This work was supported by the National Institutes of Health (R01-EB014496). The authors would like to thank Iason Apostolakis for his contribution to the experiments.

وقد وافق هذا البروتوكول من قبل لجنة رعاية واستخدام الحيوان المؤسسي من جامعة كولومبيا. تم تنفيذ جميع الحصول على البيانات ومعالجتها باستخدام بيئة ماتلاب. 1. انشاء التجريبية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ديغا في الجسم الحي الكلاب عينة الكبد السابقين خلال 90 دقيقة. وضع عينة الكبد في خزان مملوء نزع الغاز الفوسفات الحل (الشكل 1). إصلاح العينة الكبد على امتصاص الصوتية مع الإبر في الأطراف من الكبد. </li><li style=";text-align:right;direction:rtl"> إدراج 64 عنصرا، 0.32 ملم الملعب، 2.5 ميغاهيرتز تردد مركز مجموعة مراحل (التصوير) من خلال ثقب دائري يقع في وسط ل93-عنصر صفيف نصف كروية HIFU محول (العلاجي) في مركز تردد 4.5 ميغاهيرتز، 70 مم العمق البؤري و 1.7 مم × 0.4 مم حجم البؤري 11. محاذاة كل من محولات شارك في محوريا وإصلاح محول التصوير في محول العلاجي مع مسامير التكيف. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تغطية محول HIFU مع مركباتolume تسيطر عليها غشاء من مادة البولي يوريثين مليئة المياه المتدفقة نزع الغاز لتبريده. تركيب محول التجمع على 3-D مناور الكمبيوتر التي تسيطر عليها. </li></ol></li><li style=";text-align:right;direction:rtl"> قم بتوصيل محول HIFU إلى مولد وظيفة إرسال 25 هرتز السعة التضمين الموجي شرط مع 500 فولت كحد أقصى السعة. قم بتوصيل محول التصوير لنظام الموجات فوق الصوتية للبرمجة بشكل كامل باستخدام برنامج ماتلاب. ملاحظة: هناك برامج المرتبطة بنظام الموجات فوق الصوتية واستخدام بيئة ماتلاب أن تكون مثبتة على جهاز كمبيوتر متصل إلى النظام. يجب أن توضع A 50 ديسيبل مكبر للصوت RF وشبكة المطابقة بين محول HIFU ومولد وظيفة لتضخيم التوالي القوة وتطابق مقاومة. </li><li style=";text-align:right;direction:rtl"> إنشاء شبكة القطبية، وذلك باستخدام ماتلاب، بدءا من 50 مم من سطح مجموعة و 40 مم العميقة في الاتجاه شعاعي مع خطوة المكانية من 9.625 ميكرون و90 درجة في الاتجاه السمتي مع 128 خطوط والتي الأصل هو FOسلطات الجمارك من موجة متباينة. تحديد مصدر متباينة موجة 10.24 مم (نصف حجم الفتحة) وراء السطح من مجموعة، وتركزت في الاتجاه الجانبي. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إنشاء شبكة الديكارتية، وذلك باستخدام ماتلاب، بدءا من 50 مم من سطح مجموعة و 40 مم العميقة في الاتجاه المحوري مع خطوة المكانية من 9.625 ميكرون و 20 ملم واسعة في الاتجاه الجانبي مع 64 خطوط لموجة الطائرة. تحديد مصدر موجة الطائرة على سطح المصفوفة. لكل شبكة، حساب الوقت من المصدر إلى كل نقطة من الشبكة والعودة إلى كل عنصر من عناصر المصفوفة. </li></ol></li><li style=";text-align:right;direction:rtl"> أدخل &quot;ReconMat_DW&quot; لتباين التصوير موجة أو &quot;ReconMat_PW&quot; للتصوير موجة الطائرة في إطار الأوامر مطلب واضغط على &quot;دخول&quot; لإنشاء مصفوفة إعادة الإعمار المرتبطة خوارزمية تأخير ومبلغ القياسية لكل شبكة. تطبيق خوارزمية تأخير ومحصلتها إلى كل متجه أساس معيار واسترداد elemen غير الأصفارالخبر من الناتج مصفوفة 11. تخصيص عناصر غير الصفرية التي تم الحصول عليها من المصفوفة الناتجة إلى مصفوفة متفرق في الموقع المقابل. حفظ مصفوفة إعادة الإعمار على القرص الصلب لجهاز الكمبيوتر. ملاحظة: المتباينة وأساليب موجة الطائرة استخدام اثنين من مصفوفات إعادة الإعمار متميزة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يلقي مصفوفة إعادة الإعمار إلى مصفوفة GPU. أدخل &quot;SetUpP4_2Flash_4B_streaming_DW&quot; لتباين التصوير موجة أو &quot;SetUpP4_2Flash_4B_streaming_PW&quot; للتصوير موجة الطائرة في إطار الأوامر ماتلاب ثم اضغط على &quot;دخول&quot; لإنشاء ملف الإعداد للحصول على البيانات قناة الموجات فوق الصوتية باستخدام البرنامج النصي المرتبطة مع مجموعة مراحل، وقدمت من قبل الشركة المصنعة من نظام الموجات فوق الصوتية. اسم ملف الإعداد &quot;P4-2Flash_DivergingWave.mat&quot; لتباين التصوير موجة و &quot;P4-2Flash_PlaneWave.mat&quot; للتصوير موجة الطائرة. ملاحظة: حزمة البرمجيات التجارية يجب أن تكون مثبتة على جهاز الكمبيوتر رس يلقي إعادة الإعمار المصفوفة المتناثرة لمصفوفة GPU. </li></ol></li><li style=";text-align:right;direction:rtl"> مزامنة نظام الموجات فوق الصوتية مع مولد وظيفة باستخدام الزناد الخارجي بحيث الحصول على البيانات ارتفاع معدل الإطار الموجات فوق الصوتية للكبد يبدأ في نفس الوقت كما HIFU. </li><li style=";text-align:right;direction:rtl"> فتح ماتلاب. تشغيل البرنامج النصي الإعداد &quot;SetUpP4_2Flash_4B.m&quot; المقدمة من قبل الشركة المصنعة نظام الموجات فوق الصوتية لاستخدام التصوير B-الوضع. اسم ملف الإعداد التي تم إنشاؤها: &quot;P4-2Flash_4B_Bmode.mat&quot;. استخدام &quot;VSX&quot; القيادة وعندما &quot;اسم ملف .mat على العملية:&quot; إن المطالبة، أدخل اسم ملف الإعداد &quot;P4-2Flash_4B_Bmode.mat&quot;. نقل كل محولات الطاقة واستخدام شاشة عرض B-الوضع الذي ظهر على شاشة الكمبيوتر لوضعها في المنطقة المستهدفة من الكبد إلى يجتذ. استهداف المنطقة حوالي 1 سم تحت سطح الكبد لتجنب ارتفاع توهين الموجات فوق الصوتية بسبب الامتصاص. حفظ التقليدية B-وضع صورة للكبد على الكمبيوتر. ملاحظة:نحن هنا يقوم ablations HIFU في 11 مواقع مختلفة في عينتين الكبد عن طريق تحريك محولات مع مناور 3-D لكل الاجتثاث. </li></ol> 2. الموجات فوق الصوتية الحصول على البيانات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> فتح ماتلاب. استخدام &quot;VSX&quot; القيادة وعندما &quot;اسم ملف .mat على العملية:&quot; إن المطالبة، أدخل اسم ملف الإعداد &quot;P4-2Flash_DivergingWave.mat&quot; لتباين التصوير موجة أو &quot;P4-2Flash_PlaneWave.mat&quot; لموجة الطائرة التصوير. بدء HIFU وتطبيقه خلال 2min إلى المنطقة المستهدفة. </li><li style=";text-align:right;direction:rtl"> الحصول على بيانات القناة RF في 1000 لقطة في الثانية خلال 2 دقيقة باستخدام موجات متباينة. بدلا من ذلك، الحصول على بيانات القناة RF في 1000 لقطة في الثانية خلال 2 دقيقة باستخدام موجات الطائرة. </li><li style=";text-align:right;direction:rtl"> نقل البيانات إلى الكمبيوتر المضيف كل 200 لقطة عن طريق كابل صريح PCI. بدلا من ذلك، في الوقت الحقيقي الجري، الحصول على بيانات القناة RF عند 167 لقطة في الثانية خلال 2 دقيقة باستخدام موجات الطائرة وtransfإيه البيانات إلى الكمبيوتر المضيف كل 2 الإطارات. ملاحظة: وسائل التصوير مع مجموعة من 200 لقطة يوفر القرار الزماني عالية ضمن مجموعة ولكن خلق كل الفجوات بين كل مجموعة ومناسبة للمعالجة خارج الخط. طريقة التصوير في 167 إطارا في الثانية لديها من القرار الزمني أقل ولكن لا يخلق أي ثغرات في الوقت الاجتثاث بأكمله و المناسب في الوقت الحقيقي الجري. </li><li style=";text-align:right;direction:rtl"> يلقي مصفوفة بيانات القناة RF إلى واحدة مصفوفة الدقة GPU مع مطلب. ضرب مصفوفة بيانات القناة RF بالمصفوفة إعادة الإعمار للحصول على البيانات RF أعيد بناؤها 11. </li></ol> 3. التشرد التصوير <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إنشاء 6 أجل عشر بتروورث مرشح تمرير منخفض في 4 ميغاهيرتز تردد قطع باستخدام DSP نظام أدوات للمطلب. تطبيق منخفض هذا مرشح تمرير للبيانات RF أعيد بناؤها لتصفية عنصر ميغاهيرتز HIFU 4.5. </li><li style=";text-align:right;direction:rtl"> تقدير انزياح بين إطارات متتالية باستخدام 1-D تطبيع عبر الارتباطمع طول 3.1mm نافذة و 90٪ التداخل. </li><li style=";text-align:right;direction:rtl"> إنشاء 6 أجل عشر بتروورث مرشح تمرير منخفض عند 100 هرتز تردد قطع باستخدام DSP نظام أدوات للمطلب. تطبيق منخفض هذا مرشح تمرير للبيانات النزوح الزمنية باستخدام ماتلاب لاسترداد المكون تردد 50 هرتز، متذبذبة. </li><li style=";text-align:right;direction:rtl"> تحديد المنطقة ذات الاهتمام (ROI) كمنطقة محورية في -6 ديسيبل (1.7 X 0.4 مم في الماء) وتقع 70 ملم بعيدا عن سطح محول. استخراج البيانات التشريد في هذا ROI. تقدير تشريد نسبة الإشارة إلى الضوضاء (SNR د) في منطقة الوصل بعد 2 دقيقة من الاجتثاث بأنها النسبة بين يعني التشرد والانحراف المعياري للنزوح في العائد على الاستثمار. </li><li style=";text-align:right;direction:rtl"> استخراج 50 هرتز إشارة النزوح الزمني في التركيز من البيانات تشريد المصفوفة. تحويل الإشارات النزوح الزمنية في التركيز إلى صوت مسموع باستخدام ماتلاب. </li></ol>

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The authors declare that they have no competing financial interests.

في الوقت الحقيقي رصد الآفات HIFU مهم لضمان السليم والفعال تسليم الآفة. كما الأشكال الآفة، والأنسجة صمود والسعة تلقاء تحت الإثارة النقصان. تطبيق HIFU في المنطقة من نتائج الأنسجة في قوة الإشعاع الصوتية أن يدفع النزوح الأنسجة. التغير النسبي في النزوح هو بديل من التغير النس?...

High Intensity Focused Ultrasound (HIFU) is a technique that generates temperature elevation at the focal region and can be used to ablate cancerous tissue 1. Temperature elevation at the focus causes thermal lesions in the tissue 2. In order to avoid overtreating a region and to reduce treatment duration, it is imperative to reliably monitor the ablation. Magnetic resonance-guided focused ultrasound (MRgFUS) is the main technique used in clinic to guide and monitor HIFU treatment 3. MRI provides high spatial resolution images of the treated region with tissue displacement or thermal dose but has a frame rate of 0.1-1 Hz and is costly. Several ultrasound-based techniques such as B-mode imaging 4, passive acoustic mapping 5, shear wave imaging 6 and acoustic radiation force impulse 7 have been developed to guide and monitor thermal ablation. However, B-mode imaging and passive acoustic mapping do not provide imaging of mechanical properties of the ablated region which is useful to the operator to improve lesion delivery. 
Shear wave imaging and acoustic radiation force impulse can both characterize the elasticity of the tissue by measuring acoustic radiation force-induced displacements 7,8. However, in both methods, the HIFU treatment is typically interrupted to monitor the ablation. Our group has developed a technique called Harmonic Motion Imaging for Focus Ultrasound (HMIFU) which can monitor the HIFU treatment with ultrasound without stopping the ablation9,10. Briefly, a HIFU transducer sends an amplitude-modulated wave to the region to ablate while simultaneously generating an oscillatory motion in the focal region. A co-axially aligned ultrasound transducer is used to image this oscillation. The magnitude of the induced motion is related to the stiffness of the tissue.
To ensure proper lesion delivery, the temporal resolution of real-time monitoring is of key interest in ablation guidance. Recently, our group has shown real-time streaming of displacement at a frame rate up to 15 Hz, imaged with diverging waves in a narrow field of view and using a fast image reconstruction method 11. Several beamforming techniques can be used to image the displacements. A large field of view can be obtained with diverging wave imaging by changing the delay profile but the axial direction is not aligned with the HIFU beam on the lateral regions and the wave is attenuated due to geometric spreading in the lateral direction, which can affect the quality of the displacement estimation. In contrast, the lateral field of view for plane wave is upper bounded by the active aperture but the axial direction is aligned with the HIFU beam at the focus and there is no geometric spreading in the lateral direction. Depending on the type of application, one or the other imaging method can be selected. The objectives of this protocol are to show how plane wave imaging can provide real-time streaming of displacements images using HMIFU during ablation and to compare the quality of the motion estimation between diverging and plane wave imaging.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>P4-2 Phased array</td>
			<td>ATL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>H-178 HIFU transducer</td>
			<td>Sonic Concepts</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-D positioner</td>
			<td>Velmex Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AT33522A function generator</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>V-1 ultrasound system</td>
			<td>Verasonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3100L RF amplifier</td>
			<td>ENI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matching network</td>
			<td>Sonic Concepts</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Degasing system</td>
			<td>Sonic Concepts</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Programming software</td>
			<td>Matlab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Jacket software package</td>
			<td>Accelereyes</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time monitoring of high intensity focused ultrasound (hifu) ablation of in vitro canine livers using harmonic motion imaging for focused ultrasound (hmifu)

التوافقي الحركة التصوير بالموجات فوق الصوتية المركزة ل(HMIFU) هو الاسلوب الذي يمكن أن تؤدي ومراقبة عالية الكثافة (HIFU) الاجتثاث ركزت الموجات فوق الصوتية. يتم إنشاء حركة متذبذبة في محور 93 عنصر و 4.5 ميغاهيرتز تردد مركز HIFU محول عن طريق تطبيق 25 هرتز، التضمين السعة إشارة باستخدام مولد وظيفة. يتم وضع 64 عنصر و 2.5 ميغاهيرتز محول التصوير مع 68kPa ذروة الضغط confocally في وسط محول HIFU للحصول على الترددات الراديوية (RF) بيانات القناة. في هذا البروتوكول، وصفت رصد في الوقت الحقيقي الاجتثاث الحرارية باستخدام HIFU مع القوة الصوتية من 7 W على كبد الكلاب في المختبر. يتم تطبيق العلاج HIFU على النسيج خلال 2 دقيقة وتصوير المنطقة ذاب في الوقت الحقيقي باستخدام التصوير مباعدة أو موجة الطائرة تصل إلى 1000 لقطة / ثانية. يتم ضرب مصفوفة بيانات القناة RF بواسطة مصفوفة متفرق لإعادة الإعمار الصورة. مجال أعيد النظر هو 90 درجة لمدة متباينة واهاء و 20 مم للتصوير موجة الطائرة ويتم أخذ عينات البيانات في 80 ميغاهرتز. يتم تنفيذ إعادة الإعمار على وحدة المعالجة الرسومية (GPU) من أجل الصورة في الوقت الحقيقي بمعدل 4.5 إطار العرض. ويستخدم 1-D تطبيع عبر الارتباط للبيانات RF أعيد بناؤها لتقدير التشريد المحوري في المنطقة المحورية. حجم النزوح الذروة إلى الذروة في العمق البؤري يتناقص خلال الاجتثاث الحرارية التي تدل على تشنج الأنسجة نتيجة لتشكيل الآفة. وكانت نسبة النزوح إشارة إلى الضوضاء (SNR د) في منطقة محورية للموجة مستوى أعلى 1.4 مرة من لتباين موجة تبين أن التصوير موجة الطائرة تظهر لإنتاج النزوح أفضل خرائط الجودة لHMIFU من تباين التصوير الموجة.

في الوقت الحقيقي يتدفقون من HMI النزوح خلال HIFU الاجتثاث يمكن الحصول عليها باستخدام مباعدة وطائرة التصوير موجة الشكل 2 هو القبض على الشاشة فيديو يظهر الوقت الحقيقي العرض من قوة الإشعاع الصوتية النزوح الناجم عن استخدام التصوير موجة الطائرة في في المختبر ا...

Watch this Scientific Journal Video about Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU a

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

رصد في الوقت الحقيقي وارتفاع كثافة الموجات فوق الصوتية المركزة (HIFU) تذرية<em&gt; في المختبر</em&gt; الناب الأكباد عن طريق متناسق الحركة التصوير بالموجات فوق الصوتية المركزة ل(HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

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Bioengineering

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

9.9K Views.  Columbia University. This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Video: Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

الموجات فوق الصوتية على النقيض المستهدفة علاج الاورام الدبقية في الفئران عن طريق تسليم المخدرات جسيمات متناهية الصغر ، إذ تضع وتذرية الاوعية الدموية الدقيقة

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

وDecellularization Recellularization الأكباد من الجامعة

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from regulation of cell migration and proliferation, to secretion and even differentiation. The signals underlying each of these complex processes arise from the molecular interactions of extracellular matrix receptors on the surface of the cell. Integrins are the prototypic receptors and provide a mechanical link between extracellular matrix and the cytoskeleton, as well as initiating some of the adhesion-dependent signaling cascades. However, it is becoming increasingly apparent that additional transmembrane receptors function alongside the integrins to regulate both the integrin itself and signals downstream. The most elegant of these examples is the transmembrane proteoglycan, syndecan-4, which cooperates with &alpha;5&beta;1-integrin during adhesion to fibronectin. In vivo models demonstrate the importance of syndecan-4 signaling, as syndecan-4-knockout mice exhibit healing retardation due to inefficient fibroblast migration1,2. In wild-type animals, migration of fibroblasts toward a wound is triggered by the appearance of fibronectin that leaks from damaged capillaries and is deposited by macrophages in injured tissue. Therefore there is great interest in discovering strategies that enhance fibronectin-dependent signaling and could accelerate repair processes.
The integrin-mediated and syndecan-4-mediated components of fibronectin-dependent signaling can be separated by stimulating cells with recombinant fibronectin fragments. Although integrin engagement is essential for cell adhesion, certain fibronectin-dependent signals are regulated by syndecan-4. Syndecan-4 activates the Rac1 protrusive signal3, causes integrin redistribution1, triggers recruitment of cytoskeletal molecules, such as vinculin, to focal adhesions4, and thereby induces directional migration3. We have looked for alternative strategies for activating such signals and found that low-intensity pulsed ultrasound (LIPUS) can mimic the effects of syndecan-4 engagement5. In this protocol we describe the method by which 30 mW/cm2, 1.5 MHz ultrasound, pulsed at 1 kHz (Fig. 1) can be applied to fibroblasts in culture (Fig. 2) to induce Rac1 activation and focal adhesion formation. Ultrasound stimulation is applied for a maximum of 20 minutes, as this combination of parameters has been found to be most efficacious for acceleration of clinical fracture repair6. The method uses recombinant fibronectin fragments to engage &alpha;5&beta;1-integrin, without engagement of syndecan-4, and requires inhibition of protein synthesis by cycloheximide to block deposition of additional matrix by the fibroblasts., The positive effect of ultrasound on repair mechanisms is well documented7,8, and by understanding the molecular effect of ultrasound in culture we should be able to refine the therapeutic technique to improve clinical outcomes.

This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.

تحريض الإشارات التصاق التي تعتمد على استخدام الموجات فوق الصوتية المنخفضة الحدة

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

النطاق العريض للكشف عن الموجات فوق الصوتية البصرية للتطبيقات التصوير الطبي

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

عالمية باليد ثلاثي الأبعاد بصري سمعي التصوير دقق في الأنسجة العميقة تصوير الأوعية الدموية الإنسان والدراسات ما قبل السريرية وظيفية في الوقت الحقيقي

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

العين تهيج اختبار (EIT) لتحديد مخاطر العين مهيج للمواد الكيميائية باستخدام يعيد الإنسان القرنية مثل الظهارية (RHCE) الأنسجة نموذج

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

رصد في الوقت الحقيقي من بين الخلايا الصفراء حمض حيوية باستخدام حمض الصفراء الاستشعار القائمة على الحنق ترميز وراثيا

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high resolutions in live animal models. This allows for repeated imaging of the same rodent over time. This feature not only reduces the total number of rodents required in an experimental design and thereby reduces the inter-subject variation that can arise, but also allows researchers to assess longitudinal or life-long responses to an intervention. To acquire high quality images that can be processed and analyzed to more accurately quantify outcomes of bone micro-architecture, users of in vivo &#181;CT scanners must properly anesthetize the rat, and position and restrain the hind limb. To do this, it is imperative that the rat be anesthetized to a level of complete relaxation, and that pedal reflexes are lost. These guidelines may be modified for each individual rat, as the rate of isoflurane metabolism can vary depending on strain and body size. Proper technique for in vivo &#181;CT image acquisition enables accurate and consistent measurement of bone micro-architecture within and across studies.

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

This paper instructs users of in vivo micro-computed tomography (&#181;CT) scanners how to anesthetize, correctly position and restrain the hind limb of a rat for minimal movement during high-resolution imaging of the tibia. The result is high quality images that can be processed to accurately quantify bone micro-architecture.

تحديد المواقع المناسبة وضبط النفس من أطرافهم هند الفئران لتصوير مركزة عالية الدقة للعظام باستخدام الهندسة المعمارية الدقيقة في فيفو المحسوبة الدقيقة التصوير المقطعي

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

اليد التنظير السريرية نظام تصوير للوقت الحقيقي تصوير الحيوانات الصغيرة غير الغازية

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

تخطيط الاشعه الايونيه المركزة لحفر نانو-البنيات في ميكرواقطاب

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...