نشكر فيليب Denninger للمساعدة في إعداد الفيديو وشودري Bhavna لتوفير خطوط مصنع معربا عن تأكل أجهزة الاستشعار. وأيد هذا العمل من المنح المقدمة من مؤسسة العلوم الوطنية (MCB 1021677)، وزارة الطاقة (DE-FG02-04ER15542) لWBF، والمعاهد الوطنية للصحة، ومعهد هوارد هيوز الطبي لSRQGG كان مدعوما من قبل فترة طويلة EMBO الأجل الزمالة. وأيد MM من قبل مؤسسة الكسندر فون همبولت.

<ol>
	<li>Grossmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RootChip:+An+Integrated+Microfluidic+Chip+for+Plant+Science.">The RootChip: An Integrated Microfluidic Chip for Plant Science.</a> Plant Cell. 23, 4234-4240 (2011).</li><li>Unger, M. A., Chou, H. P., Thorsen, T., Scherer, A., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monolithic+microfabricated+valves+and+pumps+by+multilayer+soft+lithography.">Monolithic microfabricated valves and pumps by multilayer soft lithography.</a> Science. 288, 113-116 (2000).</li><li>Okumoto, S., Jones, A., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Imaging+with+Fluorescent+Biosensors:+Advanced+Tools+for+Spatiotemporal+Analysis+of+Biodynamics+in+Cells.">Quantitative Imaging with Fluorescent Biosensors: Advanced Tools for Spatiotemporal Analysis of Biodynamics in Cells.</a> Annu. Rev. Plant Biol. , (2012).</li><li>Loqu&#233;, D., Lalonde, S., Looger, L. L., von Wir&#233;n, N., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cytosolic+trans-activation+domain+essential+for+ammonium+uptake.">A cytosolic trans-activation domain essential for ammonium uptake.</a> Nature. 446, 195-198 (2007).</li><li>Okumoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+approach+for+monitoring+cellular+metabolites+and+ions+using+genetically+encoded+biosensors.">Imaging approach for monitoring cellular metabolites and ions using genetically encoded biosensors.</a> Curr. Opin. Biotechnol. 21, 45-54 (2010).</li><li>Fehr, M., Frommer, W. B., Lalonde, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+maltose+uptake+in+living+yeast+cells+by+fluorescent+nanosensors.">Visualization of maltose uptake in living yeast cells by fluorescent nanosensors.</a> Proc. Natl. Acad. Sci. USA. 99, 9846-9851 (2002).</li><li>Takanaga, H., Chaudhuri, B., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GLUT1+and+GLUT9+as+major+contributors+to+glucose+influx+in+HepG2+cells+identified+by+a+high+sensitivity+intramolecular+FRET+glucose+sensor.">GLUT1 and GLUT9 as major contributors to glucose influx in HepG2 cells identified by a high sensitivity intramolecular FRET glucose sensor.</a> Biochim. Biophys. Acta. 1778, 1091-1099 (2008).</li><li>Chaudhuri, B., Hörmann, F., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+glucose+flux+impedance+using+FRET+sensors+in+wild-type+Arabidopsis+plants.">Dynamic imaging of glucose flux impedance using FRET sensors in wild-type Arabidopsis plants.</a> J. Exp. Bot. 62, 2411-2417 (2011).</li><li>Meier, M., Lucchetta, E. M., Ismagilov, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+stimulation+of+the+Arabidopsis+thaliana+root+using+multi-laminar+flow+on+a+microfluidic+chip.">Chemical stimulation of the Arabidopsis thaliana root using multi-laminar flow on a microfluidic chip.</a> Lab Chip. 10, 2147-2153 (2010).</li><li>Parashar, A., Pandey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-in-chip:+Microfluidic+system+for+studying+root+growth+and+pathogenic+interactions+in+Arabidopsis.">Plant-in-chip: Microfluidic system for studying root growth and pathogenic interactions in Arabidopsis.</a> Appl. Phys. Lett. 98, 263703-26 (2011).</li><li>Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+and+the+future+of+microfluidics.">The origins and the future of microfluidics.</a> Nature. 442, 368-373 (2006).</li><li>Lee, J. N., Park, C., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solvent+Compatibility+of+Poly(dimethylsiloxane)-Based+Microfluidic+Devices.">Solvent Compatibility of Poly(dimethylsiloxane)-Based Microfluidic Devices.</a> Anal. Chem. 75, 6544-6554 (2003).</li></ol>

الإعلان عن أي تضارب في المصالح.

