A. Özcan minnetle Fen ve Mühendislik için Cumhurbaşkanlığı Erken Kariyer Ödülü (PECASE) da desteğiyle kabul Ordu Araştırma Bürosu (ARO) Genç Araştırmacı Ödülü, Ulusal Bilim Vakfı (NSF) KARİYER Ödülü, Donanma Araştırma Bürosu (ONR) Genç Araştırmacı Ödülü ve Ulusal Sağlık Enstitüleri (NIH) Müdürü, Ulusal Sağlık Enstitüleri Ofisi Direktörü sitesindeki New Yenilikçisi Ödülü DP2OD006427.

Bu bölümde, bizim cep telefonu tabanlı geniş alan floresan mikroskobu 13 ve opto-akışkan görüntüleme sitometri platformu 14 deneysel protokolleri tanıtmak. Biz bu görüntüleme platformlar sınamak için floresan boncuk ve floresan etiketli beyaz kan hücreleri kullanır. Cep telefonu A. Hazırlık Geniş alan Floresan Mikroskop ve Opto-akışkan Görüntüleme Akış Sitometresi sonucu Bir kameralı cep telefonu ve eklenti eklemek-kompakt bir opto-akışkan: cep telefonu tabanlı geniş alan floresan mikroskop ya flowsitometri iki ana bölümden oluşmaktadır. 1. Kameralı Telefon Sunulan tekniklerin hemen hemen her cep telefonu için geçerli olsa da, biz bu cihazlar için üs olarak Sony Erickson Aino seçtiniz. Bu cep telefonu üzerinde yüklü bir ~ 8 MegaPiksel RGB CMOS sensör ve bir odak uzaklığı (f 1) ~ 4.65 mm olan yerleşik bir objektife sahip. </p&gt; 2. Geniş alan Floresan Mikroskopi için Opto-akışkan Eklenti Optik eki Autodesk tarafından tasarlanmış ve ABSplus termoplastik malzeme kullanılarak 3 boyutlu yazıcı bir boyut Elite tarafından yazdırılır. Bu baskı süreci, model ve destek malzemeleri yazıcının içinde bir ekstrüzyon kafası ısıtılır ve bir modelleme kaide üzerinde tabaka tabaka yatırılır. Bu adım tamamlandıktan sonra, destek malzemesi, bizim optik bağlanma dizayn LED (~ 470 nm &#39;de orta dalga boyu, Digikey), plastik bir filtre (# NT54-46, Edmund oluşan arzu edilen prototipin bir sağlam bir 3D modeli bırakarak, çözülebilir Optik), örnek bir tepsi ve bir yüzü düz diğeri dışbükey mercek f 2 = 15 mm (# NT45-302, Edmund Optik). Tüm LED&#39;ler ve plastik filtreler kolayca floroforlar &#39;spektrumları dayalı değiştirilebilir. Bu opto-akışkan eki montajı için adımlar şunlardır: <ol><li> Kendine özgü lens kabı içindeki pozisyonlarını eki içine mercek yerleştirin. </li><li> Filtre tepsisine plastik filtre yerleştirin ve eki içine kaydırın veya bant cep telefonu kamera lensi önündeki plastik filtre. </li><li> Eki içine LED tepsiyi takın. </li><li> Örnek tepsisine örnek cam slaytlar yerleştirin. Eki içine numune tepsisini kaydırın. Örnek doğru LED&#39;ler Yüz. </li><li> Fazladan bir objektifin doğrudan cep telefonu kamera merceği ile temas halinde olduğu gibi, cep telefonu üzerine eki klipsleyin. </li><li> LED&#39;lerini açmak için eki üzerindeki düğmeyi kullanın. </li><li> Onun &quot;gece modu&quot; kullanılarak cep telefonu kamera ünitesi ile ilgi resmi örnek. </li></ol> 3. Floresan Görüntüleme Cytometry Opto-akışkan Eklenti Nadir olayların tespiti için sıvı örnekler büyük miktarlarda taranması için bir ihtiyaç olduğu zaman, optofluidic akış sitometri cihazı tercih edilebilmektedir. Biz geniş alan floresan mikroskop tasarımını değiştirmek ve bir akış sitometresinin, w haline dönüştürebilirsinizburada bir PDMS tabanlı mikro kanal sürekli olarak görüntüleme hacmi içinden geçerek sıvı örnek vermek için kullanılır. Optik eki ayrıca Autodesk tarafından tasarlanan ve Dimension tarafından Elite 3-D yazıcı yazdırılır. Ayrıca LED (~ 470 nm merkezi dalgaboyu, Digikey), bir plastik filtre (# NT54-46, Edmund Optik), örnek bir tepsi ve bir asferik lens (f = 4,5 mm) (Ürün # C230TME-A oluşur; Thorlab). Bu opto-akışkan eki montajı için adımlar şunlardır: <ol><li> Ek parçaya asferik mercek yerleştirin. </li><li> Filtre tepsisine plastik filtre yerleştirin ve eki oturtun; veya bant cep telefonu kamera objektifi önünde plastik filtre. </li><li> Aynı opto-akışkan ek parçaya mikroakışkan kanal kaydırın. </li><li> Fazladan bir objektifin doğrudan cep telefonu kamera merceği ile temas halinde olduğu gibi cep telefonu üzerine eki klipsleyin. </li><li> LED&#39;lerini açmak için eki üzerindeki düğmeyi kullanın. </li><li> Mikroakışkanlar bağlayınic şırınga pompası kanal ve sabit bir akış hızında mikroakışkan cihazın içine sıvı numune teslim. </li><li> Floresan hücre / cep telefonu kamera video modu kullanarak mikroakışkan kanal akan parçacıkların bir film çekin. </li></ol> B. Numune Hazırlama 4. Floresan Mikro-parçacık Örneklerinin Hazırlanması <ol><li> 10 mikron çapı (: Ürün # F8834 uyarma / 580nm/605nm emisyon, yeşil boncuk: kırmızı boncuklar Ürün # F8836: uyarma / emisyon 505nm/515nm) ile Floresan boncuk Invitrogen (Carlsbad, CA) satın alınır. </li><li> Yeşil floresan boncuklar, distile su, 40 ul ile kırmızı flöresanlı boncuklar 10 ul 10 ul karıştırılır. </li><li> Bir mikropipet ve bir sandviç yapı oluşturabilmek için bunun üstüne başka bir cam slayt koymak kullanarak bir cam slayt üzerine bu boncuk karışımın Place10 ul. </li><li> Örnek tepsisine Bu sandviç yapı takın ve cep telefonu eki içine kaydırın. </li&gt; </ol> 5. Floresan etiketli Akyuvarlar hazırlanması <ol><li> SYTO16 nükleik asit fluoresan etiketleme kiti (# S7578, Life Technology) ve fosfat al salin (PBS) buzdolabından dışarı arabelleğe alınmış ve oda sıcaklığına getirin. </li><li> EDTA&#39;lı kan toplama tüpü 1,5 ml polistiren tüp (# 05-408-129, Fisher Scientific) 200 ul tam kan numunesi aktarın. </li><li> İyice 200 ul tam kan numunesi ve karıştırın (# R7757, Sigma-Aldrich) 1 ml eritrosit parçalama tamponu ekleyin. </li><li> 5 dakika sonra, erimiş kan örneği santrifüjden sonra üstte yüzen madde çözeltisi çıkarın. </li><li> 200 ul PBS tamponu içine beyaz kan hücre pelletini tekrar ve yavaşça karıştırın. </li><li> Beyaz kan hücre örneği 5 ul 1 mM SYTO16 çözeltisi ekleyin. ~ 30 dakika karanlık bir ortamda alüminyum folyo ve inkübe ile örnek sarın. </li><li> Yine örnek santrifüjleyin. Süpernatant kaldırılır ve etiketlenmiş beyaz kan hücresi pelet tekrar edilirPBS tamponu içine-askıya. </li><li> Bir kapak kayma için beyaz kan hücresi sıvı örneği etiketli 5-10 ul yerleştirin ve numunenin üstüne ikinci bir kapak kayma yerleştirin. </li><li> Numune tepsisi ve görüntüyü cep telefonu floresan mikroskop kullanarak içine sıkışmış örnek slayt yerleştirin. </li></ol> Alternatif olarak <ol start="10"><li> Sürekli aynı zamanda video modunda cep telefonu kamera kullanarak akan hücre floresan mikroskobik filmi çekerken, otomatik şırınga pompa kullanarak bir mikroakışkan kanal aracılığıyla floresan etiketli beyaz kan hücreleri teslim. Ayrıca, taşınabilir bir pille çalışan bir şırınga pompası ya da yerçekimi kuvveti mikroakışkan kanalı ile akış sürmek için kullanılabilir vurgulamak gerekir. </li></ol>

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Dr Özcan hesaplamalı görüntüleme ve mikroskop araçları ticarileştirmek amaçlayan bir start-up şirketi kurucusu.

Biz geniş alan floresan mikroskopi ve cep telefonu kameraları için hafif ve kompakt opto-akışkan ekleri kullanarak opto-akışkan görüntüleme flow sitometri tabanlı cep telefonu bizim son sonuçlar sundu. Bu platform teknolojisi kullanarak biz mikro-parçacıklar ve tam kan örneklerinde etiketli beyaz kan hücreleri dahil olmak üzere floresan nesneleri görüntülendi. Bu nedenle, bu kompakt ve maliyet-etkin cep telefonu tabanlı floresan görüntüleme araç kendi CD4 HIV + hastalarda izlenmesi gibi çeşitl...

Mikroskopi ve akım sitometri yaygın biyomedikal ve bilimsel araştırma teknikleri 1-12 yanı sıra çeşitli hücre tipleri sayımı ve karakterizasyonu için klinik tanı kullanılmaktadır. Ancak, geleneksel mikroskop ve akım sitometri enstrümanlar yoğunlukla köklü merkez laboratuarlar kullanımlarını kısıtlar, hangi nispeten karmaşık ve pahalıdır. Son zamanlarda biz ümit vaat hangi bir cep telefonu, 13,14 entegre kompakt ve hafif bir floresan görüntüleme sitometri ve mikroskopi cihazı geliştirdik maliyet-etkin için kaynak kısıtlı ortamlar için floresan mikroskobu, akım sitometri ve ilgili mikro-analiz teknikleri çevirmek küresel sağlık etkileyen çeşitli teletıp uygulamaları. Optofluidic akım sitometri yapılandırmada (örn. Şekil 1C ve 1D bakın), özel tasarlanmış polydimethysiloxane (PDMS) tabanlı mikroakışkan kanal bir pozisyonda olduğunuışık yayan diyot (LED) kanalın kenarlarına popo-birleştiğinde cep telefonu kamera ünitesi önünde d. Bu mikroakışkan çip birlikte, sıvı numune içinde birlikte uyarma ışığı eşit mikro kanal içinde flüoresan etiketli örnekler pompalamak için yönlendirilir, bu gibi bir opto-akışkan düzlemsel dalga kılavuzu (örneğin PDMS-sıvı-PDMS için oluşan) oluşturur. Bu etiketli nesneleri floresans emisyon, örneğin hücreler, daha cep telefonu kamera ünitesi hemen sonra yerleştirilen ek bir lens aracılığıyla görüntülü ve cep telefonu Tamamlayıcı Metal-Oksit-Yarıiletken (CMOS) görüntü sensörünün üzerine eşleştirilir. Floresan emisyon uyarma ışık yoluna dik toplanan bu yana, ucuz bir plastik emme filtresi dağınık uyarma ışığa çıkarmak için yeterli olduğunu ve floresan görüntüleme için gerekli iyi bir karanlık alan arkaplan sağlayabilir. Bu görüntü, stat floresan nesneleri aynı zamanda, benzer bir opto-akışkan dizayn kullanarak,floresan parçacıkların iki cam arasına sıkıştırılmıştır hangi ic mod (Şekil 1A ve 1B), cep telefonu CMOS görüntü partikül sayımı için sensör ve tarafından yakalanan yerine bu floresan parçacıkların bir mikroakışkan kanal ve floresan emisyon yoluyla akan slaytlar karakterizasyonu. Farklı uygulama gereksinimleri, flow sitometri veya geniş alan floresan mikroskop İstinaden seçilebilir. Örneğin, cep telefonu akım sitometri cihazı nadir hücre veya patojenlerin tespiti için sıvı örnekler (birkaç mL gibi) büyük hacimli tarama için özellikle yararlı olabilir. Bu yazıda kompakt ve maliyet-etkin opto-akışkan ekleri kullanarak bir cep telefonu üzerinde floresan mikroskop ve görüntüleme flow sitometri araçlarının entegrasyonu bizim son bazı sonuçları gözden geçirin. Bu cep telefonu tabanlı mikro-analiz, görüntüleme sitometri ve algılama platformları çeşitli fırsatlar sağlayabilirTeletıp ve nokta-of-bakım teşhis için, özellikle dünyanın kısıtlı kaynak bölgelerinde küresel sağlık sorunları karşı mücadeleyi etkileyen.

<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Company</td>
			<td>Catalog Number </td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell-phone</td>
			<td>Sony</td>
			<td>Sony Ericsson Aino</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plano-convex lens</td>
			<td>Edmund Optics</td>
			<td># NT45-302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aspherical lens</td>
			<td>Thorlab</td>
			<td># C230TME-A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Edmund Optics</td>
			<td>#NT54-46</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Blue LED</td>
			<td>Digikey</td>
			<td>#365-1201-ND</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Battery</td>
			<td>Digikey</td>
			<td>#P032-ND</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polystyrene tube</td>
			<td>Fisher Scientific</td>
			<td>#05-408-129</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Red blood cell lysing buffer</td>
			<td>Sigma Aldrich</td>
			<td>R7757</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SYLGARD 184 SILICONE ELASTOMER KIT</td>
			<td>Dow Corning</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Red fluorescent beads (10 &mu;m)</td>
			<td>Life Technologies</td>
			<td>#F8834</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Green fluorescent beads (10 &mu;m)</td>
			<td>Life Technologies</td>
			<td>#F8836</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SYTO16 nucleic acid fluorescent labeling</td>
			<td>Life Technologies</td>
			<td># S7578</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone

Floresan mikroskobu ve akım sitometri yaygın biyomedikal araştırmalar ve klinik tanıda kullanılan araçlardır. Ancak bu cihazlar kısıtlı kaynak ayarlarında onları daha az etkin hale getirilmesi, genel olarak nispeten hantal ve pahalı. Potansiyel olarak bu sınırlamaları gidermek için, son zamanlarda kompakt, hafif ve uygun maliyetli opto-akışkan ekleri kullanarak cep telefonları geniş alan floresan mikroskop ve görüntüleme flow sitometri araçlarının entegrasyonu göstermiştir. Bizim flow sitometri tasarımı, floresan etiketli hücreleri mevcut cep telefonu kamera ünitesi yukarıda konumlandırılmış bir mikroakışkan kanal boşaltıldı. Akü ile çalışan ışık yayan diyot (LED) etkin uyarma ışık eşit floresan hedefleri heyecanlandırmak için yönlendirilir bir multi-mode levha dalga olarak görür bu mikroakışkan çip, tarafına popo-birleştiğinde vardır. Cep telefonu kamera akan floresan hücrelerinin zaman atlamalı film kaydederBu filmin dijital çerçeveleri ilgi hedef çözüm içinde etiketli hücreleri saymak için işlenir mikroakışkan kanal. Biz bu floresan etiketli hücreleri örneğin iki cam slaytlar arasındaki floresan parçacıkların sandviç ve örneğin bir uzaysal çözünürlüğü elde edebilirsiniz cep telefonu kamera kullanarak floresan görüntüler yakalayarak statik modda görüntü de, benzer bir opto-akışkan tasarımı kullanarak, ~ ~ 81 mm 2 çok geniş bir görüş alanı görüntüsü üzerine 10 mikron. Bu cep telefonu floresan görüntüleme akım sitometri dayanır ve mikroskopi platformu CD4 + HIV + hastalarda izlenmesi doğru veya içme suyu su-kaynaklı parazitler tespit için T hücrelerinin sayımı örn, özellikle kaynakların sınırlı olduğu ortamlarda yararlı olabilir.

Cep telefonu kamera akan hücrelerin bir zaman yitirmez floresan mikroskobik film kaydeder iken bizim pompalama opto-akışkan / eksitasyon şeması (Şekil 1C ve 1D) ile, floresan etiketli hücreleri sürekli bir şırınga pompası ile mikroakışkan kanal içine teslim edilebilir. Bu floresan filmler daha sonra hızlı bir şekilde floresan görüntüleme akış-sitometrisi fonksiyonu alarak, otomatik olarak tam sayı ve mikroakış kanalları üzerinden akan hücre yoğunluğu belir...

Watch this Scientific Journal Video about Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone at JoVE.com

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

Biz kompakt ve maliyet-etkin opto-akışkan ekleri kullanarak bir cep telefonu üzerinde floresan mikroskop ve görüntüleme flow sitometri araçlarının entegrasyonu bizim son sonuçlarını gözden geçirin. Bu cep telefonu tabanlı mikro-analiz cihazları gibi kaynakların sınırlı olduğu ortamlarda, örneğin su örneklerinin yüksek verimli tarama yanı sıra çeşitli hücre sayım görevleri yapmak gibi, sitometrik analizi için yararlı olabilir.

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 ...

wide-field-fluorescent-microscopy-fluorescent-imaging-flow-cytometry

Research

JoVE Journal

Bioengineering

23.5K Görüntüleme.  University of California, Los Angeles. Biz kompakt ve maliyet-etkin opto-akışkan ekleri kullanarak bir cep telefonu üzerinde floresan mikroskop ve görüntüleme flow sitometri araçlarının entegrasyonu bizim son sonuçlarını gözden geçirin. Bu cep telefonu tabanlı mikro-analiz cihazları gibi kaynakların sınırlı olduğu ortamlarda, örneğin su örneklerinin yüksek verimli tarama yanı sıra çeşitli hücre sayım görevleri yapmak gibi, sitometrik analizi için yararlı olabilir.

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

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

Biyolojik Sistemleri İyon Konsantrasyon Ölçümü için Floresan Nanopartiküller

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

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 ...

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. 
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

Bıçak kenar Tarama Mikroskopi için Numune Hazırlama, Görüntüleme ve Analiz Protokolleri

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Çoklu-spektral Görüntüleme Akım Sitometri tarafından Nanopartiküller ve Bakterilerin Hücresel İçselleştirilmesi Analizi

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Çokspektrumlu Görüntüleme Sitometrisi ile Hücreleri yerelleştirme ve Karbon nanotüpler Bağıl Kantitasyonu

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

Çekme Kuvveti Mikroskopi Hücre Kültürü Yüzey civarında yerelleştirme raportör flöresan Beads için Yeni Bir Yöntem

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

Hücreleri tarafından seçilmiş adezyon oluşumunda görselleştirme gelişmiş Disk-toplam iç yansıma floresans mikroskobu iplik

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

Multispectral görüntüleme akışı sitometrisi kullanarak In vitro micronucleus assay gerçekleştirmek için otomatik bir yöntem

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies for the detection of CTCs include flow cytometry, microfluidic devices, and real-time polymerase chain reaction (RT-PCR). Despite recent advances, methods for the detection of early ovarian cancer metastasis still lack the sensitivity and specificity required for clinical translation. Here, a novel method is presented for the detection of ovarian circulating tumor cells by photoacoustic flow cytometry (PAFC) utilizing a custom three dimensional (3D) printed system, including a flow chamber and syringe pump. This method utilizes folic acid-capped copper sulfide nanoparticles (FA-CuS NPs) to target SKOV-3 ovarian cancer cells by PAFC. This work demonstrates the affinity of these contrast agents for ovarian cancer cells. The results show NP characterization, PAFC detection, and NP uptake by fluorescence microscopy, thus demonstrating the potential of this novel system to detect ovarian CTCs at physiologically relevant concentrations.

A protocol is presented to detect circulating ovarian tumor cells utilizing a custom-made photoacoustic flow system and targeted folic acid-capped copper sulfide nanoparticles.

Fotoakustik Akış Sitometri kullanarak Yumurtalık Kanseri Algılama

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies ...

Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome formulation, liposomes may be preferentially taken up by different cell types in the body. This may influence the efficacy of the therapeutic particle as progression of different diseases is tissue- and cell-type-specific. In this protocol, we present one method for synthesizing and fluorescently labeling liposomes using DSPC, cholesterol, and PEG-2000 DSPE and the lipid dye DiD as a fluorescent label. This protocol also presents an approach for delivering liposomes in vivo and assessing cell-specific uptake of liposomes using flow cytometry. This approach can be used to determine the types of cells that take up liposomes and quantify the distribution and proportion of liposome-uptake across cell types and tissues. While not mentioned in this protocol, additional assays such as immunofluorescence and single-cell fluorescence imaging on a cytometer will strengthen any findings or conclusions made as they permit assessment of intracellular staining. Protocols may also need to be adapted depending on the tissue(s) of interest.

The goal of this protocol is to synthesize fluorescently-labeled liposomes and use flow cytometry to identify in vivo localization of liposomes at a cellular level.

Floresan Boya Etiketli Lipozomların İn Vivo Dokuya Özgü Hücresel Alımının Hazırlanması, Uygulanması ve Değerlendirilmesi

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome ...