وقد تم تمويل هذا العمل من قبل وزارة الدفاع عصر جائزة الباحث الأمل (رقم W81XWH - 05-0396) ، ثم باحثا في مركز بيو للجائزة العلوم الطبية الحيوية لبراون إدوارد الثالث باء.

1. محاذاة البصريات. المعدات الرئيسية لتجربة MP - FRAP ما يلي : وضع غير الساحلية مصدر الليزر Pockels الخلية ، (على تعديل شعاع) ، ومولد النبض ، مزدوج اللون ، عدسة موضوعية ومضان تصفية الانبعاثات ، وبوابات أنبوب مضخم للضوء ، ونظام تسجيل البيانات (الفوتون العداد والأقنية مقلحة). 2. تحديد صلاحيات مراقبة آمنة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الليزر لتخفيف من قوة ، وانخفاض ، ولكن معقولة لتوليد مضان داخل العينة. </li><li style=";text-align:right;direction:rtl"> التركيز داخل العينة. </li><li style=";text-align:right;direction:rtl"> تعيين عداد الفوتون لدمج أكثر من فترة زمنية أطول بكثير من المتوقع انتعاش مضان. التنسيق مع وحدة التخزين الثابتة التي عقدت ضمن العينة (على البرمجيات المجهر لدينا ويتحقق ذلك عن طريق فحص نقطة) وسجل عدد معقول من بيانات تعداد الفوتون. </li><li style=";text-align:right;direction:rtl"> زيادة القوة واتخاذ سلسلة أخرى من البيانات العد فوتون. كرر حتى الزيادة في تعداد الفوتون يبدو أن يقلل بشكل ملحوظ فيما يتعلق بزيادة الطاقة. </li><li style=";text-align:right;direction:rtl"> سجل مؤامرة (بحساب الفوتون) بوصفها وظيفة من السجل (السلطة). والانحراف عن منحدر اثنين يشير تبيض خلال العرض. اختيار كقوة الشاشة السلطة التي هي دون هذا قطع ، ولكن الذي يسمح للإشارة إلى نسبة الضوضاء معقولة. </li></ol> 3. تحديد معلمات الإدخال إلى مولد النبض ومقلحة الأقنية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحديد الخلفية للالمغلف في تقدير معامل الانتشار والانتعاش مرة على أساس و / أو الأدب المعادلة ستوكس آينشتاين والمعادلة نشرها. </li><li style=";text-align:right;direction:rtl"> تعيين قيم المعلمة الضرورية على مولد النبض وقشارة و. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> على مولد النبض ، وتعيين عصور ما قبل التبييض والتبييض. وهناك مجموعة جيدة من قواعد التجربة هو أن ما قبل التبييض وينبغي 1-2 مرات أكبر من الشوط الانتعاش المتوقع ، والوقت الذي ينبغي أن يكون واحدا التبييض عشر الانتعاش المتوقع half الوقت. </li><li style=";text-align:right;direction:rtl"> على قشارة ، مجموعة بن لعرض ما يقرب من نصف مدة التبييض ، وتعيين عدد من صناديق لكل سجل مثل هذا المنتج من العرض الخاص بن مختارة وصناديق لكل سجل أكبر من أو يساوي تقريبا إلى الشفاء التام من المتوقع بالاضافة الى ما قبل التبييض ، وأوقات التبييض. </li><li style=";text-align:right;direction:rtl"> عودة الى مولد النبض وضبط تردد من تسلسل pre-bleach/bleach/monitor يساوي قيمة أصغر من مجرد معكوس للمنتج من المرات عرض بن لعدد من صناديق في السجل. * </li><li style=";text-align:right;direction:rtl"> أخيرا ، تعيين عدد السجلات / المسح على مقلحة على أساس كثافة إشارة في وسعكم رصد المختار. * من المهم جدا أن الوقت لإكمال سلسلة pre-bleach/bleach/monitor (1/frequency) تعيين على مولد النبض أكبر من الوقت لجمع البيانات (عرض × بن صناديق / رقم قياسي) تعيين على قشارة. اذا لم يتم تلبية هذه المعايير ، قد يؤدي التبييض متعددة تظهر ضمن سجل واحد على قشارة. </li></ol></li><li style=";text-align:right;direction:rtl"> تعيين مراقبة السلطة إلى مستوى مقبول ، التي سبق تحديدها. تعيين قوة التبييض إلى 200 ميغاواط أو حتى أعلى من القطع تبيض تحدد خلال الاختبار رصد التبييض. هذه القوى على حد سواء كما الفولتية مختلف أنحاء الكريستال PC. </li><li style=";text-align:right;direction:rtl"> ختم المجهر ، اغلاق الاضواء ، بدوره على PMT ، تبدأ تفحص نقطة على البرامج المجهر ، ثم بدء تشغيل مولد النبض وقشارة. </li><li style=";text-align:right;direction:rtl"> تحليل منحنى الانتعاش الناجم عن رسم المنحنى ، ووضع علامات على ثلاث نقاط مهمة : 1) مضان قبل التبييض ، 2) ومضان مباشرة بعد التبييض ، و 3) ومضان في نهاية مجموعة البيانات. استخدام القيم مضان على الفور قبل وبعد التبييض لتقدير أفضل في المرة نصف الانتعاش. </li><li style=";text-align:right;direction:rtl"> مع هذا التقدير في ذلك الوقت نصف الانتعاش ، واستخدام قواعد الإبهام التي حددت من قبل لتحديد قيم جديدة للالتبييض ، وقبل التبييض ، وعرض بن لفترات. أيضا ، استخدم مضان قبل التبييض ومضان في نهاية مجموعة البيانات لتحسين تقدير الوقت اللازم لتحقيق الانتعاش الكامل. وكقاعدة عامة ، ينبغي جمع البيانات عن الفترة الزمنية التي يسمح للانتعاش الكامل ليكون أبلغ في النصف الأخير من البيانات. </li><li style=";text-align:right;direction:rtl"> ضبط المعلمات على مولد النبض وقشارة ، تأخذ منحنى جديدا ، وتستمر حتى التغيير والتبديل المعلمات المنحنى الخاص يسلك التبييض قوية والانتعاش على نحو سلس. نضع في اعتبارنا أن مدة التبييض يمكن ، ويجب خفضها الى ما دون سيادة الإبهام ، إذا أمكن ذلك. ويمكن أيضا أن تكون قوة التبييض تعديلها إذا ضحلة جدا ، أو عميقة جدا ، ويتم التوصل إلى التبييض. </li></ol> 4. اختبار لتشبع الإثارة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> رصد استخدام الصلاحيات المناسبة والتبييض ، والتي سبق تحديدها ، واتخاذ سلسلة من MP - FRAP المنحنيات. تحليل كل الانتعاش ، إلى تطبيع مضان قبل التبييض ، وذلك باستخدام الشكل المناسب الرياضية والساحات الأقل غير الخطية الخوارزمية المناسب (راجع &quot;تحليل البيانات&quot; أدناه). سجل قيمة المجهزة للمعلمة عمق التبييض.</li><li style=";text-align:right;direction:rtl"> مواصلة اتخاذ وتحليل سلسلة متعاقبة من MP - FRAP منحنيات الى زيادة قيم قوة التبييض. </li><li style=";text-align:right;direction:rtl"> سجل مؤامرة (التبييض المعلمة العمق) كدالة للتسجيل (السلطة). أي انحراف عن منحدر اثنين يشير التشبع الإثارة. اختيار السلطة التي تعطي التبييض التبييض قوية ، ولكن لا يسبب التشبع. </li></ol> 5. مواصلة اتخاذ MP - FRAP المنحنيات. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مواصلة اتخاذ المنحنيات ، كما ذكر أعلاه ، فإن جميع المعايير المحددة بشكل صحيح. </li></ol> 6. تحليل البيانات. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة البيانات قبل التبييض والتبييض من البيانات مضان تعيين وضبط مثل هذه البيانات في الوقت الذي ر = 0 يناظر قيمة مضان مباشرة بعد التبييض. </li><li style=";text-align:right;direction:rtl"> تطبيع الانتعاش مضان الناتجة فيما يتعلق مضان قبل التبييض المتوسط. </li><li style=";text-align:right;direction:rtl"> تناسب منحنى تطبيع باستخدام النموذج المناسب وخوارزمية رياضية غير خطية ، المربعات المناسب ، مثل وظيفة في lsqcurvefit MATLAB. النموذج المعروض هنا لحسابات المبالغ المستردة مضان يتأثر كلا نشرها وتدفق الحمل الحراري : <img src="/files/ftp_upload/1636/1636eq.jpg" alt="معادلة" /> حيث F o هي مضان قبل التبييض المتوسط ​​، β هو المعلمة عمق التبييض ، τ D هو السمة وقت الانتعاش ناشر ، R هي نسبة مربع من المحوري (ث ض) بعرض ل(ث ص) شعاعي لل حجم تنسيق ثنائي الفوتون ، τ VX ث = ص / ت س ، τ VY = ث ص / ت ذ ، ث = τ VZ ض / ي ت ، و ت 2 = ت × 2 + 2 + ص ت ت ض 2 هو سرعة تدفق الحمل الحراري. لتدفق يقتصر على الطائرة والتصوير ، وτ VZ → ∞ و 1 / τ اكس + 1 / τ يمكن استبدال VY مع 1 VR τ /. في غياب تدفق الحمل الحراري ، سواء في البسط exponentials انتقل إلى (1) وتقلص إلى حد كبير في التعبير ، وإلا لاثنين من المعلمات المناسب ، ودال β τ. ويتم احتساب معامل الانتشار بسهولة كما = D D ث 2 / 8τ ص. </li></ol> 7. ممثل النتائج. <img src="/files/ftp_upload/1636/1636fig1.jpg" alt="الشكل 1" /> الشكل 1. التبييض الممثل ومنحنى الانتعاش للFITC - BSA. يسلك منحنى الانتعاش القوي جيدة التبييض والانتعاش على نحو سلس ، مع مضان في استرداد كامل المبلغ عنها في النصف الأخير من البيانات. <img src="/files/ftp_upload/1636/1636fig2.jpg" alt="الشكل 2" /> الشكل 2. تطبيع منحنى الانتعاش للFITC - BSA (الحمراء) وما يرتبط بها من المربعات الصغرى تناسب (أسود). هذا يصلح تعطي معامل انتشار 52.9 ق / um2 ، بما يتفق مع الأدب.

<ol>
	<li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Biophys J. 60, 73-76 (1990).</li><li>Brown, E. B., Wu, E. S., Zipfel, W., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+molecular+diffusion+in+solution+by+multiphoton+fluorescence+photobleaching+recovery.">Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery.</a> Biophys J. 77, 2837-2849 (1999).</li><li>Sullivan, K. D., Sipprell, W. H. B. r. o. w. n., Jr, E. B., Brown, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+model+of+fluorescence+recovery+expands+the+application+of+multiphoton+fluorescence+recover+after+photobleaching+in+vivo.">Improved model of fluorescence recovery expands the application of multiphoton fluorescence recover after photobleaching in vivo.</a> Biophys J. 96, 5082-5094 (2009).</li></ol>

قوة متعددة الفوتون مضان الانتعاش بعد photobleaching تكمن في قدرته على تحقيق عينات سميكة مع القرار 3D. منذ تطوره في عام 1990 ، وقد استخدمت MP - FRAP لتحديد معامل نشر (أو المعلمات النقل المماثلة) في أجسام الخلايا ، بحكم فيفو شرائح الأنسجة سميكة ، والأنسجة في الجسم الحي والخل...

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measuring diffusion coefficients via two-photon fluorescence recovery after photobleaching

متعددة مضان الانتعاش بعد photobleaching هو أسلوب المجهري تستخدم لقياس معامل نشر (أو المعلمات النقل مماثلة) من الجزيئات ، ويمكن تطبيقها على حد سواء<em&gt; في المختبر</em&gt; و<em&gt; في الجسم الحي</em&gt; النظم البيولوجية. يتم تنفيذ متعددة مضان الانتعاش بعد photobleaching بواسطة photobleaching منطقة المصالح داخل عينة الفلورسنت باستخدام الفلاش الليزر مكثفة ، والمخففة ثم الحزم ورصد مضان كما لا تزال الفلورسنت جزيئات من خارج المنطقة لنزع فتيل الاهتمام ليحل محل جزيئات photobleached . سنبدأ مظاهرة لدينا من قبل محاذاة شعاع الليزر من خلال خلية Pockels (الليزر المغير) وعلى طول مسار بصري من خلال المسح الضوئي والليزر مربع عدسة الهدف العينة. عن البساطة ، وسوف نستخدم عينة من صبغة الفلورسنت مائي. وسوف نحدد ثم المعلمات المناسبة تجريبية لدينا عينة ، بما في ذلك رصد والقوى التبييض ، ومدة التبييض ، بعرض بن (لحساب فوتون) ، ومضان وقت الانتعاش. المقبل ، سنقوم بشرح الإجراء لأخذ منحنيات الانتعاش ، وهي عملية يمكن أن تتم بشكل آلي الى حد كبير عبر ابفيو (LabVIEW) (صكوك الوطنية ، أوستن ، تكساس) لتعزيز الإنتاجية. أخيرا ، يتم تحديد معامل نشر قبل تركيب استعادة البيانات إلى نموذج رياضي ملائم باستخدام خوارزمية تركيب المربعات الصغرى ، برمجة بسهولة باستخدام البرمجيات مثل MATLAB (وMathworks ، ناتيك ، MA).

Watch this Scientific Journal Video about Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching at JoVE.com

Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

في هذه المقالة سوف نقوم وصف الإجراء لقياس معامل الانتشار باستخدام متعدد الفوتون مضان الانتعاش بعد photobleaching. سنبدأ من خلال تنظيم ليزر على طول المسار الضوئي للعينة ، وتحديد المعايير المناسبة التجريبية ، ثم يستمر توليد وتركيب أخيرا منحنيات الانتعاش مضان.

قياس معاملات إنتشار عبر ثنائي الفوتون الاسترداد بعد الإسفار Photobleaching

Multi-fluorescence recovery after photobleaching is a microscopy technique used to measure the diffusion coefficient (or analogous transport parameters) ...

measuring-diffusion-coefficients-via-two-photon-fluorescence-recovery

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JoVE Journal

Biology

11.1K Views.  University of Rochester. في هذه المقالة سوف نقوم وصف الإجراء لقياس معامل الانتشار باستخدام متعدد الفوتون مضان الانتعاش بعد photobleaching. سنبدأ من خلال تنظيم ليزر على طول المسار الضوئي للعينة ، وتحديد المعايير المناسبة التجريبية ، ثم يستمر توليد وتركيب أخيرا منحنيات الانتعاش مضان.

Video: Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

ثنائي الفوتون axotomy والوقت الفاصل بين التصوير مبائر يعيش في الأجنة الزرد

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

الوقت الفاصل بين التصوير من الانقسام الميتوزي بعد ترنسفكأيشن سيرنا

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

ثنائي الفوتون المستندة إلى تنشيط ضوئي في الأجنة الحية اسماك الزرد

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

الكمي في الجسم الحي من التفاعلات بروتين G مستقبلات اقترن به الطيفي ثنائي الفوتون الميكروسكوب

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

Photobleaching فحوصات (FRAP و FLIP) لقياس حيوية الكروماتين البروتين في الخلايا الحية الجذعية الجنينية

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

رسم خرائط الانتشار الجزيئي في غشاء البلازما من قبل متعددة الهدف، للبحث عن المفقودين (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

الماوس حفظ بالبرودة والانتعاش الحيوانات المنوية باستخدام · لقد كرو كيت

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1&#160;was developed to enable routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely available code to measure diffusion coefficients of proteins. kICS calculates a time correlation function from a fluorescence microscopy image stack after Fourier transformation of each image to reciprocal (k-) space. Subsequently, circular averaging, natural logarithm transform and linear fits to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS.
First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope. Then, a region of interest (ROI) avoiding intracellular organelles, moving vesicles or protruding membrane regions is selected. The ROI stack is imported into a freely available code and several defined parameters (see Method section) are set for kICS analysis. The program then generates a &#34;slope of slopes&#34; plot from the k-space time correlation functions, and the diffusion coefficient is calculated from the slope of the plot. Below is a step-by-step kICS procedure to measure the diffusion coefficient of a membrane protein using the renal water channel aquaporin-3 tagged with EGFP as a canonical example.

This paper provides a step by step guide to the fluctuation analysis technique k-Space Image Correlation Spectroscopy (kICS) for measuring diffusion coefficients of fluorescently labeled plasma membrane proteins in live mammalian cells.

من السهل قياس إنتشار معاملات EGFP الموسومة البروتينات عن طريق غشاء البلازما ك الفضاء صورة الارتباط الطيفي

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

التعافي من اسماك الزرد الكبار قلوب للتطبيقات عالية الإنتاجية

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

التصوير ثنائي الفوتون من الكالسيوم داخل الخلايا<sup&gt; 2+</sup&gt; المناولة واكسيد النيتريك الإنتاج في البطانية وخلايا العضلات الملساء لعزل الجرذ الأبهر

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...