المزايا الرئيسية لأكثر من وسائل النمو RootChip التقليدية هي إعداد مينيملي للفحص المجهري، والقدرة على عكسية مرارا وتكرارا تغيير البيئة الجذرية، والقدرة على المراقبة المستمرة للأنسجة المختصة انمائيا وصحي من الناحية الفسيولوجية على مدى عدة أيام. سابقا، كانت تزرع الشتلات عموديا على وسائل الإعلام تبلور وتحويلها إلى نظام نضح فورا قبل التجربة، والتي سمح فقط قياس جذور واحدة في وقت واحد 8. وقد استخدمت أدوات ميكروفلويديك لArabidopsis، ولكن على مستوى منخفض التكامل 9 أو بدون مراقبة نضح 10. وRootChip يجمع على مستوى عال من التكامل، مع القدرة على أتمتة التجارب من خلال توجيه تدفق دقيق. ميزة أخرى من هذا المنبر، سمة من جميع أجهزة ميكروفلويديك 11، هو أن هناك حاجة إلى كميات ضئيلة فقط من سائل لتزويد جذر مع الجوز ضروريrients، حتى بالنسبة للتجارب التي تمتد عدة أيام. تم تصميم حاليا RootChip كجهاز تستخدم مرة واحدة، ولكن نظرا لأن تكاليف إنتاج رقائق منخفضة، وكميات صغيرة من الكواشف المستهلكة يجعل من رقاقة جدا لا تزال فعالة من حيث التكلفة. هناك بضع خطوات الحاسمة التي يجب اتخاذها لضمان صحة والشتلات: وحدة التخزين في المخاريط البلاستيكية غير ميكروليتر فقط 3-4، والتي سوف تبدأ لتجف عند تعرضها للهواء. وبالتالي فمن الأهمية بمكان أن يتم نقل المخاريط على الرقاقة بسرعة ويتم الاحتفاظ بها رطوبة عالية حتى الجذور قد وصلت إلى غرف المراقبة، والتي سوف تزويدها كميات كافية من المياه. يجب أن يتم تنفيذ الخطوات 4،2-4،5 بسرعة ودون انقطاع لمنع جفاف الشتلات. خطوات 3،5-3،8 وصف حضانة للرقاقة في وسائل الإعلام السائلة خلالها جذور تنمو في غرف المراقبة. قد يتم تخطي هذه الخطوة من قبل تركيب شريحة في جarrier فورا والبدء نضح مستمر مع متوسط ​​نمو. ومع ذلك، نوصي تمرغ في متوسط ​​نمو بين عشية وضحاها، كما أنها لديها بعض المزايا: 1) أنه يخلق بيئة رطبة وبالتالي فإن الشتلات هم أقل عرضة لتصبح المجفف لأنها تنمو في غرفة المراقبة، 2) وغارقة في رقاقة في السائل، وذلك وسوف التفريغ (الخطوة 6.4) يكون أسرع. من المهم استخدام وسائل الإعلام مع تركيز المذاب منخفضة. قد تكون الحلول أكثر تركيزا ويعجل تسد القنوات، وخاصة إذا تم استخدام رقاقة على مدى عدة أيام. بمجرد توصيل الجهاز إلى خط ضغط الهواء، ويتم التحكم في تدفق المتوسطة من خلال تغيير الضغط الهيدروليكي في الصمامات. لضمان إغلاق السليم للصمامات الميكرو ميكانيكية، من المهم اختيار ضغط الرقابة التي هي نحو ثلاثة أضعاف ضغط التدفق. ينبغي للضغط تدفق لا تزيد عن 15 رطل كما سيتم دفع السوائل من مداخل الجذر. ارتفاع الضغوط أماهذ أيضا أن يسبب التبطين للرقاقة، مما يجعل غير صالحة للاستعمال رقاقة. وجود قيود على RootChip هو أن PDMS يسهل اختراقها ومسعور. في حين أن مادة خاملة عمليا إلى المحاليل المائية، قد امتصاص المركبات العضوية 12. وهذا يمكن أن تتداخل مع التبادل السريع للحلول والمركبات العضوية قد تسرب من المادة حتى عندما تم إيقاف العرض من هذا المركب على مدخل. بسبب المسامية، قد تستخدم المذيبات العضوية يسبب تورم في PDMS 12. نستمر في تحسين وتوسيع RootChip فائدتها، على سبيل المثال مع جذور نباتات المحاصيل. ونحن نعتقد أن من خلال تحسين الوصول إلى جذر لعلاج والمراقبة، وأدوات ميكروفلويديك مثل RootChip سوف تفتح أبعادا جديدة للبحث جذر.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> البنود </td><td> مصدر </td><td> معلومات </td></tr><tr><td> الناقل رقاقة، والبرمجيات وغيرها من المعلومات. </td><td> مؤسسة كارنيجي - DPB </td><td> CAD والتصنيع باستخدام الحاسب الآلي الملفات لتصنيع الناقل، والبرمجيات، وتحكم على مزيد من المعلومات غير متوفرة للتنزيل من موقع <a href="http://dpb.carnegiescience.edu/technology/rootchip" target="_blank">http://dpb.carnegiescience.edu/technology/rootchip</a> يمكن أيضا لشركات الاتصالات أن يؤمر من هذا الموقع. </td></tr><tr><td> RootChip </td><td> ستانفورد مسبك </td><td> قناع التصاميم والبروتوكولات تلفيق متاحة عند الطلب. ويمكن طلب الجاهزة للاستخدام من RootChips <a href="http://www.stanford.edu/group/foundry/" target="_blank">http://www.stanford.edu/group/foundry/</a> </td></tr><tr><td> رقاقة تحكم </td><td> المنزل مدمجة</td><td> وقد وضعت في الأصل وحدة تحكم صمام النظام الآلي من قبل رافائيل غوميز سيوبيرج، مختبر لورانس بيركلي الوطني. ويمكن الاطلاع على تعليمات مفصلة كيفية بناء الخاصة بك وحدة تحكم صمام دفعتها في <a href="https://sites.google.com/a/lbl.gov/microfluidics-lab/valve-controllers" target="_blank">https://sites.google.com/a/lbl.gov/microfluidics-lab/valve-controllers</a> </td></tr></tbody></table>

time-lapse fluorescence imaging of arabidopsis root growth with rapid manipulation of the root environment using the rootchip

وظائف الجذر كما المرساة المادية للمصنع، وهي الهيئة المسؤولة عن امتصاص المواد الغذائية والمياه المعدنية مثل عناصر النيتروجين والفوسفور والكبريت والتتبع أن النباتات الحصول من التربة. إذا كنا نريد لوضع نهج مستدامة لإنتاج عالية المحصول، ونحن بحاجة إلى فهم أفضل لكيفية تطور الجذر، يتناول مجموعة واسعة من المواد الغذائية، ويتفاعل مع الكائنات التكافلية والمسببة للأمراض. لتحقيق هذه الأهداف، نحن بحاجة إلى أن تكون قادرة على استكشاف جذور بالتفصيل المجهرية على مدى فترات زمنية تتراوح ما بين دقائق إلى أيام. قمنا بتطوير RootChip، وهو polydimethylsiloxane (PDMS) - جهاز ميكروفلويديك القائم، والذي يسمح لنا أن تنمو والجذور صورة من الشتلات Arabidopsis مع تجنب أي الاجهاد البدني إلى الجذور خلال التحضير لتصوير 1 (الشكل 1). الجهاز يحتوي على هيكل قناة مشقوقة يضم صمامات الميكرو ميكانيكية لتوجيه تدفق السوائلمن مداخل حل كل دائرة من الدوائر المراقبة 8 2. هذا النظام نضح يسمح تسيطر على المكروية الجذرية وتعديل مع الدقة والسرعة. حجم الدوائر على نحو 400 NL، مما يتطلب سوى كميات ضئيلة من محلول الاختبار. هنا نقدم بروتوكول مفصل لدراسة علم الأحياء الجذر على RootChip باستخدام التصوير النهج القائمة مع القرار في الوقت الحقيقي. ويمكن تحليل الجذور على مدى عدة أيام باستخدام المجهر مرور الوقت. ويمكن perfused الجذور مع حلول المغذيات أو مثبطات، ويمكن تحليل ما يصل الى ثمانية الشتلات في نفس الوقت. هذا النظام لديه القدرة لمجموعة واسعة من التطبيقات، بما في ذلك تحليل نمو الجذور في وجود أو عدم وجود المواد الكيميائية، ومضان التحليل القائم على التعبير الجيني، وتحليل أجهزة الاستشعار، على سبيل المثال الحنق nanosensors 3.

Watch this Scientific Journal Video about Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip at JoVE.com

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

تنص هذه المادة على بروتوكول للزراعة شتلات Arabidopsis في RootChip، منصة التصوير ميكروفلويديك الذي يجمع بين التحكم الآلي من شروط النمو مع مراقبة جذر المجهرية والحنق القائم على قياس مستويات الأيض داخل الخلايا.

الوقت الفاصل بين التصوير الإسفار من نمو الجذور Arabidopsis مع التلاعب السريع للبيئة الجذر باستخدام RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

time-lapse-fluorescence-imaging-arabidopsis-root-growth-with-rapid

Research

JoVE Journal

Bioengineering

19.5K Views.  Carnegie Institution for Science. تنص هذه المادة على بروتوكول للزراعة شتلات Arabidopsis في RootChip، منصة التصوير ميكروفلويديك الذي يجمع بين التحكم الآلي من شروط النمو مع مراقبة جذر المجهرية والحنق القائم على قياس مستويات الأيض داخل الخلايا.

Video: Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but important bioinstruments capable of testing the force dependence of structural features in proteins. Scale has been a limiting parameter on how accurately researchers can peer into the nanomechanical world of molecules, such as nucleic acids, enzymes, and motor proteins that perform life-sustaining work. Atomic force microscopy (AFM) is well tuned to determine native structures of fibrous proteins with a distance resolution on par with electron microscopy. However, in AFM force studies, the forces are typically much higher than a single molecule might experience 1, 2. Optical traps (OT) are very good at determining the relative distance between the trapped beads and they can impart very small forces 3. However, they do not yield accurate absolute lengths of the molecules under study. Molecular simulations provide supportive information to such experiments, but are limited in the ability to handle the same large molecular sizes, long time frames, and convince some researchers in the absence of other supporting evidence2, 4.
The gravitational force spectrometer (GFS) fills a critical niche in the arsenal of an investigator by providing a unique combination of abilities. This instrument is capable of generating forces typically with 98% or better accuracy from the femtonewton range to the nanonewton range. The distance measurements currently are capable of resolving the absolute molecular length down to five nanometers, and relative bead pair separation distances with a precision similar to an optical trap. Also, the GFS can determine stretching or uncoiling where the force is near equilibrium, or provide a graded force to juxtapose against any measured structural changes. It is even possible to determine how many amino acid residues are involved in uncoiling events under physiological force loads 2. Unlike in other methods where there is extensive force calibration that must precede any assay, the GFS requires no such force calibration 5. By complementing the strengths of other methods, the GFS will bridge gaps in understanding the nanomechanics of vital proteins and other macromolecules.

This is a step-by step guide showing the purpose, operation, and representative results from the novel gravitational force spectrometer.

مما يدل على استخدامات مطياف الرواية قوة الجاذبية على التمدد وقياس البروتينات الليفية

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but ...

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

الذهب Nanostar التجميعي مع أسلوب بذور فضة النمو وساطة

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

إنشاء تدرجات لاصق وقابل للذوبان للهجرة الخليوي مع التصوير مضان المجهري

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Sample Drift Correction Following 4D Confocal Time-lapse Imaging

The generation of four-dimensional (4D) confocal datasets; consisting of 3D image sequences over time; provides an excellent methodology to capture cellular behaviors involved in developmental processes.&#160; The ability to track and follow cell movements is limited by sample movements that occur due to drift of the sample or, in some cases, growth during image acquisition. Tracking cells in datasets affected by drift and/or growth will incorporate these movements into any analysis of cell position. This may result in the apparent movement of static structures within the sample. Therefore prior to cell tracking, any sample drift should be corrected. Using the open source Fiji distribution 1 &#160;of ImageJ 2,3 and the incorporated LOCI tools 4, we developed the Correct 3D drift plug-in to remove erroneous sample movement in confocal datasets. This protocol effectively compensates for sample translation or alterations in focal position by utilizing phase correlation to register each time-point of a four-dimensional confocal datasets while maintaining the ability to visualize and measure cell movements over extended time-lapse experiments.

Time-lapse microscopy allows the visualization of developmental processes. Growth or drift of samples during image acquisition reduces the ability to accurately follow and measure cell movements during development. We describe the use of open source image processing software to correct for three dimensional sample drift over time.

عينة تصحيح الانجراف بعد 4D متحد البؤر التصوير الوقت الفاصل

The generation of four-dimensional (4D) confocal datasets; consisting of 3D image sequences over time; provides an excellent methodology to capture ...

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

التصوير مورفولوجيا الجذر الشعر شتلات نبات في منصة موائع جزيئية طبقتين

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

إجراء وسائط تصوير متعددة مع واحد Fluorescence المجهر

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

القياس المستمر للضوضاء البيولوجية في الإشريكية القولونية باستخدام المجهر الفاصل الزمني

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, biomimetic surfaces mimicking the microstructure of leaf surface, were used to study the effects of leaf microstructure on leaf-environment interactions. However, no such tool exists for roots. We developed a tool allowing the synthetic mimicry of the root surface microstructure into an artificial surface. We relied on the soft lithography method, known for leaf surface microstructure replication, using a two-step process. The first step is the more challenging one as it involves the biological tissue. Here, we used a different polymer and curing strategy, relying on the strong, rigid, polyurethane, cured by UV for the root molding. This allowed us to achieve a reliable negative image of the root surface microstructure including the delicate, challenging features such as root hairs. We then used this negative image as a template to achieve the root surface microstructure replication using both the well-established polydimethyl siloxane (PDMS) as well as a cellulose derivative, ethyl cellulose, which represents a closer mimic of the root and which can also be degraded by cellulase enzymes secreted by microorganisms. This newly formed platform can be used to study the microstructural effects of the surface in root-microorganism interactions in a similar manner to what has previously been shown in leaves. Additionally, the system enables us to track the microorganism&#8217;s locations, relative to surface features, and in the future its activity, in the form of cellulase secretion.

Biomimetics has been previously used as a tool to study leaf-microorganism interactions. However, no such tool exists for roots. Here, we develop a protocol to form synthetic surfaces mimicking root surface microstructure for the study of root-environment interactions.

محاكاة حيوية النسخ المتماثل من الجذر السطح المجهري باستخدام تغيير من الطباعة الحجرية لينة

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

تصوير تدفق الدم مع دوبلر فائق السرعة

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

شريحة موائع دقيقة بسيطة للنمو على المدى الطويل وتصوير Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...