يعترف المؤلفون بالدكتور مايكل ج. فريدلاندر، مدير معهد فرجينيا للتكنولوجيا كاريليون للأبحاث لتشجيعه مساعينا البحثية. وقد تم دعم هذا المشروع من خلال صناديق التنمية لS.M.M وD.F.K. وجزئيا بمبادرة نانو بيو من معهد التكنولوجيا الحرجة والعلوم التطبيقية في جامعة فرجينيا للتكنولوجيا.

1. إعداد أجهزة التقاط التقارب16
<ol><li>تنظيف رقائق السيليكون نيتريد E عن طريق احتضان لهم في 15 مل من الأسيتون لمدة 2 دقيقة تليها 15 مل من الميثانول لمدة 2 دقيقة (الشكل 1A). السماح للرقائق لتجف تحت تدفق الهواء صفح.</li>
	<li>احتضان رقائق المجففة على لوحة اثارة ساخنة (دون اثارة) لمدة 1.5 ساعة في 150 درجة مئوية، ثم السماح لهم لتبرد لدرجة حرارة الغرفة قبل الاستخدام.</li>
	<li>استخدام المحاقن هاملتون لتكوين خليط الدهون في أنابيب زجاجية صغيرة لاحتواء 25٪ الكلوروفورم، 55٪ DLPC (1،2-dilauroyl-phosphocholine) في الكلوروفورم (1 ملغ / مل) و 20٪ ني-NTA الدهون (1،2-dioleoyl-iminodiacetic حمض-سكرينيل-النيكل الملح) في الكلوروفورم (1 ملغ/مل) لحجم إجمالي قدره 40 ميكرولتر.</li>
	<li>تطبيق 1 ميكرولتر aliquot من الخليط على كل قطرة 15 ميكرولتر من الماء ميلي كيو على قطعة من بارا فيلم في طبق بيتري الرطبة (الشكل 1B). احتضان عينات على الجليد لمدة 60 دقيقة على الأقل.</li>
</ol>2. التقاط الجزيئات الكلية17
<ol><li>ضع رقاقة E مع فاصل متكامل 150 نانومتر على رأس عينة أحادية الطبقة واحتضان لمدة 1 دقيقة(الشكل 1C).</li>
	<li>رفع بلطف رقاقة الخروج من العينة وإضافة aliquot 3 ميكرولتر من له الموسومة البروتين A. حضانة لمدة 1 دقيقة في درجة حرارة الغرفة(الشكل 1D).</li>
	<li>لطخة بعيدا إسقاط الزائدة باستخدام Whatman #1 ورقة تصفية وإضافة على الفور aliquot 3 ميكرولتر من حل الأجسام المضادة. حضانة لمدة 1 دقيقة في درجة حرارة الغرفة.</li>
	<li>إزالة الحل الزائد باستخدام حقنة هاملتون وإضافة على الفور 1 ميكرولتر aliquot من DLPs فيروس الروتا (0.1 ملغ / مل) في محلول عازلة تحتوي على 50 م م HEPES (درجة الحموضة 7.5), 150 mM NaCl, 10 mM MgCl2 و 10 mM CaCl2. حضانة لمدة 2 دقيقة على الأقل في درجة حرارة الغرفة. وقد وصفت إعداد DLPs سابقا18.
</li>
</ol>3. تجميع غرفة Microfluidic وتحميل حامل عينة في الموقع
<ol><li>تحميل الرطب E-رقاقة تحتوي على عينة الفيروسية في غيض من حامل العينة microfluidic. توهج التفريغ الثانية شقة E-رقاقة لمدة دقيقة واحدة ثم تحميلها على رأس رقاقة المسافة(الشكل 2، لوحات 1-5).</li>
	<li>بدلا من ذلك، لزيادة التباين من الجزيئات البيولوجية، يمكن إضافة وصمة عار المعادن الثقيلة(على سبيل المثال 0.2٪ أورانيل formate) إلى عينات الرطب قبل وضع الشريحة E الثانية على العينة الرطب. ومع ذلك، فإن الشريحة E التي تحتوي على العينة تحتاج إلى غسلها بماء ميلي كيو قبل إضافة كاشف التباين.</li>
	<li>ساندويتش التجميع بأكمله معا لتشكيل الضميمة مختومة، التي عقدت في مكان ميكانيكيا داخل حامل من قبل 3 مسامير النحاس(الشكل 2،لوحات 6-8).</li>
	<li>بعد التجميع، يتم ضخ طرف الحامل إلى10-6 تور باستخدام محطة ضخ جافة بضخ توربو قبل وضع الحامل داخل TEM.</li>
</ol>4. التصوير في السائل باستخدام المجهر الإلكتروني انتقال
<ol><li>قم بتحميل حامل العينة في الموقع في مجهر إلكترون ناقل الحركة (FEI Company) المجهز بخيوط LaB6 ويعمل بمعدل 120 كيلو فولت.</li>
	<li>قم بتشغيل خيوط TEM وضبط الارتفاع المتمحور حول مرحلة المجهر فيما يتعلق بالعينة باستخدام وظيفة المتذبذب لإمالة العينة من -15 درجة إلى +15 درجة ذهابا وإيابا في العمود. هذا الإجراء يضبط المرحلة في الاتجاه z للمساعدة في حساب سمك السليم للغرفة السائلة. تضمن هذه الخطوة أيضا استخدام تكبير دقيق عند تسجيل الصور.</li>
	<li>تسجيل الصور على طول حواف وفي مناطق الزاوية من غرفة microfluidic أولا. تحتوي هذه المناطق عادة على الحل أنحف. تسجيل صور العينات في ظل ظروف جرعة منخفضة (1-3 الإلكترونات / Å2)باستخدام كاميرا CCD. استخدام التكبير الاسمي من 6000X - 30000X.</li>
	<li>تحديد قيمة ديفوكوس المناسبة من خلال التركيز على حافة غرفة السوائل. استخدم قيمة -1.5 ميكرومتر لتسجيل الصور عند تكبير 30,000X. إذا واجه حل سميك أو إذا لم يتم استخدام عامل التباين في إعداد العينة، استخدم قيم defocus أعلى في نطاق -2 إلى -4 ميكرومتر.</li>
	<li>لضمان احتواء الحل في غرفة microfluidic في جميع أنحاء التجارب ، ركز شعاع الإلكترون حتى تتشكل الفقاعات في السائل داخل الجهاز.</li>
</ol>

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صاحبة البلاغ، مادلين ج. ديوك، موظفة في شركة Protochips.

في عملنا المقدم ، استخدمنا نهج التقاط التقارب لربط DLPs فيروس الروتا إلى منصة microfluidic. وقد سمح ذلك بالتصوير الموقعي للمجمعات الجزيئية الكبيرة في بيئة دقيقة سائلة. نهج التقاط كبيرة فيما يتعلق تقنيات التصوير microfluidic أخرى لأنه ي توطين العينات البيولوجية إلى نافذة التصوير لنفي القض...

<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

الهدف المشترك لعلماء الأحياء والمهندسين هو فهم الأعمال الداخلية للآلات الجزيئية. المجاهر الإلكترونية انتقال (TEMs) هي أدوات مثالية لتصور هذه التفاصيل المعقدة في القرار شبه الذري1-2. من أجل الحفاظ على نظام فراغ عالية من TEM، وعادة ما تكون جزءا لا يتجزأ من العينات البيولوجية في أفلام رقيقة من الجليد الزجاجي3،والسكريات4، والأملاحالمعدنية الثقيلة5،أو مزيج من بعض من6. ونتيجة لذلك، قد تكشف صور العينات المضمنة عن لقطات محدودة فقط للعمليات الديناميكية.
قام بارسونز وزملاؤه بمحاولات مبكرة للحفاظ على العينات البيولوجية المرطبة في الغرف السائلة البيئية باستخدام مراحل ضخ تفاضلية. تم تسجيل أنماط الحيود الإلكترون من بلورات كاتالاز غير ملطخة بنجاح إلى قرار من 3 Å في حالةرطبة 7-8. بالإضافة إلى ذلك ، يمكن فحص مجالات الدهون المنفصلة مرحليا في الأغشية المائية من كرات الدم الحمراء البشرية9-10. ومع ذلك ، فإن الحركة الناجمة عن نشر السائل وتدخله في شعاع الإلكترون ، أدت إلى فقدان شديد للقرار ولم تتم محاولة إجراء المزيد من التجارب باستخدام العينات البيولوجية حتى وقت قريب.
وقد تم إدخال أصحاب العينات الدقيقة الفلورية المطورة حديثا التي تستخدم رقائق أشباه الموصلات لتشكيل غرفة بيئية صغيرة الحجم. يمكن لهذه الأجهزة الحفاظ على عينات في السائل في حين يتم وضعها في عمود TEM11-12. وقد سمح هذا الاختراق التقني في التصوير TEM الباحثين لعرض، للمرة الأولى، الأحداث التقدمية على المستوى الجزيئي13. نشير إلى هذه الطريقة الجديدة باسم"في الموقع المجهر الجزيئي" كما يمكن الآن إجراء التجارب "داخل" العمود EM14-15. الهدف العام لهذه الطريقة هو تصوير التجمعات البيولوجية في السائل من أجل مراقبة سلوكياتها الديناميكية بدقة نانومتر. والأساس المنطقي وراء هذه التقنية المتقدمة هو تسجيل الملاحظات في الوقت الحقيقي ودراسة الخصائص الجديدة للآلات البيولوجية في الحل. هذه المنهجية توسع استخدام TEMs لأغراض أوسع في البيولوجيا الخلوية والجزيئية12-16.
في مقال الفيديو الحالي ، نقدم بروتوكولا شاملا لتجميع واستخدام حامل عينة microfluidic متاح تجاريا. يستخدم هؤلاء أصحاب المتخصصة رقاقات نيتريد السيليكون المنتجة مع الفواصل المتكاملة لتشكيل غرفة السائل الذي يحيط أحجام دقيقة من الحل. يتم حفر رقيقة وشفافة النوافذ في الرقائق الدقيقة لأغراض التصوير12. نحن نثبت الاستخدام السليم لحامل microfluidic لفحص جزيئات فيروس الروتا سيميان ذات الطبقات المزدوجة (DLPs) في السائل باستخدام TEM. لضمان أن التجمعات البيولوجية ، مثل DLPs ، لا تنتشر بسرعة على مسافات كبيرة أثناء التصوير ، نستخدم نهج التقاط التقارب لربطها بسطح الغرفةالدقيقة 16. هذه الخطوة التقاط الجزيئية له ميزة كبيرة على التقنيات البديلة لتصوير العينات البيولوجية في السائل لأنه يسمح للحصول على الصور التي سيتم استخدامها لروتين معالجة المصب. هذه الخطوة التقاط المستخدمة جنبا إلى جنب مع التصوير microfluidic فريدة من نوعها لإجراءاتنا17. القراء الذين يستخدمون تطبيقات البيولوجيا الهيكلية باستخدام TEM أو غرف التصوير microfluidic قد تنظر في استخدام تقنيات التقاط تقارب عندما الملاحظات الديناميكية على المستوى الجزيئي هي الهدف النهائي.

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		<col />
		<col />
		<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

يستخدم الباحثون بانتظام المجاهر الإلكترونية الإرسال (TEMs) لفحص الكيانات البيولوجية وتقييم المواد الجديدة. هنا، ونحن نصف تطبيق إضافي لهذه الصكوك- عرض التجمعات الفيروسية في بيئة سائلة. هذه الطريقة المثيرة والجديدة لتصور الهياكل البيولوجية تستخدم حامل عينة microfluidic المتقدمة مؤخرا. يوضح مقال الفيديو الخاص بنا كيفية تجميع واستخدام حامل microfluidic لتصوير العينات السائلة داخل TEM. على وجه الخصوص، نستخدم جزيئات ثنائية الطبقات من فيروس الروتا سيميان (DLPs) كنظام نموذج لدينا. كما نقوم بوصف الخطوات اللازمة لتغطية سطح الغرفة السائلة بأغشية بيولوجية تقارب تربط DLPs بنافذة المشاهدة. هذا يسمح لنا لتصوير التجميعات بطريقة مناسبة لتحديد هيكل 3D. وهكذا، نقدم لمحة أولى عن الجسيمات شبه البيئية في بيئة سائلة أصلية.

تظهر الصور التمثيلية ل DLPs في السائل باستخدام الرقائق الإلكترونية التي تم تفريغها توهج(الشكل 3A)أقل DLPs في منطقة عرض معينة، ويفترض أن يكون ذلك بسبب الانتشار، بالمقارنة مع DLPs التي يتم إثراء على أجهزة التقاط التقارب(الشكل 3B). إضافة أورانيل formate في غرفة التصوير يعزز التباين من ...

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

هنا نصف إجراء لتصوير المجمعات الفيروسية في السائل في قرار نانومتر باستخدام المجهر الإلكتروني انتقال.

في الموقع TEM من الجمعيات البيولوجية في السائل

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

10.0K Views.  Protochips, Inc.. هنا نصف إجراء لتصوير المجمعات الفيروسية في السائل في قرار نانومتر باستخدام المجهر الإلكتروني انتقال.

Video: In situ TEM of Biological Assemblies in Liquid

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Multiparametric رسم الخرائط البصرية للقلب الأرنب Langendorff - perfused

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

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).

الخلوية لاستخراج الدهن المستهدفة التخفيف النظائر المستقرة تحليل السائل الطيف اللوني القداس

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

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.

الكمي لالعالمي الانبساطي وظيفة عن طريق تحليل يستند النمذجة-الحركية من Transmitral التدفق عبر الشكلية ملء Parametrized الانبساطي

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.

جيل من تحجيم، لامع عالية الارتفاع Nanocomposites نسبة في البيولوجية السائل المتوسطة

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.

في الموقع خارطة الخواص الميكانيكية لالأغشية الحيوية التي تتبع الجسيمات Microrheology

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.

بالتناوب المغناطيسي Microgels الجيلاتين الهجين-الميدان الاستجابة لرقابة الإصدار المخدرات

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.

تقنية جديدة لتوليد ومراقبة التوهج في إعداد البيولوجي

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.

الآلي الجمعية معالجة السائل الروبوتية أجهزة وحدات الحمض النووي

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.

ل<em&gt; في المختبر</em&gt; نموذج الثقافة الجهاز من الفئران فقرات القرص

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.

توليف حيويا الكريستال السائل المرنة كسوات كما السقالات الخليوي لزراعات الخلايا 3D المكانية

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 ...