çaplarda (5, 10 ve 20 mikron. Duke Bilimsel) polistiren mikro almak ve hepsi oda sıcaklığına getirmek. </li><li> RPMI 1640 classic sıvı ortam (Fisher Scientific) bir seyreltici olarak steril bir 5 ml polipropilen tüpe 2 mL ekleyin. </li><li> 30 dakika boyunca tam kan inkübe sonra, 10 mcL tortulaşmış eritrosit örnek pipet ve RPMI çözüm içine aktarın. </li><li> 20 mcL polistiren mikro toplam hacmi, eritrosit RPMI seyreltme içine ekleyin ve hafifçe pipetleme veya bir heyecan çubuğu ile manyetik karıştırıcı kullanılarak hücre çözümü kışkırtmak. </li><li> 5-15 mcL heterojen bir çözüm toplam hacmi, forseps kullanarak ikinci bir çözüm üzerinde, aynı kapak kayma kayma ve yerde bir kapak üzerine yerleştirin. Örnek eşit iki kapak fişleri arasında sıkışmış olmalı. </li><li> Sonra, bir vakum kalem (NT57-636, Edmund Optik) kullanarak görüntüleme sensörü dizinin aktif bölge üzerine örnek yüklemek. </li></ol> 2. Tam Kan Boyama <ol><li> % 0.1 &#39;lik bir karışım hazırlanması ile başlayan güç tipi, saf Eosin Y (MW = 691,85, Acros Organics), çinko içermeyen saf Yeni Metilen Mavi boya (MW = 347,90, Acros Organics) kullanarak Eosin Y ve seyreltilmiş Yeni Metilen Çözüm tamponlu Potasyum Oksalat Monohidrat (99.0% Reaktif ACS, Acros Organik); her çözüm 0,45 mikron gözenek boyutu Syringless Filtresi (Whatman) sulu beyaz kan hücre boyama ile arındırılır. </li><li> Sonra, yavaşça, 2 dakika boyunca bir manyetik karıştırıcı kullanılarak bir hacim oranı 1:1 bir polipropilen beher tam kan örneği ile çözünmüş sulu boyama reaktif karıştırın ve 10 dakika inkübe edin. </li><li> 5 ml polipropilen tüpe 50 mcL lekeli bir kan çözüm aktarın ve 1:200 bir hacim oranı elde etmek için RPMI 1640 classic 1.95 ml sıvı solüsyon (Fisher Scientific) ile seyreltilir. Daha sonra, 30 saniye için seyreltme girdap. Farklı bir seyreltme oranı da bu aşamada devreye alınabilir. </li><li> Kapak fişleri arasında veya bir mikroakışkan rezervuar içinde 10-100 mcL lekeli bir hücre çözümü Pipet. Sonra, bir vakum kalem kullanarak (NT57-636, Edmund Optik) numune, sensör dizisi aktif bölge üzerine yerleştirin. </li></ol> TEMSİLCİSİ SONUÇLAR ve_content &quot;&gt; <img src="/files/ftp_upload/1650/1650Fig1tmb.jpg" alt="Şekil 1" border="0" /> Şekil 1: LUCAS doğru sayma ve çeşitli hücreler ve mikro-nesneleri lensler, lazerler veya diğer hantal optik bileşenleri kullanarak 2 boyutlu holografik imzalar dayalı dijital farklılaşma sağlar. Karakteristik LUCAS imzaları çeşitli mikro-nesneleri bu resimde gösterildiği ve 40X objektif lens ile elde edilen geleneksel bir mikroskop görüntü karşılaştırılır. Lütfen Şekil 1&#39;de bir büyük halini görmek için <a href="https://www.jove.com/files/ftp_upload/1650/1650Fig1.jpg">buraya tıklayınız</a> . <img src="/files/ftp_upload/1650/1650Fig2.jpg" alt="Şekil 2" /> Şekil 2: (Sol) Ham görüntü, kırmızı kan hücreleri, 10um, 5um ve 3 um parçacıklar içeren heterojen bir karışım . Aynı alan için (Sağ) Tam otomatik LUCAS karakterizasyonu sonuçları gösterilmiştir. Karar algoritması 3um boncuklar gibi gürültü oranı yüksek yoğunluklu bölgelerinde, düşük sinyal ile parçacıkların yanı sıra karakterize sağlam olduğunu unutmayın. <img src="/files/ftp_upload/1650/1650Fig3tmb.jpg" alt="Şekil 3" border="0" /> Şekil 3: LUCAS özel arayüz gösterilmiştir. Java tabanlı LUCAS yazılımı gibi sensör piksel boyutu veya ışık dalga boyu gibi çeşitli deneysel koşullar için girdi sağlar. Resmin üzerine görüş belirli bir alan seçimi de yapılabilir ve hedef hücre modelleri, istatistiksel bir hücre gölge kütüphane oluşturmak için kullanıcı tarafından tanımlanabilir. , Elde edilen LUCAS görüntü, daha sonra bu eğitim verileri (örneğin, hücre gölge kütüphane) ve işaretlenmiş (sayılır) resim kullanıcıya görüntülenen dayalı karakterize edilebilir. Sayımı sonuçları, istatistikler, aynı zamanda daha fazla analiz için bir XML dosyası olarak depolanır.

<ol>
	<li>Seo, S., Su, T., Tseng, D. K., Erlinger, 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+Holographic+Imaging+for+On-Chip+Cytometry+and+Diagnostics".">"Lensfree Holographic Imaging for On-Chip Cytometry and Diagnostics".</a> Lab Chip. , (2009).</li><li>Su, T., Seo, S., Erlinger, 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="High-throughput+Imaging+and+Characterization+of+a+Heterogeneous+Cell+Solution+on+a+Chip"+.">"High-throughput Imaging and Characterization of a Heterogeneous Cell Solution on a Chip" .</a> Biotechnology and Bioengineering. , (2008).</li><li>Su, T., Seo, S., Erlinger, 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="Multi-color+LUCAS:+Lensfree+on-chip+cytometry+using+tunable+monochromatic+illumination+and+digital+noise+reduction".">"Multi-color LUCAS: Lensfree on-chip cytometry using tunable monochromatic illumination and digital noise reduction".</a> Cellular and Molecular Bioengineering. , (2008).</li></ol>

LUCAS platformu doğru saymak ve holografik imzaları dayalı bir çip üzerinde çeşitli micro-objects/cells tespit gösterildiği gibi, noktası bakım, tıbbi teşhis ve yüksek verimli hücre biyolojisi için umut vaat eden bir araç sağlar. Doğru kaydedilen holografik desen işlemek için geliştirilmiş özel bir LUCAS karar yazılım uygulanmaktadır. LUCAS görüntülerin eğitim tarafından oluşturulan bir istatistik kırınım görüntü veritabanını kullanır Bu algoritma, heterojen bir çözüm içinde...

<table><tbody><tr><td>Charged couple device (CCD)</td><td>KODAK</td><td>KAI-11002</td><td></td></tr><tr><td>Charged couple device (CCD)</td><td>KODAK</td><td>KAF-39000</td><td></td></tr><tr><td>Complementary Metal-Oxide-Semiconductor (CMOS)</td><td>Micron</td><td>MT9P031</td><td></td></tr><tr><td>Xenon Lamp</td><td>Newport Corp.</td><td>Cornerstone T260</td><td></td></tr><tr><td>Vacuum pen</td><td>Edmund Scientific</td><td>NT57-636</td><td></td></tr><tr><td>5, 10, and 20 &amp;mu;m Microbeads</td><td>Thermo Fisher Scientific, Inc.</td><td>4000 Series</td><td></td></tr><tr><td>RPMI</td><td>Fisher Scientific</td><td>1640</td><td></td></tr><tr><td>Pure Eosin Y</td><td>Acros Organics</td><td>MW=691.85</td><td></td></tr><tr><td>New Methylene Blue(NMB) Dye</td><td>Acros Organics</td><td>MW=347.90</td><td></td></tr><tr><td>Potassium Oxalate Monohydrate</td><td>Acros Organics</td><td>99.0% Reagent ACS</td><td></td></tr></tbody></table>

lensless on-chip imaging of cells provides a new tool for high-throughput cell-biology and medical diagnostics

Diğer lens ve optik bileşenlerin birlikte çalışan nesnel lenslerin kullanımı ile Konvansiyonel optik mikroskoplar görüntü hücreleri. Oldukça etkili olmakla birlikte, bu klasik yaklaşım Mikroakiskan sanat gelişmiş devlet ile uyumlu hale getirmek için görüntüleme platformu minyatürleştirme için bazı sınırlamalar vardır. Bu raporda, LUCAS olarak adlandırılan, bir lensless çip üzerinde görüntüleme kavramının deneysel detaylar (tanıtmak<strong<em&gt; L</em</strong&gt; Ensless<strong<em&gt; U</em</strong&gt; LTRA geniş alan görüş<strong<em&gt; C</em</strong&gt; Ell izleme<strong<em&gt; A</em</strong&gt; Rray platformu dayalı<strong<em&gt; S</em</strong, Görüntü gibi büyük açıklıklı bir ultra geniş alan heterojen bir hücre çözümü hedefleri için herhangi bir mikroskop veya diğer hantal optik bileşenleri gerektirmez&gt; hadow görüntüleme) ~ 18 cm<sup&gt; 2</sup&gt;. Ayrıca, geleneksel mikroskoplar aksine, LUCAS görüntü, ~ 9 ml numune hacmi karşılık refocusing gerek kalmadan ~ 5 mm derinlik-alan üzerinde ilgi heterojen bir hücre çözümü. Bu görüntüleme platformu, tipi, sayısı ve çözüm içindeki hücrelerin göreli konumları belirlemek, her bir hücre, görüş alanı içinde ilgi ve bu hücre gölgelerin otomatik dijital işleme (yani, lensless dijital hologramlar) gölgesinde kaydeder. Hantal herhangi bir optik bileşenleri veya mekanik tarama aşamaları gerektirmez, çünkü, aynı zamanda, bakım tanılama araçları özellikle noktası için oldukça önemlidir maliyeti, azalttığını önemli ölçüde minyatür bir platform sunmaktadır. Ayrıca, bu platformun görüntüleme hacmi yüksek verimli hücre biyoloji deneyleri için son derece değerli olabilir geleneksel optik mikroskoplar, daha iyi büyüklükte emir.

Watch this Scientific Journal Video about Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics at JoVE.com

Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics

Hücre Lensfree çip üzerinde görüntüleme ve karakterizasyonu gösterilmiştir. Bu çip üzerinde hücre görüntüleme yaklaşımı, kaynak yoksul ayarları için özellikle uygun hale, tıbbi teşhis ve yüksek verimli hücre biyolojisi uygulamaları için kompakt ve maliyet-etkin bir araç sağlar.

Hücre Lensless-çip Görüntüleme Yüksek throughput Hücre Biyolojisi ve Tıbbi Teşhis için Yeni Aracı sağlar.

Conventional optical microscopes image cells by use of objective lenses that work together with other lenses and optical components. While quite ...

lensless-on-chip-imaging-cells-provides-new-tool-for-high-throughput

Research

JoVE Journal

Biology

11.9K Görüntüleme.  University of California, Los Angeles. Hücre Lensfree çip üzerinde görüntüleme ve karakterizasyonu gösterilmiştir. Bu çip üzerinde hücre görüntüleme yaklaşımı, kaynak yoksul ayarları için özellikle uygun hale, tıbbi teşhis ve yüksek verimli hücre biyolojisi uygulamaları için kompakt ve maliyet-etkin bir araç sağlar.

Video: Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Yüksek çözünürlüklü Fiber-optik Mikroendoskopi In situ Hücresel Görüntüleme

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Biyomühendislik

Lensless Fluorescent Microscopy on a Chip

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for high-throughput screening applications. This emerging technology platform has the potential to eliminate the need for bulky and/or costly optical components through the help of novel theories and digital reconstruction algorithms. Along the same lines, here we demonstrate an on-chip fluorescent microscopy modality that can achieve e.g., &lt;4&mu;m spatial resolution over an ultra-wide field-of-view (FOV) of &gt;0.6-8 cm2 without the use of any lenses, mechanical-scanning or thin-film based interference filters. In this technique, fluorescent excitation is achieved through a prism or hemispherical-glass interface illuminated by an incoherent source. After interacting with the entire object volume, this excitation light is rejected by total-internal-reflection (TIR) process that is occurring at the bottom of the sample micro-fluidic chip. The fluorescent emission from the excited objects is then collected by a fiber-optic faceplate or a taper and is delivered to an optoelectronic sensor array such as a charge-coupled-device (CCD). By using a compressive-sampling based decoding algorithm, the acquired lensfree raw fluorescent images of the sample can be rapidly processed to yield e.g., &lt;4&mu;m resolution over an FOV of &gt;0.6-8 cm2. Moreover, vertically stacked micro-channels that are separated by e.g., 50-100 &mu;m can also be successfully imaged using the same lensfree on-chip microscopy platform, which further increases the overall throughput of this modality. This compact on-chip fluorescent imaging platform, with a rapid compressive decoder behind it, could be rather valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

A lensless on-chip fluorescent microscopy platform is demonstrated that can image fluorescent objects over an ultra-wide field-of-view of e.g., &#62;0.6-8 cm2 with &#60;4&#956;m resolution using a compressive sampling based decoding algorithm. Such a compact and wide-field fluorescent on-chip imaging modality could be valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

Bir Chip Lensless Floresan Mikroskopi

On-chip lensless imaging in general aims to replace bulky lens-based optical microscopes with simpler and more compact designs, especially for ...

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

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. 1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.
Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7 LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of &lt;1 &mu;m x &lt;1 &mu;m x &lt;3 &mu;m in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3, and can be particularly useful for lab-on-a-chip platforms.

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of &lt;1 &mu;m &times; &lt;1 &mu;m &times; &lt;3 &mu;m in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

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 ...

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 &mu;m over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.

Bir Cep telefonu ile ilgili Geniş alan Floresan Mikroskopi ve Floresan Görüntüleme Flow Sitometri

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

Tek Hücrelerinin yönlü Kimyasal Analiz için mikroakışkan Chip

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 &#181;m x 500 &#181;m region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

Fotonik Çip Kullanarak Yüksek İşLeç Toplam İç Yansıma Floresansı ve Direkt Stokastik Optik Rekonstrüksiyon Mikroskobu

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

Biyokimya

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Yüksek Verimli Mikroakışkan Cihaz Kullanarak Dinamik ÇevreSel Koşulların Üretilmesi

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience comorbidity related to chronic inflammation despite antiretroviral therapy. However, these inflammatory signaling has not been fully characterized. The role of early entry events on the activation of cellular signaling events and downstream gene expression has not been captured at the single-cell level. Here the authors describe a method that applies principles of live-cell fluorescence microscopy to an automated single-cell platform that cultures and images cells over user-customized time courses, allowing for high-throughput analysis of dynamic cellular processes. This assay can track single-cell live fluorescence microscopy of early events that immediately follow HIV-1 infection, notably the influx of calcium that accompanies exposure to the virus and the development of productive infection using a fluorescent reporter virus. MT-4 cells are loaded with a calcium-sensitive dye and cultured in isolated pens on a nanofluidic device. The cultured cells are infected with an HIV-1 reporter virus (HIV-1 NLCI). A fluorescence microscope positioned above the nanofluidic device measures calcium influx over an 8-min time course following acute HIV-1 exposure. HIV-1 productive infection is measured in those same cells over a 4-day interval. Imaging data from these time courses are analyzed to define virus-host receptor interactions and signaling pathway dynamics. The authors present an integrated, scalable alternative to traditional imaging methods using a novel optofluidic platform capable of single-cell sorting, culturing, imaging, and software automation. This assay can measure the kinetics of events under various conditions, including cell type, agonist, or antagonist effect, while measuring an array of parameters. This is the first established method for nanofluidic high-throughput longitudinal single-cell culture and imaging: This technique can be broadly adapted to study cellular signaling kinetics and dynamic molecular interactions.

Here, we present a protocol in which single cells are monitored for acute events and productive HIV-1 infection on a nanofluidic device. Imaging data define virus-host receptor interactions and signaling pathway dynamics. This is the first method for nanofluidic high-throughput longitudinal single-cell culture and imaging to study signaling kinetics and molecular interactions.

Multiparametre Optofluidik Platform Kullanılarak Kalsiyum Akını ve HIV-1 Enfeksiyonunun Tek Hücreli Karakterizasyonu

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience ...

İmmünoloji ve Enfeksiyon

Microfabrication of Implantable Optics Integrated in a Microstructured Imaging Window for Advanced In Vivo Imaging

In the context of biomaterials and drug testing in animal models, this study presents a streamlined protocol for fabricating a novel implantable integrated imaging window. The micro-device comprises a sophisticated system of microlenses coupled with micro-scaffolds specifically designed for in vivo quantification of the immune response using advanced non-linear excitation microscopy. The protocol is based on two-photon polymerization (2PP) of the biocompatible photoresist SZ2080, which enables the fabrication of micro-scaffolds and micro-lenses in a continuous sequence to enhance manufacturing efficiency and precision. To further improve speed, accuracy, and structural integrity, a hybrid optics fabrication approach was implemented, involving the 2PP of the microlens outer shell followed by UV bulk crosslinking of the inner core. This innovative technique optimizes the optical properties of the microlenses while streamlining the production process. The resulting micro-device demonstrates high reproducibility and mechanical stability, making it an effective method for prototyping microscale optical systems for a range of biomedical applications.

This protocol describes the fabrication of an implantable integrated imaging window using 3D laser printing. The window consists of a system of microlenses coupled with micro-scaffolds. The method involves two-photon polymerization (2PP) of the biocompatible photoresist SZ2080 in a continuous sequence, optimizing manufacturing efficiency and alignment between the different components.

Gelişmiş In Vivo Görüntüleme için Mikroyapılı Görüntüleme Penceresine Entegre Edilmiş İmplante Edilebilir Optiklerin Mikrofabrikasyonu

In the context of biomaterials and drug testing in animal models, this study presents a streamlined protocol for fabricating a novel implantable ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

Dinamik için objektif-ücretsiz Video mikroskobu ve kantitatif analiz yapisan hücre kültürü

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...