نشكر مركز التنمية التصوير البيولوجي (BIDC) جامعة كاليفورنيا في سان فرانسيسكو لاستخدام Imaris، XT Imaris ومطلب. نشكر V. Kharazia للمساعدة التقنية وAT هنري، LK Floren، L. Daitch لمساهماتها في التحرير من المخطوطة. وأيد هذا العمل من خلال تمويل من الدولة للأبحاث الطبية في كاليفورنيا الإسراف في تناول الكحول والمخدرات من خلال UCSF لSEB، والمعاهد الوطنية للصحة: ​​1R21DA029966 NIH-01 وجائزة المسار السريع لفحص جمع MLSMR لUCSF SEB مدرسة، الصيدلة ( مكتب العميد والصيدلة الإكلينيكية) وكلية الطب (علم الأدوية السريري والعلاج التجريبي) لCLHK.

1. تحليل ثلاثي الأبعاد من المظهرية وحيدة الخلية التغييرات المظهرية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وتقاس الكلى الجنينية البشرية (HEK293) مع خلايا هيماغلوتينين (HA)-الموسومة كورتيكوتروبين الإفراج عن عامل مستقبلات-2 (CRF-R2)، وهو بروتين G-يقترن مستقبلات (GPCR) كما هو موضح سابقا 4، 5. </li><li style=";text-align:right;direction:rtl"> تركت دون علاج الخلايا (أي علاج، NT)، مع حفز يجند الذاتية CRF-R2، كورتيكوتروبين عامل الإفراج، CRF (1 ميكرومتر، 30 دقيقة)، أو سابقة التجهيز مع خصم CRF-R2 انتقائية، ومكافحة sauvagine 30 (AS -30، 1 ميكرومتر، 30 دقيقة) قبل العلاج ناهض. </li><li style=";text-align:right;direction:rtl"> تم إصلاحها ثم الخلايا، وتعامل مع permeabilized HA للسامية. كان CRF-R2 تصور باستخدام اليكسا 594 نانومتر المتقارن المضادة للماوس (مفتش 1) الأجسام المضادة. وقد استخدم دابي لتصور المرحلة التفتلي نوى. </li><li style=";text-align:right;direction:rtl"> للحد من الذاتية مجرب، لم تكن معروفة في الظروف التجريبية حتى بعد تم الحصول عليها من الصور وتحليلها. </li><li style=";text-align:right;direction:rtl"> حصلنا على صورةS من الخلايا HEK293 الثابتة باستخدام خطة لامزيغ-63x/1.4 الهدف DIC النفط وزايس LSM المجهر متحد البؤر META 510 متصلة مترابطة متكاملة نظام ليزر ثنائي الفوتون تتكون من الليزر فيردي-V5 و900-F ميرا نظام ليزر. </li><li style=";text-align:right;direction:rtl"> أثناء عملية الحصول على البيانات، ومجزأة الخلايا كل من باجتزاء متعددة الأطياف، NM 488، و 790 نانومتر (نانومتر ~ تحويلة 350 2ph.) وZ-تقسيم (0.5 ميكرومتر الزيادات) لتشمل البيانات من الغشاء النووي لمستقبلات للخارج الخلية الخارجي الأطراف. </li><li style=";text-align:right;direction:rtl"> تمت معالجة البيانات أولا باستخدام مضان Imaris، والذي يسمح التصور وتجزئة بيانات المجهر 3D، وتم إنشاء نموذج 3D تتألف من voxels مكعب للتحليل المظهرية. ثم، تم استخدام وحدة لImaris XT Imaris التفاعل مع الكمبيوتر MATLAB لغة البرنامج لتحديد إحداثيات المكان ملحقات GPCR. على أن تأخذ في الاعتبار التباين الخلوية، وحصلنا على تحليل الصور مضان تاكEN من 22 الخلايا: يوجد علاج (NT) (ن = 7)، ناهض (CRF) العلاج (ن = 8) وقبل العلاج مع خصم (AS-30) قبل العلاج ناهض (ن = 7) (الشكل 2) . <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يجب المنطقة الاهتمام (ROI) تشمل خلية واحدة غير موجود في المرحلة التفتلي بالموقع وليس على مقربة من الخلايا الأخرى. بهذه الطريقة، سوف تشمل تحليل الخلايا لا مع نواة واحدة فقط وملحقات مستقبلات مضطرب من جراء قربها من الخلايا الأخرى. </li><li style=";text-align:right;direction:rtl"> أعيد بناؤها أولا هيكل 3D من البيانات الخلوية متعددة الأطياف باستخدام مضان Imaris (v.7.1.1). </li><li style=";text-align:right;direction:rtl"> بعد الخوارزمية التي صممها Imaris، واستخدم لأول مرة لتمثيل تقديم السطحية الغشاء النووي. سوف Imaris تحديد إذا كان هناك أكثر من واحد في النواة ROI. </li><li style=";text-align:right;direction:rtl"> ثم تم استخدام خوارزمية البقع-إنشاء ملحق لتحديد موقع CRF-R2. واستخدم البقع لأنه كشف يعوض عن الضوضاء الخلفية وكثافة غير النظامية للشبكة معقدة غير متبلور على شكل الخلايا. </li><li style=";text-align:right;direction:rtl"> لتحقيق أقصى قدر من إدراج كل وحدة من كشف مضان من CRF-R2، أنشئت قطر للبقع &#39;إلى 0.2 ميكرون، وهو أصغر وحدة في الصورة لاستقراء المعلومات متميزة في شكل كثافة يقاس باستخدام فلتر التمويه. تأسست تصفية بقعة في إنشاء المواقع عملية آلية. البرنامج، ومع ذلك، يعطي المستخدم المرونة اللازمة لاستخدام الفلاتر لتحديد المعلمات. </li><li style=";text-align:right;direction:rtl"> لتجنب اقتطاع البيانات، تم تحويل مجموعة من البيانات 8 بت (غير موقعة) نقطة ثابتة، إلى 32 بت عشرية. </li><li style=";text-align:right;direction:rtl"> وتبادل البيانات فوكسل شدة على الفور باستخدام إحداثيات البيانات Imaris XT حدة تفاعل مع MATLAB والمكان المحدد المكاني للكل بقعة تم تحديد عن طريق إجراء التحول المسافة باستخدام الغشاء النووي كنقطة مرجعية (الشكل 3). </li></ol></li><li style=";text-align:right;direction:rtl"> يمكن كميا البيانات الناتجة وعرضها في شكل رسوم بيانية لياليtatistical التحليل. وأجريت مقارنات بين المجموعات باستخدام اتجاهين ANOVA والبعدي Bonferroni. يتم عرض البيانات كما يعني ± SD. وتعتبر فروق ذات دلالة إحصائية في * 0،05 &lt;P. وأدلى حسابات مع بريزم GraphPad 5.02 (الشكل 4). </li></ol> 2. ممثل النتائج للتدليل على قوة نهجنا، ونحن كميا التغيرات الخلوية التي تنتج عن التفاعل بين مستقبلات البروتين يقترن G (GPCRs) والموجهة القشرية عامل الإفراج عن مستقبلات-2 (CRF-R2) مع يجند لها CRF الذاتية في الخلايا HEK293 بالنقل. نعرض التي تقع CRF-R2 المستقبلات في الغشاء البلازمي والمشروع من مناطق محدودة من غشاء الخلايا (الشكل 2A والفيلم 1). باستخدام التحليل التقليدي 2D، فمن الممكن الكشف عن هذه المجموعة الفرعية من مستقبلات CRF-R2 خارج الخلية إلا إذا حللنا مستقبلات الالتصاق بورجات على زجاج يغطي. وبالتالي نفقد أي معلومات أخرى مستمدة من البيانات Z-مكدسة متعددة الأطياف (الشكل 5). عندما يتم التعامل مع الخلايا CRF، وتقلص إلى حد كبير المستقبلات خارج الخلية، ويتضح ذلك من انخفاض في المسافة من البقع من غشاء البلازما. وإعادة توزيعها أيضا أنها من مواقع محدودة في المقام الأول إلى عدد من المواقع المتميزة (2B الشكل والفيلم 2). يتم منع تأثير على توزيع CRF الغشاء مستقبلات من المعالجة المسبقة مع خصم CRF-R2 محددة، antisavagine 30 (AS-30) ونجد أن ملحقات CRF-R2 لا تتغير (الشكل 2C والفيلم 3). توزيع البعيدة من البقع، وتآمر في فترات 5 ميكرومتر لون الطيف مشفرة، ويستخدم لتصور المسافة بين voxels من الغشاء النووي. ويوجد علاج مضاد pretreatmالأنف والحنجرة (AS-30، 1 ميكرومتر، 30 دقيقة) قبل بدء العلاج ناهض (CRF، 1 ميكرومتر، 30 دقيقة) لا تظهر أي مهمة (م) الفرق في الانكماش GPCR. علاج الخلايا مع ناهض (CRF، 1 ميكرومتر، 30 دقيقة) يقلل تدريجيا من عدد CRF-R2 المحتوية على voxels بالمقارنة مع أي علاج، 0-5 ميكرومتر (م)، 6-15 ميكرون (** p &lt; 0.01) و&gt; 15 ميكرون (*** p &lt;0.005)، أو مقارنة AS-30 معاملة، 0-10 ميكرون (NS)، 11-15 ميكرومتر (** p &lt;0.01) و&gt; 15 ميكرون (*** p &lt;0.005) (الشكل 4). <img alt="الشكل 1" src="/files/ftp_upload/4233/4233fig1.jpg" /> الشكل 1. الرسم البياني من التقنيات المتاحة حاليا والحد من أجل تحليل الصور مضان. <a href="http://www.jove.com/files/ftp_upload/4233/4233fig1large.jpg" target="_blank">اضغط هنا لمشاهدتها بشكل اكبر شخصية</a> . <img alt="الشكل 2" src="/files/ftp_upload/4233/4233fig2.jpg" /> <strong&gt; الشكل 2. 3D الصور المتعددة الأطياف مضان من الخلايا المصابة بالعدوى بالنقل مع HEK293 HA-CRF-R2 قبل وبعد العلاج ناهض وخصم. الصور المدمجة تبين HA-CRF-R2-بحثها استخدام الألغام المضادة للHA وتصور باستخدام الأجسام المضادة مترافق اليكسا 488 الثانوية المضادة للماوس (مفتش 1)، وكان يستخدم لتصور دابي نوى. تم الحصول على الصور مبائر المجهر مع الليزر (CLS) المسح. مقياس بار 5 ميكرون. <img alt="الشكل 3" src="/files/ftp_upload/4233/4233fig3.jpg" /> الشكل 3. إعادة بناء نموذج 3D من الخلايا المصابة بالعدوى بالنقل مع HEK293 HA-CRF-CLS R2 من الصور باستخدام برامج Imaris. تقديم سطح نواة وخلق بؤر تصف ملحقات GPCR تحويلها إلى حويصلات صغيرة. تمت معالجة البيانات أولا باستخدام مضان Imaris الذي يسمح للتجزئة والتصور 3D المجهر من مجموعة البيانات. ثم، تم استخدام Imaris XT لImaris التفاعل مع MATLAB. تم تبادل شدة فوكسل فيبقعة الإحداثيات. البقع الطيف ترميز الألوان (الأزرق 0-5 ميكرون، الأخضر، 6-10 ميكرون، ميكرومتر 11-15 الأصفر والأحمر&gt; ميكرون 15) تمثل النموذج المسافة الغشاء النووي. مقياس تقييم من 5 ميكرون. <img alt="الشكل 4" src="/files/ftp_upload/4233/4233fig4.jpg" /> ويستخدم الشكل 4. تمثيل بياني لتوزيع البعيدة من البقع المرسومة في لون الطيف مشفرة في 5 ميكرومتر فترات لتصور المسافة بين voxels من الغشاء النووي. يوجد علاج والمعالجة المسبقة مع خصم (AS-30، 1 ميكرومتر، 30 دقيقة) قبل بدء العلاج ناهض (CRF، 1 ميكرومتر، 30 دقيقة) لا تظهر أي مهمة (م) الفرق في الانكماش GPCR. علاج الخلايا مع ناهض (CRF، 1 ميكرومتر، 30 دقيقة) يقلل تدريجيا المسافة بين عدد من CRF-R2 المحتوية على voxels بالمقارنة مع أي علاج 0-5 ميكرون (NS)، 6-15 ميكرون (ع ** &lt;0.01) و&gt; 15 ميكرون (*** p &lt;0.005)، أو AS-30 ميكرومتر العلاج 0-10 (م)، 11-15<html> ميكرومتر (** p &lt;0.01) و&gt; 15 ميكرون (*** P &lt;0.005). <img alt="الشكل 5" src="/files/ftp_upload/4233/4233fig5.jpg" /> الشكل 5. الحد من تحليل المظهرية 2D من الخلايا transfected مع HEK 293 HA-CRF-R2. تصور المقطع منتصف الطائرة من الخلايا (3-4μm فوق ساترة الزجاج) تظهر وسط النوى مع دابي وHA-R2-CRF بحثها استخدام الألغام المضادة للHA وتصور باستخدام اليكسا 488nm مترافق المضادة للماوس (مفتش 1) الثانوية والأجسام المضادة المكتسبة مع CLS لا يظهر أي فرق بين عدم المعالجة (A) (NT) و (B) ناهض (CRF، 1 ميكرومتر، 30 دقيقة)، في حين أن نقطة التصاق مستقبلات مختلفة بشكل كبير. الفيلم 1. الدورية بحرية نموذج 3D في وضع &quot;فق&quot; من HEK 293 الخلايا transfected مع CRF-HA-R2، أي علاج، لتقييم الاختلافات بين النمط الظاهري الخلوية البروتينات المستقبلة. مقياس بار 5 حتي 20 ميكرون./ files/ftp_upload/4233/4233movie1.avi &quot;الهدف =&quot; _blank &quot;&gt; اضغط هنا لمشاهدة الفيلم. فيلم 2. الدورية بحرية نموذج 3D في وضع &quot;فق&quot; من HEK 293 الخلايا transfected مع HA-CRF-R2، والعلاج ناهض، CRF (CRF، 1 ميكرومتر، 30 دقيقة) لتقييم الاختلافات بين النمط الظاهري الخلوية البروتينات المستقبلة. شريط نطاق 5 حتي 20 ميكرون. <a href="http://www.jove.com/files/ftp_upload/4233/4233movie2.avi" target="_blank">انقر هنا لمشاهدة الفيلم.</a> الفيلم 3. بحرية 3D الدورية في وضع &quot;فق&quot; من HEK 293 الخلايا transfected مع CRF-HA-R2، المعالجة مع خصم (AS-30، 1 ميكرومتر، 30 دقيقة) قبل بدء العلاج ناهض (CRF، 1 ميكرومتر، 30 دقيقة ) لتقييم الاختلافات بين النمط الظاهري الخلوية البروتينات المستقبلة. شريط نطاق 5 حتي 20 ميكرون. <a href="http://www.jove.com/files/ftp_upload/4233/4233movie3.avi" target="_blank">انقر هنا لمشاهدة الفيلم.</a>

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الإعلان عن أي تضارب في المصالح.

أثبتنا أن يسببها العلاج CRF تغييرا كبيرا في التشكل وموقع CRF-R2. وقد تحول دون التغيير في CRF-R2 عن طريق العلاج خصم انتقائية. أثبتنا أن التعديلات لم يتم اكتشاف مستقبلات ويمكن أن لا تقاس باستخدام معيار تقنيات متعددة الأطياف 2D. القدرة على دراسة الصور 3D المعقدة أمر بالغ الأهمية ?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> كتالوج رقم </td><td> التعليقات (اختياري) </td></tr><tr><td> الجنينية البشرية الكلى (HEK293) </td><td> كوكتيل الأمريكية نوع الثقافة </td><td> CRL-1573 </td><td></td></tr><tr><td> Dulbecco في معدلة النسر متوسطة (DMEM) </td><td> إينفيتروجن </td><td> 11965118 </td><td></td></tr><tr><td> الجنين المصل البقري (FBS) </td><td> إينفيتروجن </td><td> SH30070.03 </td><td></td></tr><tr><td> AlexaFluor-488 (IgG2b) </td><td> إينفيتروجن </td><td> A-11001 </td><td></td></tr><tr><td> وحيدة النسيلة المضادة للHA.11 (مفتش 1) </td><td> كوفانس </td><td> 16B12 </td><td></td></tr><tr><td> دابي </td><td> ناقلات المختبرات </td><td> H-1200 </td><td>؛ </td></tr><tr><td> CRF </td><td> سيجما </td><td> C2917 </td><td></td></tr><tr><td> Antisauvagine-30 (AS-30) </td><td> سيجما </td><td> A4727 </td><td></td></tr></tbody></table>

an analytical tool that quantifies cellular morphology changes from three-dimensional fluorescence images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

Watch this Scientific Journal Video about An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images at JoVE.com

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

وضعنا منصة البرمجيات التي تستخدم Imaris العلوم العصبية، وImarisXT MATLAB لقياس التغيرات في مورفولوجية شكل غير معروف مأخوذ من ثلاثي الأبعاد من الخلايا مبائر مضان واحد. ويمكن استخدام هذا النهج الجديد لقياس التغييرات في شكل الخلية بعد تفعيل مستقبلات وبالتالي يمثل أداة ممكنة إضافية لاكتشاف المخدرات.

أداة تحليلية يحدد مقدار التغييرات الخلوية الصرف من التقاط صور ثلاثية الأبعاد الإسفار

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

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Research

JoVE Journal

Biology

14.5K Views.  University of California, San Francisco. وضعنا منصة البرمجيات التي تستخدم Imaris العلوم العصبية، وImarisXT MATLAB لقياس التغيرات في مورفولوجية شكل غير معروف مأخوذ من ثلاثي الأبعاد من الخلايا مبائر مضان واحد. ويمكن استخدام هذا النهج الجديد لقياس التغييرات في شكل الخلية بعد تفعيل مستقبلات وبالتالي يمثل أداة ممك?...

Video: An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

رقاقة المستندة إلى خلية ثقافة ثلاثي الأبعاد في المفاعلات الصغيرة Perfused

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

التصنيع الدقيق للرقاقة الحجم السقالات لزراعة الخلايا ثلاثي الأبعاد

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

أسلوب الإنتاجية العالية عالميا لدراسة مورفولوجية عضية في البيرة س.

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

التجمع ، والتحميل ، والمحاذاة لخلية نموذج تحليلي نابذة فائقة السرعة

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

التغيرات في الغدة الثديية المورفولوجيا واحتمال الاصابة بسرطان الثدي في الفئران

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

عزل الخلايا الظهارية الثديية من ثلاثي الأبعاد الخلايا المختلطة الجسم الشبه الكروي ثقافة المشارك

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

التحليل السريع واستكشاف الإسفار الميكروسكوب الصور

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

الحنق التصوير في الهلاميات المائية ثلاثية الأبعاد

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

أديبو--واضحة: منديل مسح طريقة لتصوير ثلاثي الأبعاد للأنسجة الدهنية

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

المناهج الكمية لدراسة الهياكل الخلوية ومورفولوجيا الأعضاء في الكنورهابدية

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...