البرتو Guiggiani لتطوير نظام مراقبة الوقت الحقيقي، إيفيلينا Chieregatti وهانكو تسوشيما للمناقشات بصيرة، جياكومو Pruzzo واليساندرو بارودي لتطوير الالكترونيات والبرمجيات المخصصة، وكلوديا Chiabrera ومارينا ناني عن مشورة الخبراء والمساعدة في إعداد ثقافة الخلية.

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يعلن الكتاب أنه ليس لديهم مصالح مالية المتنافسة.

نفيدكم في هذا العمل أسلوب كمي لمقارنة التصاق محوار إلى الركيزة ثقافة، في وقت واحد عن طريق إجراء القياس الطيفي قوة الليزر خلال الناجم عن آفة الخلية. ويرتبط الإفراج قياس التوتر لدرجة التصاق الخلية إلى الركيزة: يجب أن الخلايا تحتوي على عدد أكبر من الالتصاقات البؤرية ال?...

المجهر الضوئي هو واحد من نظام التصوير أقل الغازية المتاحة لمراقبة الخلايا الحية. مع استغلال تأثيرات مثل ضغط الإشعاع (كما في ملاقط بصرية 1)، أو ذات الطاقة العالية تدفق الفوتون (كما هو الحال في الليزر مشرح 2)، تم تمديد هذه التكنولوجيا لنانو التلاعب. نظام التصوير الضوئي وتقدم لمراقبة دقيقة لتصور والتلاعب أهداف الخلوية الفرعية 3. في الوقت نفسه، وذلك بفضل لمعايرة دقيقة من قوة الليزر تسليمها، أدوات بصرية إنجاز إما لينة أو الغازية التلاعب عينة مع استنساخ لم يسبق لها مثيل. عدة مختبرات متكاملة، في نفس الإعداد التجريبية، ملاقط بصرية ومشرح الليزر ليجتذ العضيات 4، لتلتحم مع بعضها خلايا مختلفة 5، أو لتحفيز الخلايا عن طريق مدفوعة بصريا الشحنات 6،7. بينما ملاقط بصرية، بعد معايرة لصلابة البصرية، تسمح للسيطرة القوة المطبقة على الخلية على نطاق piconewton، ويمكن لنظم تشريح الليزر تعدل التلاعب البصرية، والتي تتراوح من غشاء الصور poration إلى الاجتثاث من العضيات واحد أو تشريح هياكل شبه الخلوية. ومع ذلك، يعتمد الليزر معايرة تشريح على التقييم النوعي للكيان من التلاعب البصرية فيما يتعلق الطاقة تسليمها إلى عينة، تقوم أساسا على تحليل الصور التي توضح التغيرات المورفولوجية التي لحقت عينة 8. في الطريقة المعروضة، ونحن لشرح كيفية تنفيذ القياس الطيفي قوة أثناء تشريح محور عصبي الليزر من الخلايا العصبية النامية، على القياس، وعلى نطاق وpiconewton، القوة التي ينتجها التوازن تغير في بنية الهيكل الخلوي من حجرة شبه الخلوية 9. الخلايا العصبية مثقف التمسك الركيزة، واستقطاب خلال التنمية. وتحدث هذه المرحلة الاستقطاب خلال الأيام الخمسة الأولى في المختبر. في المرحلة الثانية من الاستقطاب، واحدة من extrudneurites التي جي يصبح أطول، وأنها سوف تفرق أن تصبح محور عصبي 10. استطالة محور عصبي ردا على قوة سحب في مخروط النمو قد صيغت من قبل نموذج Dennerl في 11. في الآونة الأخيرة، وقد تم تمديد هذا النموذج ليشمل 12 دور التصاق محوار إلى ركائز المصفوفة خارج الخلية. وأظهر هذا النموذج الفيزيائية الحيوية، اقترح بعد الملاحظات التجريبية 13، أن سحب القوات على مخروط النمو، نشر على طول محوار، هي عن طريق التضمين الالتصاقات البؤرية إلى الركيزة. وبالمثل، آفة محور عصبي ينتج الافراج المحلية من التوتر نشر نحو خلايا الجسم. وبالتالي، اقترحنا أن قياس مثل التوتر الذي صدر في مكان على طول محور عصبي بين الآفة وسوما الخلية توفر إمكانية لتقييم نتائج الملطف من الالتصاقات البؤرية تتأثر. نحن معايرة اللازمة طاقة الفوتون-الجريان للمشرح ليزر للتحكم في مدى ل damag محور عصبي ألحقته، من عانى من قطع كامل للآفة جزئية. بعد المعايرة، كررنا آفة جزئية إلى محاور عصبية من الخلايا العصبية عدة التفريق وضعت بروتوكول لقياس الافراج عن التوتر، وبالتالي الحصول على معلمة الكمي لتقدير التصاق من محور عصبي إلى الركيزة 14. في العمل الحالي، ونحن تصف بالتفصيل بروتوكول المتقدمة، وهو ما يمثل الإجراء التجريبي دقيقة لتقييم ومقارنة مع piconewton حساسية الالتصاق محور عصبي إلى الركيزة في ظروف تجريبية مختلفة مثل المعالجة الكيميائية 14، أو أنواع مختلفة من الدعم ثقافة الخلية.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Polymer microspheres, &Oslash; 4 &mu;m, COOH coated</td>
			<td>Bangs laboratories</td>
			<td>PC05N/6700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PolyLink Protein Coupling Kit</td>
			<td>Polyscience</td>
			<td>19539</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>IR laser</td>
			<td>IPG Laser GmbH</td>
			<td>YLM-5-SC-LP</td>
			<td>ytterbium continuous wave (CW) fiber laser operating at 1064 nm, with linear polarization</td>
		</tr>
		<tr>
			<td>Spatial light modulator</td>
			<td>Hamamatsu</td>
			<td>LCOS-SLM 10468-07</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Blue-tweezers software</td>
			<td>Optics group, University of Glasgow</td>
			<td>Free downloadable software</td>
			<td><a href="http://www.physics.gla.ac.uk/Optics/projects/tweezers/slmcontrol/" target="_blank">http://www.physics.gla.ac.uk/Optics/projects/tweezers/slmcontrol/</a></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td>Free downloadable software</td>
			<td><a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/ </a></td>
		</tr>
		<tr>
			<td>QPD</td>
			<td>Thorlabs</td>
			<td>S5980 with C5460SPL 6041 board</td>
			<td>Four quadrant photo-diode to measure x, y trapped probe displacement</td>
		</tr>
		<tr>
			<td>PD</td>
			<td>Teem Photonics</td>
			<td>PDA100A-EC</td>
			<td>Photodiode to measure z trapped probe displacement</td>
		</tr>
		<tr>
			<td>nano-Pulse UV laser</td>
			<td>AA-optoelctronics</td>
			<td>PNV-001525-040</td>
			<td>Pulsed UVA laser, pulse length 400 ps</td>
		</tr>
		<tr>
			<td>Acoustic Optic Modulator</td>
			<td>Olympus</td>
			<td>MQ110-A3-UV, 355 nm fused silica</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Andor</td>
			<td>BX51</td>
			<td>Equipped with a 60X, 0.9 NA, water dipping objective</td>
		</tr>
		<tr>
			<td>CCD</td>
			<td>Warner Instruments</td>
			<td>V887ECSUVB EMCCD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Peltier device</td>
			<td>Physic Instruments</td>
			<td>QE1 resistive heating with TC-344B dual channel heater controller</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope stage: micro+piezo stage</td>
			<td>National Instruments</td>
			<td>Three linear stages M-126.CG1 carrying a separate 3-axis piezoelectric nano-positioning stage P-733.3DD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Daq</td>
			<td>&nbsp;</td>
			<td>NI PCI-6229</td>
			<td>Acquiring the x, y, z position of the trapped probe, and sending feedback loop signals to microscope stage</td>
		</tr>
		<tr>
			<td>Linux Real Time Application Interface (RTAI) machine</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Real time feedback loop system, to control stage position, developed on a dedicated PC desktop</td>
		</tr>
	</tbody>
</table>

measurement of tension release during laser induced axon lesion to evaluate axonal adhesion to the substrate at piconewton and millisecond resolution

ويتأثر تشكيل اتصالات وظيفية في شبكة الخلايا العصبية النامية من خلال اشارات خارجية. هو نمو محوار من الخلايا العصبية النامية عرضة للإشارات الكيميائية والميكانيكية، والآليات التي الحواس ويستجيب لإشارات الميكانيكية غير مفهومة جيدا. سوف توضيح دور القوات في نضوج الخلية تمكين تصميم السقالات التي يمكن أن تعزز التصاق الخلايا واقتران هيكل الخلية إلى الركيزة، وبالتالي تحسين قدرة من أنواع الخلايا العصبية المختلفة لتجديد بعد الاصابة. هنا، نحن تصف طريقة لتطبيق في وقت واحد قياسات التحليل الطيفي خلال قوة الليزر التي يسببها الآفة الخلية. نقيس الافراج عن التوتر في محور عصبي lesioned جزئيا من خلال تتبع التداخل في وقت واحد من اجراء تحقيق المحاصرين بصريا انضمت إلى غشاء من محور عصبي. لدينا بروتوكول تجريبي بالكشف عن الافراج عن التوتر مع piconewton حساسية، والديناميكية لإطلاق سراح التوتر فيالقرار وقت ميلي ثانية واحدة. ولذلك، فإنه يوفر وسيلة عالية الدقة لدراسة كيفية اقتران الميكانيكية بين الخلايا وركائز يمكن أن يكون عن طريق التضمين العلاج الدوائي و / أو عن طريق الخواص الميكانيكية متميزة من الركيزة.

الخلية يولد قوات الجر على الركيزة التي كتبها الالتصاقات البؤرية لها. القوة المتولدة من قبل عناصر هيكل الخلية هي في حالة توازن مع القوة مواجهة الركيزة الثقافة. بعد الليزر التي يسببها آفة من محوار، تتعطل بعض من الكابلات الهيكل الخلوي المتوترة ويتم تحريرها التوتر على م...

Watch this Scientific Journal Video about Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution at JoVE.

Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution

قمنا بقياس الافراج عن التوتر في محور عصبي التي تم lesioned جزئيا مع مشرح ليزر من قبل في وقت واحد القياس الطيفي قوة أجريت على اجراء تحقيق بصريا-المحاصرين انضمت إلى غشاء من محور عصبي. بروتوكول التجريبية المتقدمة بتقييم التصاق محوار إلى الركيزة الثقافة.

قياس الافراج عن التوتر أثناء المستحثة بالليزر اكسون الآفات لتقييم التصاق محور عصبي إلى الركيزة في Piconewton والقرار ملي

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is ...

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Research

JoVE Journal

Bioengineering

10.2K Views.  National Research Council of Italy. قمنا بقياس الافراج عن التوتر في محور عصبي التي تم lesioned جزئيا مع مشرح ليزر من قبل في وقت واحد القياس الطيفي قوة أجريت على اجراء تحقيق بصريا-المحاصرين انضمت إلى غشاء من محور عصبي. بروتوكول التجريبية المتقدمة بتقييم التصاق محوار إلى الركيزة الثقافة.

Video: Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

تصميم مفاعل حيوي لضغط الدوري السابقين فيفو دراسة صمامات القلب الأورطي

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1. The main difficulty of this assay is the perceived cost ($25-100K) and technical expertise required for implementing a laser ablation system2,3. However, solid-state pulse lasers of modest costs (&lt;$10K) can provide robust performance for laser ablation in transparent preparations where target axons are "close" to the tissue surface. Construction and alignment of a system can be accomplished in a day. The optical path provided by light from the focused condenser to the ablation laser provides a convenient alignment guide. An intermediate module with all optics removed can be dedicated to the ablation laser and assures that no optical elements need be moved during a laser ablation session. A dichroic in the intermediate module allows simultaneous imaging and laser ablation. Centering the laser beam to the outgoing beam from the focused microscope condenser lens guides the initial alignment of the system. A variety of lenses are used to condition and expand the laser beam to fill the back aperture of the chosen objective lens. Final alignment and testing is performed with a front surface mirrored glass slide target. Laser power is adjusted to give a minimum size ablation spot (&lt;1um). The ablation spot is centered with fine adjustments of the last kinematically mounted mirror to cross hairs fixed in the imaging window. Laser power for axotomy will be approximately 10X higher than needed for the minimum ablation spot on the target slide (this may vary with the target you use). Worms can be immobilized for laser axotomy and time-lapse imaging by mounting on agarose pads (or in microfluidic chambers4). Agarose pads are easily made with 10% agarose in balanced saline melted in a microwave. A drop of molten agarose is placed on a glass slide and flattened with another glass slide into a pad approximately 200 um thick (a single layer of time tape on adjacent slides is used as a spacer). A "Sharpie" cap is used to cut out a uniformed diameter circular pad of 13mm. Anesthetic (1ul Muscimol 20mM) and Microspheres (Chris Fang-Yen personal communication) (1ul 2.65% Polystyrene 0.1 um in water) are added to the center of the pad followed by 3-5 worms oriented so they are lying on their left sides. A glass coverslip is applied and then Vaseline is used to seal the coverslip and prevent evaporation of the sample.

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse imaging is a sensitive way to assay the effects of mutations in C. elegans on axon regeneration. A high quality, but inexpensive, laser ablation system can be easily added to most microscopes. Time lapse imaging over 15 hours requires careful immobilization of the worm.

بناء منخفض الميزانية Axotomy نظام ليزر لدراسة التجديد في أكسون C. ايليجانس</em

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1.  The main difficulty of this assay ...

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

شفط Micropipette من خلايا الركيزة المرفقة لتقدير تصلب الخليوي

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

باستخدام الممانعة الخلوي الركيزة ويعيش التصوير خلية قياس التغييرات في الوقت الحقيقي في التصاق الخلوية ودي التصاق المستحث بواسطة مصفوفة تعديل

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of exosomes to determine if they carry disease discriminatory biomarkers. For performing exosomal analysis, it is necessary to develop a method for extracting and analyzing exosomes from target biofluids without damaging the internal content.
Electric field-induced release and measurement (EFIRM) is a method for specifically extracting exosomes from biofluids, unloading their cargo, and testing their internal RNA/protein content. Using an anti-human CD63 specific antibody magnetic microparticle, exosomes are first precipitated from biofluids. Following extraction, low-voltage electric cyclic square waves (CSW) are applied to disrupt the vesicular membrane and cause cargo unloading. The content of the exosome is hybridized to DNA primers or antibodies immobilized on an electrode surface for quantification of molecular content.
The EFIRM method is advantageous for extraction of exosomes and unloading cargo for analysis without lysis buffer. This method is capable of performing specific detection of both RNA and protein biomarker targets in the exosome. EFIRM extracts exosomes specifically based on their surface markers as opposed to size-based techniques.
Transmission electron microscopy (TEM) and assay demonstrate the functionality of the method for exosome capture and analysis. The EFIRM method was applied to exosomal analysis of 9 mice injected with human lung cancer H640 cells (a cell line transfected to express the exosome marker human CD63-GFP) in order to test their exosome profile against 11 mice receiving saline controls. Elevated levels of exosomal biomarkers (reference gene GAPDH and protein surface marker human CD63-GFP) were found for the H640 injected mice in both serum and saliva samples. Furthermore, saliva and serum samples were demonstrated to have linearity (R = 0.79). These results are suggestive for the viability of salivary exosome biomarkers for detection of distal diseases.

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM

Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves.

الكشف عن Exosomal العلامات البيولوجية التي يسببها الميدانية الكهربائية الإصدار والقياس (EFIRM)

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

تسليم ينترادوكتال إلى الأرنب الغدة الثديية

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior of these materials. In the past, it has not been possible to satisfactorily measure the softening of thin films under conditions mimicking body environment without substantial effort. This publication presents a new and simple method that allows dynamic mechanical analysis (DMA) of polymers in solutions, such as phosphate buffered saline (PBS), at relevant temperatures. The use of environmental DMA allows measurement of the softening effects of polymers due to plasticization in various media and temperatures, which therefore allows a prediction of the materials behavior under in vivo conditions.

To allow reliable predictions of the softening of polymeric substrates for neural implants in an in vivo environment, it is important to have a reliable in vitro method. Here, the use of dynamic mechanical analysis in phosphate buffered saline at body temperature is presented.

تحليل الميكانيكية الحيوية البيئية للتنبؤ بسلوك تليين يزرع العصبية

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...

Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor

Molecular tension transmitted by integrin-ligand bonds is the fundamental mechanical signal in the integrin pathway that plays significant roles in many cell functions and behaviors. To calibrate and image integrin tension with high force sensitivity and spatial resolution, we developed an integrative tension sensor (ITS), a DNA-based fluorescent tension sensor. The ITS is activated to fluoresce if sustaining a molecular tension, thus converting force to fluorescent signal at the molecular level. The tension threshold for ITS activation is tunable in the range of 10&#8211;60 pN that well covers the dynamic range of integrin tension in cells. On a substrate grafted with an ITS, the integrin tension of adherent cells is visualized by fluorescence and imaged at submicron resolution. The ITS is also compatible with cell structural imaging in both live cells and fixed cells. The ITS has been successfully applied to the study of platelet contraction and cell migration. This paper details the procedure for the synthesis and application of the ITS in the study of integrin-transmitted cellular force.

Integrin tension plays important roles in various cell functions. With an integrative tension sensor, integrin tension is calibrated with picoNewton (pN) sensitivity and imaged at submicron resolution.

التصوير إنتغرين التوتر وقوة الخلوية في القرار Submicron مع جهاز استشعار توتر التكاملية

Molecular tension transmitted by integrin-ligand bonds is the fundamental mechanical signal in the integrin pathway that plays significant roles in many ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

قياس إمكانات العمل البصري أحادي الخلية في خلايا القلب المشتقة من الخلايا الجذعية المتعددة القدرات

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical for regulating many processes ranging from development to immunology. Therefore, developing the tools to study these forces at the molecular scale is critical. Our group developed a suite of molecular tension sensors to quantify and visualize the forces generated by cells and transmitted to specific ligands. The most sensitive class of molecular tension sensors are comprised of nucleic acid stem-loop hairpins. These sensors use fluorophore-quencher pairs to report on the mechanical extension and unfolding of DNA hairpins under force. One challenge with DNA hairpin tension sensors is that they are reversible with rapid hairpin refolding upon termination of the tension and thus transient forces are difficult to record. In this article, we describe the protocols for preparing DNA tension sensors that can be "locked" and prevented from refolding to enable "storing" of mechanical information. This allows for the recording of highly transient piconewton forces, which can be subsequently "erased" by the addition of complementary nucleic acids that remove the lock. This ability to toggle between real-time tension mapping and mechanical information storing reveals weak, short-lived, and less abundant forces, that are commonly employed by T cells as part of their immune functions.

This paper describes a detailed protocol for using DNA-based tension probes to image the receptor forces applied by immune cells. This approach can map receptor forces &gt;4.7pN in real-time and can integrate forces over time.

مجسات توتر الحمض النووي لرسم خريطة لقوى مستقبلات بيكونيوتن العابرة بواسطة الخلايا المناعية

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical ...