Yazarlar, Virginia Tech Carilion Araştırma Enstitüsü Direktörü Dr. Michael J. Friedlander'ı araştırma çabalarımızı teşvik etmek için kabul ediyorlar. Bu proje S.M.M ve D.F.K.'ye geliştirme fonları ve kısmen Virginia Tech Kritik Teknoloji ve Uygulamalı Bilim Enstitüsü'nün Nano-Bio girişimi tarafından desteklendi.

1. Benzeşim Yakalama Cihazları Hazırlayın16
<ol><li>Silikon nitrür E-çiplerini 2 dakika boyunca 15 ml aseton ve ardından 2 dakika boyunca 15 ml metanol(Şekil 1A)inkübe ederek temizleyin. Talaşların laminer hava akışı altında kurumasına izin verin.</li>
	<li>Kurutulmuş talaşları 150 °C'de 1,5 saat boyunca ısıtılmış bir karıştırma plakasında (karıştırmadan) kuluçkaya bırakın, ardından kullanmadan önce oda sıcaklığına soğumalarını bekleyin.</li>
	<li>%25 kloroform içermek için küçük cam tüplerde lipit karışımları oluşturmak için Hamilton şırınnalarını kullanın, Kloroformda %55 DLPC (1,2-dilauroyl-fosfokolin) ve toplam 40 μl hacim için kloroformda (1 mg/ml) %20 Ni-NTA lipid (1,2-dioleoyl-iminodiacetic acid-succinyl-nikel tuzu).</li>
	<li>Nemli bir Petri kabındaki bir Parafilm parçasına her 15 μl Milli-Q su damlasına karışımın 1 μl'lik bir aliquotunu uygulayın (Şekil 1B). Örnekleri en az 60 dakika boyunca buz üzerinde kuluçkaya yatır.</li>
</ol>2. Makromolekülleri Yakalayın17
<ol><li>Monolayer örneğinin üzerine 150 nm entegre ara parça içeren bir E-çip yerleştirin ve 1 dakika kuluçkayayatırın (Şekil 1C).</li>
	<li>Çipi numuneden yavaşça kaldırın ve oda sıcaklığında 1 dakika boyunca 3 μl'lik bir Protein A. Incubate ekleyin (Şekil 1D).</li>
	<li>Whatman #1 filtre kağıdını kullanarak fazla damlayı blot ve hemen 3 μl aliquot antikor çözeltisi ekleyin. Oda sıcaklığında 1 dakika kuluçkaya yatır.</li>
	<li>Bir Hamilton şırıngı kullanarak fazla çözeltiyi çıkarın ve hemen 50 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM MgCl 2 ve 10 mM CaCl2içeren tampon çözeltiye 1 μl'lik bir rotavirüs DLPs (0.1 mg /ml) ekleyin. Oda sıcaklığında en az 2 dakika kuluçkaya yatır. DLL'lerin hazırlanması daha önce açıklanmıştır18.
</li>
</ol>3. Mikroakışkan Hazneyi Monte Edin ve Yerinde Numune Tutucuyu Yükleyin
<ol><li>Viral örneği içeren ıslak E-çipi mikroakışkan numune tutucunun ucuna yükleyin. 1 dakika boyunca ikinci bir düz E-çipi kızdırma deşarjı, daha sonra ara parça yongasının üzerine yüklenir(Şekil 2, paneller 1-5).</li>
	<li>Alternatif olarak, biyolojik makromoleküllerin kontrastını artırmak için, ıslak numuneye ikinci E çipi yerleştirmeden önce ıslak örneklere ağır metal lekesi(örneğin% 0,2 uranil format) eklenebilir. Bununla birlikte, numuneyi içeren E-çipin kontrast reaktifini eklemeden önce Milli-Q suyu ile yıkanması gerekecektir.</li>
	<li>3 pirinç vida ile tutucu içinde mekanik olarak tutulan kapalı bir muhafaza oluşturmak için tüm montajı bir araya getirin(Şekil 2, paneller 6-8).</li>
	<li>Montajdan sonra, tutucuyu TEM içine yerleştirmeden önce turbo pompalı kuru pompa istasyonu kullanılarak tutucunun ucu10 -6 Torr'a pompalanır.</li>
</ol>4. İletim Elektron Mikroskobu Kullanarak Sıvıda Görüntüleme
<ol><li>Yerinde numune tutucuyu LaB6 filamenti ile donatılmış ve 120 kV'da çalışan bir İletim Elektron Mikroskobuna (FEI Company) yükleyin.</li>
	<li>TEM filamentini açın ve numuneyi sütunda -15° ila +15° ileri geri eğmek için yalpalama işlevini kullanarak mikroskop aşamasının ösantrik yüksekliğini numuneye göre ayarlayın. Bu prosedür, sıvı haznesinin uygun kalınlığını hesaba katmaya yardımcı olmak için z yönünde aşamayı ayarlar. Bu adım ayrıca görüntüleri kaydederken doğru bir büyütmenin kullanılmasını sağlar.</li>
	<li>Önce mikroakışkan odanın kenarları boyunca ve köşe bölgelerinde görüntüleri kaydedin. Bu alanlar genellikle en ince çözümü içerir. CCD kamera kullanarak düşük doz koşullarında (1-3 elektron/ş2)örneklerin görüntülerini kaydedin. 6.000X - 30.000X nominal büyütme kullanın.</li>
	<li>Akışkan odanın kenarına odaklanarak uygun defokus değerini belirleyin. Görüntüleri 30.000X büyütmede kaydetmek için -1,5 μm değerini kullanın. Kalın çözelti ile karşılaşılırsa veya numune hazırlanmasında kontrast maddesi kullanılmazsa, -2 ila -4 μm aralığında daha yüksek defokus değerleri kullanın.</li>
	<li>Deneyler boyunca çözeltinin mikroakışkan haznede bulunduğundan emin olmak için, kabarcıklar cihaz içindeki sıvıda oluşana kadar elektron ışınını odaklayın.</li>
</ol>

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Improved environmental chambers make it possible to examine wet specimens easily.</a> Science. 186, 407-414 (1974).</li><li>Parsons, D. F., Matricardi, V. R., Moretz, R. C., Turner, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopy+and+diffraction+of+wet+unstained+and+unfixed+biological+objects.">Electron microscopy and diffraction of wet unstained and unfixed biological objects.</a> Adv. Biol. Med. Phys. 15, 161-270 (1974).</li><li>Hui, S. W., Parsons, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+diffraction+of+wet+biological+membranes.">Electron diffraction of wet biological membranes.</a> Science. 184, 77-78 (1974).</li><li>Hui, S. W., Parsons, D. 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Yazar Madeline J. Dukes, Protochips, Inc.'in bir çalışanıdır.

Sunduğumuz çalışmada, rotavirüs DLP'lerini mikroakışkan bir platforma bağlamaya yönelik benzeşim yakalama yaklaşımını çalıştık. Bu, sıvı bir mikroçevrinde makromoleküler komplekslerin yerinde görüntülenmesini sağladı. Yakalama yaklaşımı, diğer mikroakışkan görüntüleme teknikleri açısından önemlidir, çünkü görüntüleri sıvı olarak kaydederken ortaya çıkan büyük difüzif sorunları reddetmek için biyolojik örnekleri görüntüleme penceresine lok...

<a href="https://app.jove.com/t/6605">This corrects</a> the article <a href="https://dx.doi.org/10.3791/50936">10.3791/50936</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/50936">In situ TEM of Biological Assemblies in Liquid</a>. The Representative Results and References sections were&#160;updated.

Erratum: In situ TEM of Biological Assemblies in Liquid

Biyologların ve mühendislerin ortak amacı moleküler makinelerin iç işleyişini anlamaktır. İletim Elektron Mikroskopları (TEM'ler), bu karmaşık ayrıntıları atoma yakın çözünürlük1-2'degörselleştirmek için ideal araçlardır. Bir TEM'in yüksek vakum sistemini sürdürmek için, biyolojik numuneler tipik olarak vitreus buz 3 ,şekerler4,ağır metal tuzları5veya6'nınbir kombinasyonunun ince filmlerine gömülür. Sonuç olarak, gömülü örneklerin görüntüleri dinamik süreçlerin yalnızca sınırlı anlık görüntülerini ortaya çıkabilir.
Çevresel sıvı odalarında nemlendirilmiş biyolojik örneklerin korunması için erken girişimler Parsons ve meslektaşları tarafından diferansiyel pompalama aşamaları kullanılarak gerçekleştirilendir. Lekesiz katalaz kristallerinin elektron kırınım desenleri, hidratlı bir durumda 3 şçözünürlüğe başarıyla kaydedildi7-8. Ek olarak, faza ayrılmış lipit alanları insan eritrositlerinin hidratlı zarlarında incelenebilir9-10. Bununla birlikte, sıvının yayılmasından ve elektron ışınlarına müdahalesinden kaynaklanan hareket, ciddi çözünürlük kaybına neden oldu ve yakın zamana kadar biyolojik örnekler kullanılarak daha fazla deney yapılmaya çalışılamamıştır.
Mikro ölçekli bir çevre odası oluşturmak için yarı iletken mikroçipleri kullanan yeni geliştirilen mikroakışkan numune tutucular tanıtıldı. Bu cihazlar, numuneleri bir TEM sütunu11-12'yeyerleştirilmişken sıvı halde muhafaza edebilir. TEM görüntülemedeki bu teknik atılım, araştırmacıların ilk kez moleküler düzeydeki ilerici olayları görüntülemelerini sağladı13. Deneyler artık EM sütunu14-15içinde gerçekleştirilebildiği için bu yeni modaliteyi"yerinde moleküler mikroskopi" olarak adlandırıyoruz. Bu yöntemin genel amacı, nanometre çözünürlüğünde dinamik davranışlarını gözlemlemek için biyolojik montajları sıvı içinde görüntülemektedir. Geliştirilen tekniğin arkasındaki mantık, gerçek zamanlı gözlemleri kaydetmek ve biyolojik makinelerin yeni özelliklerini çözümde incelemektir. Bu metodoloji, hücresel ve moleküler biyolojide daha geniş amaçlar için TEM kullanımını genişletir12-16.
Mevcut video makalesinde, piyasada bulunan bir mikroakışkan numune tutucuyu birleştirmek ve kullanmak için kapsamlı bir protokol sunuyoruz. Bu özel tutucular, dakika hacimlerini içeren bir sıvı odası oluşturmak için entegre aralayıcılarla üretilen silikon nitrür mikroçiplerini kullanır. İnce, şeffaf pencereler görüntüleme amacıyla mikroçiplere kazınır12. Bir TEM kullanarak sıvıdaki simian rotavirüs çift katmanlı partikülleri (DLL'ler) incelemek için mikroakışkan tutucunun doğru kullanımını gösteriyoruz. DLP'ler gibi biyolojik montajların görüntüleme sırasında büyük mesafelerde hızla yayılmasına izin vermek için, onları mikroakışkan odanın yüzeyine bağlamak için Benzeşim Yakalama yaklaşımını16. Bu moleküler yakalama adımı, biyolojik örneklerin sıvı içinde görüntülenmesi için alternatif tekniklere göre büyük bir avantaja sahiptir, çünkü aşağı akış işleme rutinleri için kullanılacak görüntülerin elde edilmesine izin verir. Mikroakışkan görüntüleme ile birlikte kullanılan bu yakalama adımı prosedürlerimize özgüdür17. TEM veya mikroakışkan görüntüleme odalarını kullanarak yapısal biyoloji uygulamaları kullanan okuyucular, moleküler düzeyde dinamik gözlemler nihai hedef olduğunda benzeşim yakalama tekniklerinin kullanımını düşünebilirler.

<table border="0" cellpadding="0" cellspacing="0" width="832">
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		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:237px;">E-chips, spacer chip</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;">EPB-52TBD</td>
			<td style="width:167px;">400 &#956;m x 50 &#956;m window</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:237px;">E-chip, top chip</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;">EPT-45W</td>
			<td style="width:167px;">400 &#956;m x 50 &#956;m window</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Ni-NTA lipid</td>
			<td style="width:183px;">Avanti Polar Lipids</td>
			<td style="width:245px;">790404P</td>
			<td style="width:167px;">Powder form</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">DLPC (12:0) lipid</td>
			<td style="width:183px;">Avanti Polar Lipids</td>
			<td style="width:245px;">850335P</td>
			<td style="width:167px;">Powder form</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Volumetric flasks&#160;</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">20-812A; 20-812C</td>
			<td style="width:167px;">1 ml; 5 ml&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Hamilton Syringes</td>
			<td style="width:183px;">Hamilton Co.</td>
			<td style="width:245px;">80300, 80400</td>
			<td style="width:167px;">1-10 &#956;l; 1-25 &#956;l</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Whatman #1 filter paper</td>
			<td style="width:183px;">Whatman</td>
			<td style="width:245px;">1001 090</td>
			<td style="width:167px;">100 pieces, 90 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass Petri dishes</td>
			<td style="width:183px;">Corning&#160;</td>
			<td style="width:245px;">70165-101</td>
			<td style="width:167px;">100 mm x 15 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass Pasteur pipettes</td>
			<td style="width:183px;">VWR</td>
			<td style="width:245px;">14673-010</td>
			<td style="width:167px;">14673-010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass culture tubes</td>
			<td style="width:183px;">VWR</td>
			<td style="width:245px;">47729-566</td>
			<td style="width:167px;">6 mm x 50 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Acetone</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">&#160;A11-1</td>
			<td style="width:167px;">1 L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Methanol</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">&#160;A412-1</td>
			<td style="width:167px;">1 L</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">Chloroform&#160;</td>
			<td style="width:183px;">Electron Microcopy Sciences</td>
			<td style="width:245px;">12550</td>
			<td style="width:167px;">100 ml&#160;</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">His-tagged Protein A</td>
			<td style="width:183px;">Abcam, Inc.</td>
			<td style="width:245px;">ab52953</td>
			<td style="width:167px;">10 mg</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">Milli-Q water system</td>
			<td style="width:183px;">EMD Millipore Corp.</td>
			<td style="width:245px;">Z00QSV001</td>
			<td style="width:167px;">Ultrapure Water</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">HEPES</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">BP310-500</td>
			<td style="width:167px;">500 g</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Equipment&#160;</td>
			<td style="width:183px;"></td>
			<td style="width:245px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Poseidon In situ specimen holder</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;"></td>
			<td>&#160;FEI compatible</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">FEI Spirit BioTwin TEM</td>
			<td style="width:183px;">FEI Co.</td>
			<td style="width:245px;"></td>
			<td>120 kV</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Eagle 2k HS CCD camera</td>
			<td style="width:183px;">FEI Co.</td>
			<td style="width:245px;"></td>
			<td>10&#160;&#197;/pixel sampling at 30,000X</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:237px;">Gatan 655 Dry pump station</td>
			<td style="width:183px;">Gatan, Inc.</td>
			<td style="width:245px;"></td>
			<td>Pump holder tip to 10-6&#160;range</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:237px;">PELCO easiGlow, glow discharge unit</td>
			<td style="width:183px;">Ted Pella, Inc.</td>
			<td style="width:245px;"></td>
			<td>&#160;Negative polarity mode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Isotemp heated stir plate</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;"></td>
			<td>Heat to 150 &#186;C for 1.5 hr</td>
		</tr>
	</tbody>
</table>

in situ tem of biological assemblies in liquid

Araştırmacılar, biyolojik varlıkları incelemek ve yeni malzemeleri değerlendirmek için düzenli olarak İletim Elektron Mikroskoplarını (TEM' ler) kullanırlar. Burada, bu aletler için ek bir uygulama açıklıyoruz - viral montajları sıvı bir ortamda görüntülemek. Biyolojik yapıları görselleştirmenin bu heyecan verici ve yeni yöntemi, yakın zamanda geliştirilen mikroakışkan bazlı bir numune tutucuyu kullanır. Video makalemiz, bir TEM içindeki sıvı örnekleri görüntülemek için bir mikroakışkan tutucunun nasıl birleştirılacağını ve kullanılacağını göstermektedir. Özellikle simian rotavirüs çift katmanlı partikülleri (DLP' ler) model sistemimiz olarak kullanıyoruz. Ayrıca, sıvı odasının yüzeyini GÖRÜNTÜLEME penceresine DLP'leri bağlayan benzeşim biyofilmleri ile kaplama adımlarını da açıklıyoruz. Bu, montajları 3D yapı belirlemeye uygun bir şekilde görüntü oluşturmamızı sağlar. Böylece, doğal bir sıvı ortamda subviral parçacıkların ilk bakışını sunuyoruz.

Parlama deşarjı yapılan E-çipler kullanılarak sıvıdaki DLL'lerin temsili görüntüleri (Şekil 3A), benzeşim yakalama cihazlarında zenginleştirilmiş DLL'lere kıyasla, muhtemelen difüzyon nedeniyle belirli bir görüntüleme alanında daha az DLL göstermektedir (Şekil 3B). Görüntüleme odasına uranil format eklenmesi, numunenin kontrastını ve dolayısıyla çözeltideki bireysel DLL'lerin görünürlüğünü arttırır(Şekil 3C, üst panel). Daha iy...

Watch this Scientific Journal Video about In situ TEM of Biological Assemblies in Liquid at JoVE.com

In situ TEM of Biological Assemblies in Liquid

Burada, viral kompleksleri bir iletim elektron mikroskobu kullanarak nanometre çözünürlüğünde sıvı olarak görüntüleme prosedürünü açıklıyoruz.

Yerinde Sıvıda Biyolojik Montajlar TEM

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

in-situ-tem-of-biological-assemblies-in-liquid

Research

JoVE Journal

Bioengineering

In situ TEM of Biological Assemblies in Liquid

9.9K Görüntüleme.  Protochips, Inc.. Burada, viral kompleksleri bir iletim elektron mikroskobu kullanarak nanometre çözünürlüğünde sıvı olarak görüntüleme prosedürünü açıklıyoruz.

Video: In situ TEM of Biological Assemblies in Liquid

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

Hücresel Lipid Ekstraksiyon Hedefli Kararlı İzotop Seyreltme Sıvı Kromatografi-Kütle Spektrometresi Analizi

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Biyomühendislik

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Parametrized Diyastolik Dolum Biçimcilik yoluyla Transmitral Akışının Modellenmesi Kinematik tabanlı Analizi ile Küresel Diyastolik Fonksiyonu Kantitasyonu

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

Biyolojik Sıvı Ortamda Ölçeklenebilir, Metalik Yüksek Aspect Ratio Nanokompositlerin Üretimi

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Parçacık izleme Microrheology tarafından Biyofilmler Mekanik Özelliklerinin Yerinde Haritalama

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Kontrollü İlaç Release için Manyetik Alan-Duyarlı Hibrid Jelatin mikroj Alternatif

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

Biyolojik ortamda oluşturuluyor ve Gözlem Kemilüminesans için Yeni Bir Teknik

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Robot sıvı işleme derleme modüler DNA cihazların otomatik

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

bir<em&gt; İn Vitro</emKemirgen İntervertebral Disk&gt; Organ Kültürü Modeli

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

3D Mekansal Hücre Kültürleri için hücre yapı iskeleleri olarak biyo-uyumlu sıvı kristal Elastomer Köpüklerin sentezi

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Biyolojik Dokuların Uyarılması için Elektrikli ve Manyetik Alan Cihazları

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...