بدعم الصناديق المؤسسية الداخلية والبيولوجي برنامج علماء العلوم في جامعة ميشيغان هذا العمل.

<ol>
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الإعلان عن أي تضارب في المصالح.

وهناك اعتبار رئيسي للتصوير مرور الزمن يعيش هو الحفاظ على سلامتها وسلامة الخلية والكائن في. دبي انترناشيونال كابيتال المجهري يوفر الفوائد التي لم يتم كشفها نموذج للأشعة فوق البنفسجية والحرارة المفرطة من مصدر الإضاءة. هي مناسبة تماما للكشف المجهري DIC الهجرة الخلية ويتغير شكل الخلية مثل الحرائك الخلوية والتعرف على بعض الهياكل التحت خلوية ، مثل مغزل الإنقسامية والغشاء النووي. التصوير مضان من البروتينات مراسل يكمل المجهري مدينة دبي للإنترنت عن طريق تحديد الأجزاء التحت خلوية إضافية والسماح التصور للبروتينات محددة. للتخفيف من أخطار وأضرار الضيائية إلى الجنين أثناء التصوير مضان ، ونحن عادة زيادة الربح من الكاميرا الرقمية للتعويض عن انخفاض كثافة الأشعة فوق البنفسجية ومدة الإضاءة. خافت المعدلة وراثيا للصحفيين الفلورسنت ، خوارزميات deconvolution إلى إعادة تعيين من خارج تركيز الضوء أو deblurring خوارزميات للحد من الخلفية يمكن تحسين جودة الصور الملتقطة. بينما المجاهر المتقدمة مثل نظم مبائر multiphoton أو في بعض الأحيان ضرورية ، وجدنا أنه يمكن للصحفيين الفلورسنت تصوير العديد من استخدام هذا الإعداد epifluorescent المجهر ، مما أسفر عن صور ذات جودة عالية. مدير الدقيقة (http://www.micro-manager.org) ، بالإضافة إلى كونها مجانية ، يحفظ الملفات مباشرة إلى تنسيق TIFF بدلا من تنسيقات الملفات الشخصية لكثير من حزم البرامج التجارية. هذا يبسط كثيرا من تحليلات البيانات المتوفرة لدينا من خلال القضاء على خطوة تحويل ملف اللازمة لقراءة ملفات الصور في ImageJ ، فوتوشوب وغيرها من برامج تحرير الصور.

اوليمبوس BX61 المجهر مع الكاميرا أوكرا - ER هاماماتسو. 10X فلوريت خطة (NA 0.3) وPlanAchromat 60x (NA 1.35) هدف العدسات مدير الدقيقة 1.3.46 (http://www.micro-manager.org) صورة J 1.43 (http://rsbweb.nih.gov/ij) loci_tools.jar (http://www.loci.wisc.edu) Agarose -- البيولوجيا الجزيئية / هلام الصف الكهربائي مجهر شرائح الصيادين Superfrost / بلاس (كتالوج # 12-550-15) مختبر الشريط (دليل فيشر # 15-901 السلسلة) 18 ملم x 18 ملم 1.5 # Coverslips M9 (242 ملي KH 2 ص 4 ، 40 مم نا 2 هبو 4 ، 9 ملم ز كلوريد الصوديوم ، و 19 ملي NH 4 الكلورين MgSO 4) (http://130.15.90.245/wormlab_recipe_book.htm). البيض العازلة (118 ملي مول كلوريد الصوديوم ، و 48 ملي بوكل ، CaCl 2 مم 2 ، 2 مم MgCl 2 و 25 HEPES ملم ، ودرجة الحموضة 7.3) 25. فازلين فرشاة / عصا شفرات حلاقة حرارة كتلة تشريح المجهر الديدان 22 سلك البلاتين اختيار 22 27G1 / 2 دقيقة الإنزلاق الإبرة (بيكتون ديكنسون) أو مشرط صغير بشفرة

imaging c. elegans embryos using an epifluorescent microscope and open source software

العمليات الخلوية ، مثل العزل الصبغي ، والتجمع ، والحرائك الخلوية ، هي بطبيعتها ديناميكية. الوقت الفاصل بين التصوير من الخلايا الحية ، وذلك باستخدام البروتينات الفلورية المسمى الصحفي أو الفرق المقابل تدخل (DIC) المجهري ، ويسمح لدراسة تطور الزمني لهذه الأحداث الديناميكية التي هي على خلاف ذلك يستدل من تحليل عينات 1،2 الثابتة. وعلاوة على ذلك ، ودراسة لوائح التنموية يتطلب إجراء العمليات الخلوية الوقت الفاصل بين التجارب على كائن حي سليمة خلال التنمية. الجنين ايليجانس Caenorhabiditis خفيفة وشفافة لها ، ثابتة السريع البرنامج الإنمائي مع خلية معروفة النسب 3 ، مما يوفر تجربة نموذج مثالي لدراسة المسائل في بيولوجيا الخلية والتنمية 4،5 6-9. جيم ايليجانس هو قابلة للتعديل الوراثي للتلاعب من قبل الوراثة إلى الأمام (على أساس الطفرات العشوائية 10،11) ، وعلم الوراثة العكسية لاستهداف جينات معينة (على أساس رني بوساطة التدخل واستهدفت الطفرات 12-15). بالإضافة ، يمكن بسهولة خلق حيوانات معدلة وراثيا في التعبير عن بروتينات أو الموسومة fluorescently صحفيين 16،17. هذه الصفات مجتمعة تجعل من السهل التعرف على المسارات الجينية التي تنظم العمليات الخلوية الأساسية والتنموية في الجسم الحي 18-21. في هذا البروتوكول نقدم أساليب التصوير المباشر لجيم ايليجانس أجنة باستخدام البصريات DIC أو مضان GFP على مجهر epifluorescent المجمع. نبدي السهولة التي يمكن أيضا المجاهر متاحة بسهولة ، وتستخدم عادة لتصوير نموذج ثابت ، تطبق على الوقت الفاصل بين التحليل باستخدام برمجيات المصدر المفتوح لأتمتة عملية التصوير.

Watch this Scientific Journal Video about Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software at JoVE.com

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

و<em&gt; C. ايليجانس</em&gt; الجنين هو نظام قوي لدراسة بيولوجيا الخلايا والتنمية. نقدم بروتوكولا للتصوير حي ل<em&gt; C. ايليجانس</em&gt; استخدام أجنة البصريات مدينة دبي للإنترنت أو استخدام مضان المجاهر epifluorescent متاحة بسهولة والبرمجيات مفتوحة المصدر.

التصوير C. ايليجانس الأجنة باستخدام مجهر Epifluorescent وبرمجيات المصدر المفتوح

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

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Biology

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

27.7K Views.  University of Michigan. و C. ايليجانس الجنين هو نظام قوي لدراسة بيولوجيا الخلايا والتنمية. نقدم بروتوكولا للتصوير حي ل C. ايليجانس استخدام أجنة البصريات مدينة دبي للإنترنت أو استخدام مضان المجاهر epifluorescent متاحة بسهولة والبرمجيات مفتوحة المصدر.

Video: Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

توليد مستقرة ايليجانس جيم المعدلة وراثيا عن طريق Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

لإعداد ذبابة الفاكهة الأجنة لايف التصوير باستخدام بروتوكول إسقاط الشنق

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging

This paper aims to instruct the reader in the assembly and operation of an infrared near-field microscope for imaging beyond the diffraction limit.  The apertureless near-field microscope is a light scattering-type instrument that provides infrared spectra at circa 20 nm resolution. A complete list of components and a step-by-step protocol for use is provided.  Common errors in assembly and instrument tuning are discussed.  A representative data set that shows the secondary structure of an amyloid fibril is presented.  

The assembly of a nearfield infrared microscope for imaging protein aggregates is described.

التجمع ، وضبط واستخدام المجهر حقل قرب Apertureless الأشعة تحت الحمراء للتصوير البروتين

This paper aims to instruct the reader in the assembly and operation of an infrared near-field microscope for imaging beyond the diffraction limit.  The ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

استخدام الميكروسكوب الفاصل الزمني لتصور نقص الأكسجين الناجم عن تعطل الحركة في<em&gt; C. ايليجانس</em&gt; الأجنة

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

C. ايليجانس الانجذاب الكيميائي الفحص

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

تلوين الأجسام المضادة في<em&gt; C. ايليجانس</em&gt; عن طريق &quot;تجميد تكسير&quot;

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

قياس آثار البكتيريا على<em&gt; C. ايليجانس</em&gt; السلوك باستخدام مقايسة الاحتفاظ البيض

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

نموذج نقص الأكسجين المجاعة لنقص التروية / ضخه في<em&gt; C. ايليجانس</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time.

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

This protocol describes the setup of a light sheet microscope and its implementation for in vivo imaging of C. elegans embryos.

إعداد بسيط ضوء مجهر ورقة ل<em&gt; في توتو</em&gt; التصوير من<em&gt; C. ايليجانس</em&gt; التنمية

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces ...

Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators.
Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software1&#160;to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

Presented is a flexible informatics workflow enabling multiplexed image-based analysis of fluorescently labeled cells. The workflow quantifies nuclear and cytoplasmic markers and computes marker translocation between these compartments. Procedures are provided for perturbation of cells using siRNA and reliable methodology for marker detection by indirect immunofluorescence in 96-well formats.

سير العمل عالية المحتوى، الكمي خلية فردية من نيون علامات من المجاهر البيانات العالمي، بدعم من برمجيات المصدر المفتوح

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly ...