This work was supported in part by the National Institutes of Health (P41-EB015903, R21EY023043, K25EB015885), National Science of Foundation (CBET-0853773) and Human Frontier Science Program (Young Investigator Grant).

<ol>
	<li>Brillouin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+de+la+lumiere+et+des+rayonnes+X+par+un+corps+transparent+homogene;+influence+del'agitation+thermique.">Diffusion de la lumiere et des rayonnes X par un corps transparent homogene; influence del'agitation thermique.</a> Ann. Phys. (Paris) . 17, 88-122 (1922).</li><li>Scarcelli, G., Yun, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multistage+VIPA+etalons+for+high-extinction+parallel+Brillouin+spectroscopy.">Multistage VIPA etalons for high-extinction parallel Brillouin spectroscopy.</a> Opt. Exp. 19 (11), 10913-10922 (2011).</li><li>Scarcelli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+Brillouin+microscopy+for+three-dimensional+mechanical+imaging.">Confocal Brillouin microscopy for three-dimensional mechanical imaging.</a> Nat. Phot. 2 (1), 39-43 (2008).</li><li>Nichols, A. J., Evans, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video-rate+Scanning+Confocal+Microscopy+and+Microendoscopy.">Video-rate Scanning Confocal Microscopy and Microendoscopy.</a> J. Vis. Exp. (56), e3252 (2011).</li><li>Steelman, Z., Meng, Z., Traverso, A. J., Yakovlev, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brillouin+spectroscopy+as+a+new+method+of+screening+for+increased+CSF+total+protein+during+bacterial+meningitis.">Brillouin spectroscopy as a new method of screening for increased CSF total protein during bacterial meningitis.</a> J. Biophoton. 8 (5), 1-7 (2014).</li><li>Koski, K. J., Yarger, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brillouin+imaging.">Brillouin imaging.</a> Appl. Phys. Lett. 87 (6), 061903 (2005).</li><li>Faris, G. W., Jusinski, L. E., Hickman, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+stimulated+Brillouin+gain+spectroscopy+in+glasses+and+crystals.">High-resolution stimulated Brillouin gain spectroscopy in glasses and crystals.</a> J. Opt. Soc. Am. B. 10 (4), 587-599 (1993).</li><li>Scarcelli, G., Yun, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Brillouin+optical+microscopy+of+the+human+eye.">In vivo Brillouin optical microscopy of the human eye.</a> Opt. Exp. 20 (8), 9197-9202 (2012).</li><li>Scarcelli, G., Besner, S., Pineda, R., Kalout, P., Yun, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Biomechanical+Mapping+of+Normal+and+Keratoconus+Corneas.">In Vivo Biomechanical Mapping of Normal and Keratoconus Corneas.</a> Jama Ophtalmol. , (2015).</li><li>Scarcelli, G., Kim, P., Yun, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+In+Vivo+Measurement+of+Age-Related+Stiffening+in+the+Crystalline+Lens+by+Brillouin+Optical+Microscopy."> In Vivo Measurement of Age-Related Stiffening in the Crystalline Lens by Brillouin Optical Microscopy.</a> Biophys. J. 101 (6), 1539-1545 (2011).</li><li>Antonacci, G., Foreman, M. R., Paterson, C., T&#246;r&#246;k, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+broadening+in+Brillouin+imaging.">Spectral broadening in Brillouin imaging.</a> Appl. Phys. Lett. 103 (22), 221105 (2013).</li><li>Meng, Z., Traverso, A. J., Yakovlev, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Background+clean-up+in+Brillouin+microspectroscopy+of+scattering+medium.">Background clean-up in Brillouin microspectroscopy of scattering medium.</a> Opt. Exp. 22 (5), 5410-5415 (2014).</li><li>Reiss, S., Burau, G., Stachs, O., Guthoff, R., Stolz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+resolved+Brillouin+spectroscopy+to+determine+the+rheological+properties+of+the+eye+lens.">Spatially resolved Brillouin spectroscopy to determine the rheological properties of the eye lens.</a> Biomed. Opt. Exp. 2 (8), 2144-2159 (2011).</li><li>Scarcelli, G., Kling, S., Quijano, E., Pineda, R., Marcos, S., Yun, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brillouin+microscopy+of+collagen+crosslinking:+noncontact+depth-dependent+analysis+of+corneal+elastic+modulus.">Brillouin microscopy of collagen crosslinking: noncontact depth-dependent analysis of corneal elastic modulus.</a> Invest. Ophthalmol. Vis. Sci. 54 (2), 1418-1425 (2013).</li></ol>

Giuliano Scarcelli and Seok H. Yun hold patents related to Brillouin spectroscopy technology.

وهناك ميزة التصميم الأساسية لهذا التكوين مطياف هي أن المرحلتين يمكن محاذاة بشكل مستقل. عندما انزلق على ETALON VIPA من مسار بصري، والعدسات المتبقية من المرحلة مطياف تشكل 1: نظام التصوير 1، بحيث يتم تصوير نمط الأطياف من كل مرحلة على كاميرا CCD. وبالتالي، فإنه واضح وصريح للذهاب...

نثر Brillouin، وصف لأول مرة من قبل ليون برويون 1 في عام 1922، هو نثر غير مرن الضوء من وسائط الصوتية الحرارية في الحالة الصلبة ومن تقلبات الكثافة الحرارية في سائل أو غاز. التحول الطيفي للضوء متناثرة، وعادة في شبه غيغاهرتز المدى، يوفر المعلومات حول التفاعل بين الضوء الساقط والفونونات الصوتية في العينة. ونتيجة لذلك، يمكن أن توفر معلومات مفيدة عن الخصائص اللزجة من المواد التي تم فحصها. في نسخته عفوية، Brillouin نثر له عموما المقاطع العرضية في ترتيب رامان نثر، مما أدى في إشارة ضعيفة جدا. بالإضافة إلى ذلك، والتحولات تردد Brillouin هي أوامر من حجم أصغر من التحولات رامان. ونتيجة لذلك، منتشرة مطاطيا ضوء (من رايلي أو مي نثر)، الضوء الشارد، والخلفية انعكاسات الخروج من عينة يمكن بكل يسر وسهولة تلقي بظلالها على البصمة الطيفية Brillouin. لذلك السببيحتاج مطياف Brillouin ليس فقط لتحقيق غيغاهرتز الفرعي قرار الطيفي ولكن أيضا على النقيض طيفية عالية أو الانقراض. في الطيف Brillouin التقليدية يتم الوفاء بهذه المتطلبات التي كتبها monochromators-صريف المسح الضوئي، وأساليب الضرب البصرية، والأكثر شعبيا، تداخل متعددة تمريرة مسح فابري بيرو 2. هذه الأساليب تقيس كل بالتتابع المكون الطيفي. هذا النهج يؤدي إلى اكتساب مرات لطيف Brillouin واحد تتراوح بين بضع دقائق إلى عدة ساعات، اعتمادا على الصك وعلى العينة. وVIPA مطياف مرحلتين، تم إنشاؤها باستخدام هذا البروتوكول، لديه القدرة على جمع كافة المكونات الطيفية في أقل من ثانية مع توفير ما يكفي من الانقراض (&gt; 60 ديسيبل) لقمع فعالية إشارات زائفة أخرى 2. دمج etalons VIPA هو العنصر الرئيسي لهذا الطيف. A VIPA هو ETALON متينة مع ثلاثة ج مختلفةoating المناطق: في السطح الأمامي، ضيق الشريط المضادة للانعكاس طلاء يسمح للضوء بالدخول إلى VIPA، في حين أن بقية السطح تحتوي على عاكس للغاية (HR) الطلاء؛ في السطح الخلفي، طلاء عاكسة جزئيا يسمح جزء صغير (~ 5٪) من الضوء لإرسالها. عندما تركز على المدخل الضيق للVIPA يميل قليلا، ويحصل ينعكس شعاع الضوء إلى مكونات فرعية مع فرق الطور ثابت داخل VIPA 2. التداخل بين المكونات الفرعية يحقق المنشود تشتت طيفية عالية. التوفيق بين اثنين VIPAs بالتتابع في تكوين محور عبر يدخل تشتت الطيفي في الاتجاهات المتعامدة 3. تشتت الطيفي في الاتجاهات المتعامدة يفصل مكانيا القمم Brillouin من الحديث المتبادل غير المرغوب فيها، الأمر الذي يجعل من الممكن لالتقاط سوى إشارة Brillouin الشكل 1 يعرض التخطيطي لVIPA مطياف مرحلتين. الأسهم أقل من العناصر البصرية تشير إلى درجةري الحرية التي ينبغي أن تكون موجهة المراحل متعدية. <img alt="الشكل 1" class="jove_content" p="" src="/files/ftp_upload/53468/53468fig1.jpg" /> الشكل 1. الإعداد دور فعال. يسلم الألياف البصرية تناثر Brillouin في مطياف. عدسة أسطوانية C1 (و = 200 مم) وتركز الضوء إلى مدخل VIPA الأول (VIPA1). آخر عدسة اسطوانية C2 (و = 200 مم) خرائط تشتت الزاوي الطيفي إلى الانفصال المكاني في المستوى البؤري من C2. في هذه الطائرة، ويستخدم قناع العمودي لتحديد الجزء المطلوب من الطيف. يتبع تكوين مماثل، مائلا بزاوية 90 درجة. شعاع يمر من خلال عدسة كروية S1 (و = 200 مم) ويتركز في فتحة مدخل VIPA الثاني (VIPA2). عدسة كروية S2 (و = 200 مم) يخلق ثنائي الأبعاد نمط فصل طيفيا في طائرتها التنسيق، حيث يتم وضع قناع آخر أفقي. في هورتم تصوير قناع izontal على الكاميرا EMCCD باستخدام زوج عدسة مصاب بعمى الألوان. <a href="https://www.jove.com/files/ftp_upload/53468/53468fig1large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> طالب الجامعي مع بعض الدورات الدراسية البصريات والخبرة التوفيق الأساسية يجب أن يكون قادرا على بناء واستخدام هذا مرحلتين مطياف. وقد تم مؤخرا أظهرت مطياف لتكون متوافقة مع مجموعة متنوعة من تحقيقات البصرية القياسية 3،4،5 (على سبيل المثال، المجهر متحد البؤر، المنظار، شق مصباح منظار العين). هنا، يتم توصيل مطياف لالمجهر متحد البؤر. يتم محاذاة ضوء الليزر إلى بحث مقلوب المجهر نظام موحد بعد دمج الخائن 90:10 شعاع. ويقترن ضوء ارتداد مبعثر من العينة إلى الألياف وضع واحد، مما يجعل متحد البؤر المجهر.

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high speed sub-ghz spectrometer for brillouin scattering analysis

والهدف من هذا البروتوكول هو بناء مواز عالية الانقراض وعالية الدقة Brillouin البصرية مطياف. Brillouin الطيفي هو وسيلة قياس عدم الاتصال التي يمكن استخدامها للحصول على قراءات مباشرة لخصائص المواد اللزجة. فقد كان أداة مفيدة في توصيف المواد، ورصد الهيكلي والاستشعار البيئي. في الماضي، وقد Brillouin الطيفي عادة المستخدمة المسح etalons فابري بيرو لأداء التحليل الطيفي. هذه العملية تتطلب طاقة عالية الإضاءة وأوقات اكتساب طويلة، مما يجعل هذه التقنية غير مناسبة للتطبيقات الطبية الحيوية. A مطياف رواية أدخلت مؤخرا يتغلب على هذا التحدي من خلال توظيف اثنين VIPAs في تكوين محور عرضي. هذا الابتكار يمكن (غيغاهرتز) التحليل الطيفي الدقة جيجاهيرتز من الباطن مع اكتساب الوقت الفرعي الثاني وقوة الإضاءة في حدود سلامة الأنسجة البيولوجية. تطبيقات جديدة متعددة سهلت هذا التحسن هي مكعبrrently التي يجري استكشافها في الأبحاث البيولوجية والتطبيق السريري.

<html> ويبين الشكل 3 ممثل أطياف Brillouin ونوبات بهم لمواد مختلفة. وVIPAs حد سواء لديها سمك 5 ملم مما يؤدي الى FSR ما يقرب من 20 غيغاهيرتز. كانت المرة التكامل لهذه القياسات 100 مللي ثانية. أخذت 100 القياسات وبلغ متوسط. وقد اتخذ واحدة قياس المعايرة قبل الحصول على الأطي?...

Watch this Scientific Journal Video about High Speed Sub-GHz Spectrometer for Brillouin Scattering Analysis at JoVE.com

High Speed Sub-GHz Spectrometer for Brillouin Scattering Analysis

هنا نقدم بروتوكول لبناء مطياف Brillouin السريع. المتتالية تصوير تقريبا مجموعة مرحلة (VIPA) etalons تحقيق سرعة قياس أكثر من 1000 مرة أسرع من المسح التقليدي الطيف فابري بيرو. يوفر هذا التحسن وسيلة لتحليل Brillouin من الأنسجة والمواد الحيوية عند مستويات منخفضة الطاقة في الجسم الحي.

عالية السرعة غيغاهرتز الفرعي مطياف لتحليل Brillouin نثر

The goal of this protocol is to build a parallel high-extinction and high-resolution optical Brillouin spectrometer. Brillouin spectroscopy is a ...

high-speed-sub-ghz-spectrometer-for-brillouin-scattering-analysis

Research

JoVE Journal

Bioengineering

14.8K Views.  University of Maryland.  هنا نقدم بروتوكول لبناء مطياف Brillouin السريع. المتتالية تصوير تقريبا مجموعة مرحلة (VIPA) etalons تحقيق سرعة قياس أكثر من 1000 مرة أسرع من المسح التقليدي الطيف فابري بيرو. يوفر هذا التحسن وسيلة لتحليل Brillouin من الأنسجة والمواد الحيوية عند مستويات منخفض...

Video: High Speed Sub-GHz Spectrometer for Brillouin Scattering Analysis

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

[ميكروأرس] البوليمر لاكتشاف إنتاجية عالية من المواد الحيوية

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

تقييم توصيل الجينات البوليمرية الجسيمات النانوية من تحليل تتبع جسيمات متناهية الصغر وعالية الإنتاجية قياس التدفق الخلوي

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

عالية الدقة تحليل الزمانية المكانية من مستقبلات حيوية من قبل جزيء واحدة الإسفار الميكروسكوب

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells.

This article provides an in depth guide for the assembly and operation of a structured illumination microscope operating with total internal reflection fluorescence illumination (TIRF-SIM) to image dynamic biological processes with optical super-resolution in multiple colors.

دليل لهيكلة الإضاءة TIRF المجهري بسرعة عالية مع الألوان متعددة

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

إعداد ألياف البروتين خارج الخلية مصفوفة لBrillouin الطيفي

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

وإنتاجية عالية منصة ميكروأري الخليوي لتحليل مترابط من تمايز الخلايا وقوات الجر

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes have the potential to serve as biomarkers for disease diagnosis and to monitor disease progression and treatment response. However, most exosome analysis procedures require exosome isolation and purification steps, which are usually time-consuming and labor-intensive, and thus of limited utility in clinical settings. This report describes a rapid procedure to analyze specific biomarkers on the outer membrane of exosomes without requiring separate isolation and purification steps. In this method, exosomes are captured on the surface of a slide by exosome-specific antibodies and then hybridized with nanoparticle-conjugated antibody probes specific to a disease. After hybridization, the abundance of the target exosome population is determined by analyzing low-magnification dark-field microscope (LMDFM) images of the bound nanoparticles. This approach can be easily adopted for research and clinical use to analyze membrane-associated exosome biomarkers linked to disease.

Clinical translation of exosome-derived biomarkers for diseased and malignant cells is hindered by the lack of rapid and accurate quantification methods. This report describes the use of low-magnification dark-field microscope images to quantify specific exosome subtypes in small volume serum or plasma samples.

باستخدام نانوبلازمون-تعزيز تشتت والتصوير المجهر منخفضه التكبير لقياس الأورام المشتقة من الورم

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes ...

Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used method, but has the drawback that it requires fluorescent labeling, which can interfere with the activity of the molecules. Also, photobleaching and photodamage are concerns. In the case of microtubules, we have found that images of similar quality to TIRF can be obtained using interference reflection microscopy (IRM). This suggests that IRM might be a general technique for visualizing the dynamics of large biomolecules and oligomers in vitro. In this paper, we show how a fluorescence microscope can be modified simply to obtain IRM images. IRM is easier and considerably cheaper to implement than other contrast techniques such as differential interference contrast microcopy or interferometric scattering microscopy. It is also less susceptible to surface defects and solution impurities than darkfield microscopy. Using IRM, together with the image analysis software described in this paper, the field of view and the frame rate is limited only by the camera; with a sCMOS camera and&#160;wide-field&#160;illumination&#160;microtubule length can be measured with precision up to 20 nm with a bandwidth of 10 Hz.&#160;

This protocol is a guide for implementing interference reflection microscopy on a standard fluorescence microscope for label-free, high-contrast, high-speed imaging of microtubules using in vitro surfaces assays.

تنفيذ مجهريانعكاس التداخل للتصوير عالي السرعة وخالٍ من الملصقات من الأنابيب الدقيقة

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used ...

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Because manual analysis of microscopy data is very time consuming, subjective, and prohibits quantitative analyses, automation of bioimage analysis is becoming almost unavoidable. We built an informatics workflow called Substructure Analyzer to fully automate signal analysis in bioimages from fluorescent microscopy. This workflow is developed on the user-friendly open-source platform Icy and is completed by functionalities from ImageJ. It includes the pre-processing of images to improve the signal to noise ratio, the individual segmentation of cells (detection of cell boundaries) and the detection/quantification of cell bodies enriched in specific cell compartments. The main advantage of this workflow is to propose complex bio-imaging functionalities to users without image analysis expertise through a user-friendly interface. Moreover, it is highly modular and adapted to several issues from the characterization of nuclear/cytoplasmic translocation to the comparative analysis of different cell bodies in different cellular sub-structures. The functionality of this workflow is illustrated through the study of the Cajal (coiled) Bodies under oxidative stress (OS) conditions. Data from fluorescence microscopy show that their integrity in human cells is impacted a few hours after the induction of OS. This effect is characterized by a decrease of coilin nucleation into characteristic Cajal Bodies, associated with a nucleoplasmic redistribution of coilin into an increased number of smaller foci. The central role of coilin in the exchange between CB components and the surrounding nucleoplasm suggests that OS induced redistribution of coilin could affect the composition and the functionality of Cajal Bodies.

We present a freely available workflow built for rapid exploration and accurate analysis of cellular bodies in specific cell compartments in fluorescence microscopy images. This user-friendly workflow is designed on the open-source software Icy and also uses ImageJ functionalities. The pipeline is affordable without knowledge in image analysis.

محلل البنية الفرعية: سير عمل سهل الاستخدام للاستكشاف السريع والتحليل الدقيق للأجسام الخلوية في صور المجهر الفلوري

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in ...

Imaging and Quantification of the Area of Fast-Moving Microbubbles Using a High-Speed Camera and Image Analysis

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental technique and image analysis protocol presented here can also be applied for imaging microscopic bubbles in other fields of research; therefore, it has a wide range of applications. We apply this to image cavitation around dental ultrasonic scalers. It is important to image cavitation to characterize it and to understand how it can be exploited for various applications. Cavitation occurring around dental ultrasonic scalers can be used as a novel method of dental plaque removal, which would be more effective and cause less damage than current periodontal therapy techniques. We present a method for imaging the cavitation bubble clouds occurring around dental ultrasonic scaler tips using a high-speed camera and a zoom lens. We also calculate the area of cavitation using machine learning image analysis. Open source software is used for image analysis. The image analysis presented is easy to replicate, does not require programming experience, and can be modified easily to suit the application of the user.

Cavitation microbubbles are imaged using a high-speed camera attached to a zoom lens. The experimental setup is explained, and image analysis is used to calculate the area of the cavitation. Image analysis is done using ImageJ.

التصوير والتدين الكمي لمنطقة الزبلات الدقيقة سريعة الحركة باستخدام كاميرا عالية السرعة وتحليل الصور

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental ...