J.O.B. and A.M.R. were supported by predoctoral fellowships from the National Science Foundation.

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<li>Caplan, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chloroplast+stromules+runction+during+innate+immunity%5BTitle%5D)+AND+%22Dev.+Cell%22%5BJournal%5D)">Chloroplast stromules runction during innate immunity.</a> Dev. Cell. 34 (1), 45-57 (2015).</li>
<li>Hanson, M. R., Sattarzadeh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stromules%3A+recent+insights+into+a+long+neglected+feature+of+plastid+morphology+and+function%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Stromules: recent insights into a long neglected feature of plastid morphology and function.</a> Plant Physiol. 155 (4), 1486-1492 (2011).</li>
<li>Gray, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plastid+stromules+are+induced+by+stress+treatments+acting+through+abscisic+acid%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">Plastid stromules are induced by stress treatments acting through abscisic acid.</a> Plant J. 69 (3), 387-398 (2012).</li>
<li>Brunkard, J., Runkel, A., Zambryski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chloroplasts+extend+stromules+independently+and+in+response+to+internal+redox+signals%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Chloroplasts extend stromules independently and in response to internal redox signals.</a> Proc. Natl. Acad. Sci. USA. 112 (32), 10044-10049 (2015).</li>
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<li>Schattat, M., Barton, K., Baudisch, B., Klösgen, R. B., Mathur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plastid+stromule+branching+coincides+with+contiguous+endoplasmic+reticulum+dynamics%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Plastid stromule branching coincides with contiguous endoplasmic reticulum dynamics.</a> Plant Physiol. 155 (4), 1667-1677 (2011).</li>
<li>Erickson, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Agrobacterium-derived+cytokinin+influences+plastid+morphology+and+starch+accumulation+in+Nicotiana+benthamiana+during+transient+assays%5BTitle%5D)+AND+%22BMC+Plant+Biol%22%5BJournal%5D)">Agrobacterium-derived cytokinin influences plastid morphology and starch accumulation in Nicotiana benthamiana during transient assays.</a> BMC Plant Biol. 14 (1), 127(2014).</li>
<li>Ho, J., Theg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+formation+of+stromules+in+vitro+from+chloroplasts+isolated+from+Nicotiana+benthamiana%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">The formation of stromules in vitro from chloroplasts isolated from Nicotiana benthamiana.</a> PLoS One. 11 (2), e0146489(2016).</li>
<li>Brunkard, J. O., Burch-Smith, T. M., Runkel, A. M., Zambryski, P. Investigating plasmodesmata genetics with virus-induced gene silencing and an Agrobacterium-mediated GFP movement assay. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBrunkard%2C+JO+%22Method.+Mol.+Biol%22">Method. Mol. Biol</a>. , 185-198 (2015).</li>
<li>Schattat, M. H., Klösgen, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+stromule+formation+by+extracellular+sucrose+and+glucose+in+epidermal+leaf+tissue+of+Arabidopsis+thaliana%5BTitle%5D)+AND+%22BMC+Plant+Biol%22%5BJournal%5D)">Induction of stromule formation by extracellular sucrose and glucose in epidermal leaf tissue of Arabidopsis thaliana.</a> BMC Plant Biol. 11, 115(2011).</li>
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<li>Waters, M. T., Fray, R. G., Pyke, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stromule+formation+is+dependent+upon+plastid+size%2C+plastid+differentiation+status+and+the+density+of+plastids+within+the+cell%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">Stromule formation is dependent upon plastid size, plastid differentiation status and the density of plastids within the cell.</a> Plant J. 39 (4), 655-667 (2004).</li>
</ol>

The authors have nothing to disclose.

عند التحقيق stromules، يجب النظر في ثلاثة عوامل هامة من خلال: (أ) التلاعب في الأنسجة النباتية يجب أن تبقى إلى أدنى حد ممكن، (ب) يجب أن يبقى النظام التجريبي متسقة، و (ج) استراتيجيات أخذ العينات يجب أن يتم التخطيط بعناية ل ضمان قوية، ويتم تحليل البيانات استنساخه. Stromules دينامية ملحوظة: يمكن أن يمتد ويتقلص بسرعة أمام عينيه مراقب تحت المجهر. وعلاوة على ذلك، تردد stromule يختلف اختلافا كبيرا في استجابة لمجموعة واسعة من العلاجات، بما في ذلك المثيرات التي لا يمكن تجنبها من أجل رؤية stromules (مثل جرح ورقة). الحل الأساسي لهذه المشكلة التي تناولها مع البروتوكولات المذكورة هنا، هو لإجراء التجارب التي تسيطر عليها بشكل جيد، وحفظ جميع المتغيرات ثابتة. وهذا يشمل، على سبيل المثال، وإعداد كل عينة لرؤية مباشرة قبل الوصول بها إلى المجهر. لا ينبغي أن يكون معطوبا النباتاتحتى قبيل التصور. الحل الثاني لهذه المشكلة هو تقليل تعقيد البروتوكولات: من الناحية المثالية، ينبغي تطبيق أي علاج مباشرة إلى محطة دون إزالة ورقة أو التسبب في أي ضرر. وقد تم دراسة Stromules في مجموعة مذهلة من الأنواع وأنواع الخلايا، بما في ذلك الشعر القمح الجذر، ثمار الطماطم، وشتلات التبغ etiolated 8،19. هذه الدراسات الرائدة تقدمت فهم ديناميات stromule خلال تطوير المصنع وبحثت مجموعة من الأنشطة stromule. مقارنات من هذه النظم التجريبية المختلفة، ومع ذلك، يمكن أن تؤدي إلى تفسيرات خاطئة، حيث يتم تنطوي على أنواع مختلفة من البلاستيدات في العمليات البيولوجية المختلفة إلى حد كبير. على سبيل المثال، البلاستيدات الخضراء تجعل stromules ردا على الاكسدة الناجمة عن سلسلة الإلكترون الضوئي، ولكن منذ بلاستيدات عديمة اللون تفتقر إلى آلية التمثيل الضوئي، فهي ليست حساسة لنفس المحفزات 9. يمكن استخدام أي نظام تجريبي للتحقيق stromules، ولكن يجب أن تبقى على نظام تجريبي ثابت طوال فترة الدراسة إن وجدت المقارنات التي يمكن استخلاصها. العوامل في الاعتبار تشمل الأنواع، ونوع من الخلايا، ومرحلة النمو أو عمر النبات، وطريقة stromules تلطيخ (سواء من خلال التعبير مستقرة التحوير والتعبير التحوير عابرة، أو fluorophore خارجي)؛ حتى تكون هناك اختلافات طفيفة من هذه العوامل يمكن أن يربك تفسير لاحق من النتائج. وأخيرا، فإن أي دراسة البيولوجيا stromule يجب استخدام استراتيجية قوية أخذ العينات يمكن الاعتماد عليها للتأكد من أن النتائج استنساخه وذات الصلة من الناحية البيولوجية. أخذ العينات مرارا وتكرارا من مصنع واحد، على سبيل المثال، التقاط الصور من عدة مناطق من ورقة واحدة، وعلى ما يبدو تحسين القدرة الإحصائية، لكن يمكن أن يكون مضللا. كما هو مبين في بيانات تمثيلية أعلاه، تردد stromule من ورقة واحدة قد تختلف بشكل كبير من وتيرة stromule متوسطعبر العديد من الأوراق: في تلك الورقة، التي كانت تصور أثناء النهار، وجعلت 37.9٪ من البلاستيدات الخضراء stromules، على الرغم من أن وتيرة stromule نفسه عبر الأوراق أثناء النهار وكان ما يقرب من نصف ذلك، فقط 20.8٪. وعلاوة على ذلك، نظرا لمدى لا يعرف الكثير عن ديناميات stromule في هذا الوقت، أي تجربة يجب أن تتكرر تماما ثلاث مرات على الأقل، وأكثر من ذلك إن أمكن، لضمان أن متغيرات غير متوقعة ليست مسؤولة عن أي تغيرات في النشاط stromule التي يتم ملاحظتها. قبل كل شيء، منذ مجال stromule البيولوجيا هو مجرد بداية للاقلاع، يجب على الباحثين أن توثق بعناية وتفيد جميع جوانب التصميم التجريبي، بما في ذلك الانحرافات الإبداعية من بروتوكول بسيط المذكورة هنا.

Chloroplasts are dynamic organelles in plant cells responsible for photosynthesis and a host of other metabolic processes. Signaling pathways from the chloroplast also exert significant influence on plant physiology and development, coordinating plant responses to environmental stress, pathogens, and even leaf shape1-6. Recently, biologists have gained interest in a poorly understood aspect of chloroplast structure: stromules, very thin stroma-filled tubules that extend from the surface of the chloroplast7.
The biological functions of stromules remain unknown, although stromule frequency is known to vary in response to environmental stimuli7-9, and stromules may be capable of transmitting signaling molecules between organelles6. All types of plastids (not only the green, photosynthetic chloroplasts, but also clear leucoplasts, starch-filled amyloplasts, and pigmented chromoplasts, to name a few types of plastids) make stromules, and stromules are found in all land plant species that have been examined to date. Stromules can extend and retract dynamically, appearing or disappearing within seconds, or they can remain relatively stationary for long times. One of the major hurdles facing stromule biologists is that stromules are often studied using dramatically different methods, tissues, and species, making comparisons across the stromule biology literature difficult. Going forward, standard practices and thorough descriptions of the experimental systems used to study stromules will be critical to discovering the function of these ubiquitous features of chloroplast morphology.
Here we describe methods for visualizing stromule formation in the epidermal chloroplasts of Nicotiana benthamiana leaves. In the mesophyll, chloroplasts are densely packed into large, three-dimensional cells, which makes it difficult to accurately and rapidly visualize stromules by confocal microscopy. By contrast, epidermal cells are relatively flat, contain fewer chloroplasts, and are at the surface of the leaf, allowing for easy and rapid visualization of stromules. N. benthamiana is an ideal model system for these experiments because, unlike many plant species, all cells in the epidermis of N. benthamiana make chloroplasts10. In the epidermis of most plants, including Arabidopsis thaliana, only the stomatal guard cells have chloroplasts, while other epidermal cells have "leucoplasts", plastids that are clear, relatively amorphous, and nonphotosynthetic9,11,12. Thus, whereas a single field of view of an A. thaliana epidermis might show only a handful of chloroplasts in a pair of guard cells, a field of view of an N. benthamiana epidermis will include dozens or even hundreds of chloroplasts. All of the methods described here, however, can be modified to investigate other questions in stromule biology; for example, we have used the same approach to study leucoplast stromules of A. thaliana9.

<table><tbody><tr><td>Hepes</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>NaOH</td><td>Fischer-Scientific</td><td>S320-1</td><td></td></tr><tr><td>Sorbitol</td><td>Sigma-Aldrich</td><td>S1876</td><td></td></tr><tr><td>EDTA</td><td>Fischer-Biotech</td><td>BP121</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>221279</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M0250</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9625</td><td></td></tr><tr><td>Laser Scanning Confocal Microscope&amp;nbsp;</td><td>Carl Zeiss Inc</td><td>Model: LSM710</td><td></td></tr><tr><td>Carboxyfluorescein diacetate (CFDA)</td><td>Sigma-Aldrich</td><td>21879&amp;nbsp;</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>EMD</td><td>MX1458-6</td><td></td></tr><tr><td>Waring blender</td><td>Waring&amp;nbsp;</td><td>Model: 31BL92</td><td></td></tr><tr><td>Fiji</td><td>fiji.sc</td><td></td><td>Open-source software for analyzing biological images</td></tr></tbody></table>

visualizing stromule frequency with fluorescence microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

وقد استخدم هذا البروتوكول لتصور تردد stromule في النهار والليل في النبتات من N. الشباب الشتلات benthamiana. تم دمج شرائح من كومة Z- في صورة واحدة (الشكل 1A). لأغراض البصرية، كانت تلك الصورة ثم غير المشبعة ومقلوب بحيث يظهر سدى الأسود (الشكل 1B). وصفت البلاستيدات الخضراء إما عدم وجود stromules (النجمة الخضراء) أو وجود stromule واحد على الأقل (أرجواني النجمة). من البلاستيدات الخضراء البشرة 87 تصور، 33 ديهم stromules. وهكذا، فإن تردد stromule في هذه الورقة هو 37.9٪. باستخدام هذا التحليل، وتكرر البروتوكول لمدة 21 محطات أخرى (ورقة واحدة للنبات) خلال النهار، وما مجموعه 24 محطات في الليل. في هذا اليوم، كان متوسط ​​تردد stromule 20.8 ± 1.8٪، في الليل، كان متوسط تردد stromule 12.8 ± 0.9٪ (الشكل2). وكان تردد Stromule أعلى بكثير خلال النهار، على النحو الذي يحدده اختبار ر ولش (ن ≥ 22، ص &lt;0.0005). لاحظ أنه على الرغم من أن أكثر من 23،000 البلاستيدات الخضراء وعدة مئات من الخلايا لوحظت، وحجم العينة، ن، وكما ذكرت 22، فحص عدد من محطات مستقلة. باستخدام بروتوكول الموصوفة هنا، وقد لوحظت stromules بلاستيدات الخضراء في المختبر بعد عزلة من أوراق N. benthamiana معربا عن استهداف صانعة GFP (الشكل 3A)، أو بعد استخدام CFDA وصمة عار البلاستيدات الخضراء المعزولة من N. benthamiana (الشكل 3B) أو Spinacia OLERACEA (الشكل 3C). <img alt="شكل 1" src="/files/ftp_upload/54692/54692fig1.jpg" /> الشكل 1. تحديد مقدار التردد stromule في N. benthأوراق amiana. (A) A Z -stack من حقل جزئي نظر البشرة من N. تم دمج benthamiana معربا عن انسجة GFP لإظهار كل البلاستيدات الخضراء البشرة وstromules في صورة واحدة. (ب) تم تحويل هذه الصورة إلى الأبيض والأسود ومقلوب لأغراض البصرية. يشار إلى البلاستيدات الخضراء مع stromules من قبل النجمة أرجواني. يشار إلى البلاستيدات الخضراء دون stromules من قبل النجمة الخضراء. تمثل الحانات نطاق 10 ميكرون. تعديل من Brunkard وآخرون. 9 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54692/54692fig1large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> <img alt="الشكل 2" src="/files/ftp_upload/54692/54692fig2.jpg" /> الشكل 2. مقارنة تردد stromule عبر الظروف. وقد استخدم هذا البروتوكول لتحديد شارعتردد omule في 22 مصنعا في اليوم و 24 محطات في الليل. وكان تردد Stromule أعلى بكثير خلال النهار من الليل (اختبار ر ولش، ن ≥ 22، ص &lt;0.0005). أشرطة الخطأ تشير إلى الخطأ المعياري. تعديل من Brunkard وآخرون. 9 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54692/54692fig2large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a> <img alt="الشكل (3)" src="/files/ftp_upload/54692/54692fig3.jpg" /> الشكل 3. تصور stromules بعد استخراج البلاستيدات الخضراء سليمة من الأوراق. (A) البلاستيدات الخضراء من N. المعدلة وراثيا benthamiana يترك GFP أعرب (الأخضر) التي تستهدف تم عزل باستخدام البروتوكولات المذكورة هنا بلاستيدات الخضراء. تم عزل (ب) البلاستيدات الخضراء من النوع البري N. benthamiana أوراق وملطخة CFDA، التي فلووresces فقط داخل سليمة، البلاستيدات الخضراء قابلة للحياة (كما هو موضح باللون الأصفر). (C) البلاستيدات الخضراء ويمكن أيضا أن تكون معزولة من الأنواع النباتية الأخرى، مثل السبانخ (س. OLERACEA)، ومن ثم ملطخة CFDA (الصفراء). ويرد الكلوروفيل تألق ذاتي باللون الأحمر. تعديل من Brunkard وآخرون. 9

Watch this Scientific Journal Video about Visualizing Stromule Frequency with Fluorescence Microscopy at JoVE.com

Visualizing Stromule Frequency with Fluorescence Microscopy

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

تصور التردد Stromule مع مضان المجهر

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

visualizing-stromule-frequency-with-fluorescence-microscopy

Nanyang Technological University

Research

JoVE Journal

Biology

8.0K Views.  USDA. Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Video: Visualizing Stromule Frequency with Fluorescence Microscopy

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins.
Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

مراقبة المكانية الفصل في استعمار سطح الميكروبية السكان

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

Genetics

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

تعيش خلية من التصوير<emالعصوية الرقيقة> و العقدية الرئوية الآلي باستخدام الميكروسكوب مرور الزمن

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Immunology and Infection

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 &#181;m x 500 &#181;m region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

عاليه الانتاجيه الكلي الداخلية انعكاس الفلورية والتوجيه العشوائي البصرية أعاده البناء المجهري باستخدام رقاقه فوتوسونيك

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

Biochemistry

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

تصور المجمعات الجزيئية واحدة في فيفو استخدام الميكروسكوب الإسفار متقدم

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. 
Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series.

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Streptomyces are characterized by a complex life cycle that has been experimentally challenging to study by cell biological means. Here we present a protocol to perform fluorescence time-lapse microscopy of the complete life cycle by growing Streptomyces venezuelae in a microfluidic device.

مضان الوقت الفاصل بين التصوير من كامل<em&gt; S. venezuelae</em&gt; دورة الحياة عن طريق جهاز ميكروفلويديك

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of ...

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

أدوات الموائع الدقيقة للتحقيق في التفاعلات الفطرية الميكروبية على المستوى الخلوي

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...

Bioengineering

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

جيل من تفرقوا Presomitic الأديم المتوسط ​​الثقافات خلية التصوير من اسماك الزرد الإنقسام على مدار الساعة في خلايا واحدة

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

الكمي الزمانية التعبير MAPK المستحثة في خلايا الخميرة واحدة

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

طريقة لتصنيف النصوص في الخلايا الفردية الإشريكيّة القولونية للأسفار جزيء واحد في الموقع تجارب التهجين

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

تصور التردد Stromule مع مضان المجهر

Summary

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مراقبة المكانية الفصل في استعمار سطح الميكروبية السكان

تعيش خلية من التصوير و العقدية الرئوية الآلي باستخدام الميكروسكوب مرور الزمن

عاليه الانتاجيه الكلي الداخلية انعكاس الفلورية والتوجيه العشوائي البصرية أعاده البناء المجهري باستخدام رقاقه فوتوسونيك

تصور المجمعات الجزيئية واحدة في فيفو استخدام الميكروسكوب الإسفار متقدم

مضان الوقت الفاصل بين التصوير من كامل

أدوات الموائع الدقيقة للتحقيق في التفاعلات الفطرية الميكروبية على المستوى الخلوي

جيل من تفرقوا Presomitic الأديم المتوسط الثقافات خلية التصوير من اسماك الزرد الإنقسام على مدار الساعة في خلايا واحدة

الكمي الزمانية التعبير MAPK المستحثة في خلايا الخميرة واحدة

طريقة لتصنيف النصوص في الخلايا الفردية الإشريكيّة القولونية للأسفار جزيء واحد في الموقع تجارب التهجين

التصوير خيوط متوسطة وميكروتوبوليس مع الفحص المجهري 2-الأبعاد التعمير البصرية العشوائية المباشرة

تصور التردد Stromule مع مضان المجهر

Summary

Explore More Videos

مراقبة المكانية الفصل في استعمار سطح الميكروبية السكان

تعيش خلية من التصوير و العقدية الرئوية الآلي باستخدام الميكروسكوب مرور الزمن

عاليه الانتاجيه الكلي الداخلية انعكاس الفلورية والتوجيه العشوائي البصرية أعاده البناء المجهري باستخدام رقاقه فوتوسونيك

تصور المجمعات الجزيئية واحدة في فيفو استخدام الميكروسكوب الإسفار متقدم

مضان الوقت الفاصل بين التصوير من كامل

أدوات الموائع الدقيقة للتحقيق في التفاعلات الفطرية الميكروبية على المستوى الخلوي

جيل من تفرقوا Presomitic الأديم المتوسط ​​الثقافات خلية التصوير من اسماك الزرد الإنقسام على مدار الساعة في خلايا واحدة

الكمي الزمانية التعبير MAPK المستحثة في خلايا الخميرة واحدة

طريقة لتصنيف النصوص في الخلايا الفردية الإشريكيّة القولونية للأسفار جزيء واحد في الموقع تجارب التهجين

التصوير خيوط متوسطة وميكروتوبوليس مع الفحص المجهري 2-الأبعاد التعمير البصرية العشوائية المباشرة

جيل من تفرقوا Presomitic الأديم المتوسط الثقافات خلية التصوير من اسماك الزرد الإنقسام على مدار الساعة في خلايا واحدة