Biz araştırma desteği, NIH NIBIB İntramural Araştırma Programı tarafından kabul ediyorsunuz.

Örnek Hücre Montaj ve Tork <ol><li> Biz birlikte 2 pencere meclisleri koyarak örnek hücre yapısı başlar. Pencere tutucu bir pencere conta yerleştirin. Pozisyon gemisi açılış sahibinin kaması karşısında pencere pencere tutucu içinde astar (ya da bazen bir pencere yastık denir) böylece. Hafif bir açıyla, pencere pencere pencere sahibinin anahtar yolu ile katip işareti hizalayarak tutucuya yerleştirin. Pencerenin her iki tarafta çok kenarlarına hafifçe bastırın. </li><li> Uygun sızdırmazlık ve örnek hücrenin doğru tork sağlamak için, biz hafifçe kat Spinkote yağ ile konut conta ve vida halkası. Spinkote başparmak ve işaret parmağı arasında çok küçük bir miktar sürün. Spinkote ince, görünmez bir film vida halka konuları Coat. Aynı şekilde, kat konut conta. Herhangi bir görünür yağ silin. </li><li> Kama konut anahtar ile hizalayarak hücre gövdesi içine, size doğru bakan pencere, bir pencere montaj sürgülü örnek hücre montaj başlayın. Merkezinde kaması konut anahtar ile aynı hizaya getirin ve yavaşça hücre içinde pencere montaj üstüne düşürdüm. </li><li> Merkezinde hücre gövde içine itmek için her türlü aracı kullanmak. Bu deneyler sırasında sızıntısı sonucu CP kalıcı hasara neden olabilir. Pencere sizden uzağa merkezinde doğru bakacak şekilde çevirin ikinci pencere montaj, konut anahtar ile Kama merkezinde üstüne hizalayın ve aşağı doğru kaydırın. Üstünde bir konut conta yerleştirin, sonra bir vida halka &quot;Out&quot; sözcüğü görünür. Parmaklar ve bir hizalama aracı kullanarak, el vida halka sıkın. CP konut varil kolayca slayt değilse, ilk hizalayın, daha sonra bunun üstüne ikinci bir pencere montaj yerleştirin. Pencere montaj hafifçe aşağı doğru basınç uygulayarak, aynı zamanda hücre gövde içine hem slayt. Bu şekilde, doğrudan CP basmaktan kaçının. Şimdi toz, tüy bırakmayan ve parmak izi için hücre içine bakmak için iyi bir zaman olabilir. Ayrıca Newton&#39;un CP ve pencereler arasında hava hala gösterir Yüzüklerin göreceksiniz. Bu tork uygulanarak sonra kaybolur. </li><li> Vida halka ve görünür &quot;out&quot; kelimesi ile tork stand hücre tork Halkalı örnek hücre içindeki tüm yol aşağı yerde. Tork stand sap üzerinde sabit bir basınç uygulayarak hücre tutun. Biri sürekli hareket, vida halka-lbs 120-140 arasında sıkın. Ayarlanabilir bir mikrometre tork anahtarı kullanarak, / kg 120-140 arasında ayarlayın ve ayarlanan tork belirten anahtarı &quot;tıklama&quot; ulaşıncaya kadar vida halka sıkın. Tork standı kolunu serbest bırakın ve pencereleri dokunmadan dikkatlice örnek hücre kaldırmak. </li></ol> Örnek Hücresi Yükleme ve Sızdırmazlık <ol start="6"><li> Sedimantasyon hızı deneyleri için, rutin 200ul pipet kullanarak 12mm merkezinde her bir sektör haline 400ul hacmi yükleyin. Üst ve vida halka görünür &quot;out&quot; sözcüğü yükleme delikleri ile örnek hücre yerleştirin. SOL merkezinde sektörünün yükleme deliklerden içine pipet yarı yolda aşağıya doğru kaydırın ve yavaş yavaş referans çözüm 200ul dağıtmak. 400ul toplam bir kez tekrarlayın. Aynı pipet ve aynı tekniği kullanarak, dikkatli ve yavaş SAĞ merkezinde sektöründe örnek çözüm 400ul ile doldurun. 150-180 ul Birimleri sedimantasyon denge deneyleri için kullanılan ama aynı özen ve teknik takip edilmelidir. Mümkün olan en yakın zamanda, referans ve örnek çözümler hacimleri maç için zorunludur. Yükleme kabarcıkları önlemek ve hem de çözümleri, mümkün olduğunca eşit olarak hacimleri tutarak tüm yardım için aynı pipet kullanarak, yavaş yavaş çözümler yükleme ve dağıtım, pipet pipet üzerine sıkıca takılması. </li><li> Seal, 2 yeni kırmızı konut fiş contalar merkezi parça ve alüminyum gövdesi arasında konumlandırılmış emin olmak için her yükleme deliğin içine rahatça koyarak hücre. Her bir konut fişi ile kaplayın. El küçük bir düz tornavida ile sıkın. </li></ol> Rotor örnek Hücre Hizalama <ol start="8"><li> Rotor yüklemeden önce, karşı örnek hücre sızıntıları durumda ters pozisyonda örnek hücre daha fazla 0,5 g ÇAKMAK emin olun. Sıfır bakiye ile başlayın. İlk örnek hücre CB karşısında konumda olacak tartın. Dara örnek hücre kilo çıkarmak için denge, daha sonra, CB tartmak. Ekle veya ağırlığı dengelemek eki delikten çıkarmak. Ağırlıkları üst çıkıntı yok emin olun. Dengelemek son ağırlığı 0,5 g içinde ters örnek hücre daha hafif olduğunu sağlamak için Reweigh. </li><li> 4-yer rotor kullanarak, beyaz ok görünür olduğunu, bu nedenle pozisyon 4 dengelemek yeri ve t işaretmerkezkaç kuvveti o yönde. Rotor içindeki tüm yol aşağı emin olun. Dengelemek yerine sıkıca kadar sadece beyaz ok karşısında dengelemek üstünde bulunan ayar vidası, hafifçe sıkın, ama yine de uyumlu hale getirilmesi mümkün. Gövde ile dengeli bir örnek hücre konumundaki rotor merkezi ve vida halka görünür &quot;out&quot; sözcüğü ile karşı karşıya fişler. </li><li> Rotor altında bakıldığında, iç katip işareti (rotor merkezine yakın işareti), bir hizalama aracını kullanarak dengelemeye katip işareti ile aynı hizaya getirin. Yerinde uyumlu dengelemek kilit vidasını sıkın. Aynı şekilde, rotor pozisyon 2 içinde katip işareti ile örnek hücre katip işareti aynı hizaya getirin. </li></ol> Daha fazla bilgi için bkz. <a href="https://sedfitsedphat.nibib.nih.gov/default.aspx">https://sedfitsedphat.nibib.nih.gov/default.aspx</a>

<ol>
	<li>Brown, P. H., Balbo, A., Schuck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+protein-protein+interactions+by+sedimentation+velocity+analytical+ultracentrifugation.">Characterizing protein-protein interactions by sedimentation velocity analytical ultracentrifugation.</a> Curr Protoc Immunol. Chapter 18, Unit 18.15-Unit 18.15 (2008).</li><li>Balbo, A., Brown, P. H., Braswell, E. H., Schuck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+protein-protein+interactions+by+equilibrium+sedimentation.">Measuring protein-protein interactions by equilibrium sedimentation.</a> Curr Protoc Immunol. Chapter 18, Unit 18.8-Unit 18.8 (2007).</li><li>. Home - Workshops [Internet] Available from: <a target="_blank" href="https://sedfitsedphat.nibib.nih.gov/workshop/default.aspx">https://sedfitsedphat.nibib.nih.gov/workshop/default.aspx</a></li></ol>

Rotor uygun hücre montajı, örnek ekleme ve hücre hizalama yüksek kaliteli EAA deneyler için kritik öneme sahiptir. Bu makromoleküler sedimantasyon profilleri elde edilen veriler çok ayrıntılı bir analiz gerektiren deneyler için, özellikle de doğrudur, örneğin, protein etkileşimleri eğitim ve eser agrega miktarda belirlenmesi. Genellikle deneysel kurulum kalite, doğruluk ve hassasiyet için EAA çalışmada elde edilen sonuçlar sınırlayıcı. 3 - ve pratik tasarımı EAA deneyler daha da yürütme hakkında ayrıntılı bilgi için, Referanslar 1 bakın. Atölyeler EAA deneylerin pratik kurulum, teori ve veri analizi kapsayan Ulusal Sağlık Enstitüleri bizim laboratuvarda yılda iki kez yapılıyor [4].

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<td>4-place Ti analytical rotor</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>361964</td>
<td>&nbsp;</td>
<tr>
<td>Torque stand assembly</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>361318</td>
<td>&nbsp;</td>
<tr>
<td>Cell alignment tool</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>362340</td>
<td>&nbsp;</td>
<tr>
<td>Spinkote Lubricant</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>306812</td>
<td>&nbsp;</td>
<tr>
<td>Counterbalance</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>360219</td>
<td>&nbsp;</td>
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<td>Cell Housing kit</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>334602</td>
<td>&nbsp;</td>
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<td>Window gasket</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>327021</td>
<td>&nbsp;</td>
<tr>
<td>Window liner</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>362329</td>
<td>&nbsp;</td>
<tr>
<td>Window, sapphire</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>307177</td>
<td>&nbsp;</td>
<tr>
<td>Window, quartz</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>301730</td>
<td>&nbsp;</td>
<tr>
<td>Window holder</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>305037</td>
<td>&nbsp;</td>
<tr>
<td>12mm double sector Epon centerpiece</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>306493</td>
<td>&nbsp;</td>
<tr>
<td>Cell plug gaskets</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>327022</td>
<td>&nbsp;</td>
<tr>
<td>Cell housing plug	</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>362327</td>
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assembly, loading, and alignment of an analytical ultracentrifuge sample cell

Analitik ultrasantrifüjdeki (EAA), bize yüksek hızda santrifüj sırasında makromoleküler sedimantasyon profilleri kaydetmenize olanak verir bir güçlü biyofiziksel bir araçtır. Düzgün bir şekilde planlandığı ve yürütüldüğü, EAA sedimantasyon hızı veya sedimantasyon denge deneyi bir boyut ve şekil bakımından protein, örnek saflığı, sedimantasyon katsayısı, oligomerization devletler ve protein-protein etkileşimleri hakkında çok şey ortaya çıkarabilir. Bu teknik, ancak sıkı bir düzeyde teknik dikkat gerektirir. Örnek hücreleri iki pencere meclisleri arasında sıkışmış bir sectored merkezi parçası tutun. Onlar / lbs yaklaşık 120-140 tork basıncı ile mühürlenir. Hızlarda dönerken optik algılama sistemleri, her merkezinde sektör kanalıyla aynı radyal yolu orta hat örnek ve referans tampon tarama Referans tampon ve böylece örnek merkezinde sektörleri içine yüklenir ve sonra sızdırmazlık sonra, hücrelerin tam bir titanyum rotor hizalanır 60, 000 rpm ve çok yüksek vakum altında Sadece uygun örnek hücre montaj kritik, örnek hücre parçaları çok pahalı ve deneyler sırasında kaçak ve kırılmaları önlemek için en iyi çalışma koşullarını sağlamak için dikkat edilmesi gerekir. Hatta en ufak bir çatlak veya çizik santrifüj kırılmaya neden olabilir, dikkatli bir şekilde pencereler taşıyınız. Merkezinde ve pencereler arasında iletişim mümkün olduğunca sıkı olmalıdır; tork basınç uygulandıktan sonra, yani hiçbir Newton halkaları görünür olmalıdır. Toz, tüy bırakmayan, çizilmelere ve windows veya merkezinde tüm bu iletişim uzlaşma ve ya yağlar çok kolay bir sektörün başka bir çözüm sızıntı ya da merkezinde hep birlikte dışarı sızdıran yol açabilir. Değerli örnekleri bir deney sırasında çözümleri ile ilgili sızan bilgiler, kayıp, camları kırık ve centerpieces sık sık yol açan hücre basınç bir dengesizlik neden kalmaz. Buna ek olarak, fiş contalar ve konut fişler santrifüj odasında yüksek vakum yükleme deliklerden merkezinde sektörünün çıkardı çözümler önlemek için güvenli olmalıdır. Pencere gömlekleri ve contalar, hareket, kırık pencereleri neden olabilir tatili ve çatlaklar ücretsiz olmalıdır. Bu video örnek hücre montaj prosedürleri göstermek, yardımcı olmak için tork, yükleme ve rotor hizalama bileşeni, mükemmel bir EAA deney sırasında hasar, sızıntı çözüm ve kırılmaları en aza indirir.

Watch this Scientific Journal Video about Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell at JoVE.com

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

Analitik ultrasantrifüjdeki (EAA) örnek hücre örnek ve referans tampon ve deneyler sırasında tutar ve yüksek vakum ve 60.000 rpm rotor hızlarına maruz kalmaktadır. Bu video EAA bir deney bu çok önemli bir bileşeni montaj, yükleme ve uyum için gerekli ayrıntısına kadar titiz bir dikkat gösterecektir.

Kurul, Yükleme ve Analitik ultrasantrifüjdeki Örnek Hücre Hizalama

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

assembly-loading-alignment-an-analytical-ultracentrifuge-sample

Research

JoVE Journal

Biology

22.5K Görüntüleme.  Dynamics of Macromolecular Assembly, Laboratory of Bioengineering and Physical Science. Analitik ultrasantrifüjdeki (EAA) örnek hücre örnek ve referans tampon ve deneyler sırasında tutar ve yüksek vakum ve 60.000 rpm rotor hızlarına maruz kalmaktadır. Bu video EAA bir deney bu çok önemli bir bileşeni montaj, yükleme ve uyum için gerekli ayrıntısına kadar titiz bir dikkat gösterecektir.

Video: Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

Organizatör ve Hayvan Kutbu eksplantlar Xenopus laevis Embriyolar ve Hücre Yapışma Testi Meclisi diseksiyonu

Biyoloji

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

Tendon ex vivo Mekanik Yükleniyor

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Vitro Nükleer Meclisi fraksiyonlara Xenopus Yumurta ayıklar kullanma

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging

This paper aims to instruct the reader in the assembly and operation of an infrared near-field microscope for imaging beyond the diffraction limit.  The apertureless near-field microscope is a light scattering-type instrument that provides infrared spectra at circa 20 nm resolution. A complete list of components and a step-by-step protocol for use is provided.  Common errors in assembly and instrument tuning are discussed.  A representative data set that shows the secondary structure of an amyloid fibril is presented.  

The assembly of a nearfield infrared microscope for imaging protein aggregates is described.

Kurul, Tuning ve Protein Görüntüleme için bir Apertureless Yakın Alan Kızılötesi Mikroskop Kullanımı

This paper aims to instruct the reader in the assembly and operation of an infrared near-field microscope for imaging beyond the diffraction limit.  The ...

Cargo Loading onto Kinesin Powered Molecular Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport.
These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles 1. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available.
Building on the classic inverted motility assay 2, the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo 3. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface.
The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA4, quantum dots 5 or a wide variety of antigens via biotinylated antibodies 4-6.

Molecular shuttles consisting of functionalized microtubules gliding on surface-adhered kinesin motor proteins can serve as a nanoscale transport system. Here, the assembly of a typical shuttle system is described.

Kinesin Powered Moleküler minibüsleri üzerine Kargo Yükleniyor

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

Üç boyutlu Floresans Images Hücresel Morfoloji Değişiklikleri rakamlarla Analitik Aracı

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

Optimize STORM Süper Çözünürlük Mikroskopi Testi Örnekleri

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Tek Virion Atomik Kuvvet Mikroskopisi ve Süper Çözünürlüklü Floresan Görüntüleme için Numune Hazırlama

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

Plazmid vektörler oluşturulması Gibson tertibatı kullanılarak insan uzama faktör-1α bir promotörün regülasyonu altında FLAG-etiketli Proteinlerinin İfade

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

TEM analizi için düşük Hücre sayısı ile örnek hazırlanması için alternatif bir yaklaşım

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...