وأيد هذا العمل في جزء من المعاهد الوطنية للصحة منح HL-085887، HL-092992، S10RR027535، وMOP-93676.

1. تشريح السفن والتصوير <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الحصاد الأنسجة من الخنازير الأحداث المحلية كما هو موضح في مارتنز وآخرون 27. وضع تحصد البطينين الأيمن الخنازير في polydimethylsiloxane (PDMS) طبق تشريح الجزء السفلي تحتوي على HEPES مخزنة محلول ملحي الفسيولوجية (PSS). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مع المعونة من stereomicroscope، تشريح وإزالة جزء من الأمامي الأيسر النازل الشريان التاجي (~ 8 مم طول، و 0.5 مم) من الأنسجة المحيطة باستخدام ملقط ومقص الربيع عن طريق إزالة جزء من السفينة المحيطة طبقات أنسجة القلب. ملاحظة: الحرص على عدم ثقب جدار الوعاء الدموي. </li><li style=";text-align:right;direction:rtl"> وضع كتلة PDMS (1 × 0.5 × 0.5 سم) في طبق تشريح، واستخدام إبرة ليعلقون عليه إلى أسفل. قطع قطرها سلك التنغستن حوالي 40 ميكرون الى اثني عشر قطاعات ~ 0.3 سم طول، وتشكيل micropins، ووضع هذه في الطبق. باستخدام ملقط، وتأمين واحدة من نهاية الجزء السفينة إلى كتلة مع micropiن. </li><li style=";text-align:right;direction:rtl"> إدراج بعناية مقص الربيع صغير في تجويف السفينة، وقطع طولي أسفل جانب واحد من السفينة لفتحه تماما. توجيه جزء السفينة فتحت مع البطانة المباراة. </li><li style=";text-align:right;direction:rtl"> استخدام micropins المتبقية لتأمين حدود قطاع سفينة فتح لمنع مثل هذه أن السفينة تشكل مستطيل مسطح. ملاحظة: يجب أن تكون عازمة قمم Micropin إلى 90 درجة وإدراجها حتى الاحمرار مع سطح الكتلة. ينبغي توخي الحذر لتمتد إعداد السفينة دون إرهاق؛ عرض النهائي ينبغي ~ 1.5X عرض المتمدد البدء. </li><li style=";text-align:right;direction:rtl"> إعداد وحدة تخزين صغيرة (~ 1 مل) من فلوو-4 الحل AM التحميل عن طريق خلط فلوو-4 AM (10 ميكرومتر) الذائبة في DMSO مع بلورونيك (0.03٪) في HEPES مخزنة PSS (تحتوي في ملي: 134 كلوريد الصوديوم، 6 بوكل ، 1 MgCl، 10 HEPES، 10 الجلوكوز)، وإدراج كتلة بأكملها في حل التحميل لحوالي 40 دقيقة في درجة حرارة الغرفة في الظلام. </li><li style=";text-align:right;direction:rtl"> بعد التحميل، وغسل كتلة في HEPES مخزنةPSS لمدة 5-10 دقيقة. </li><li style=";text-align:right;direction:rtl"> جبل كتلة 50-100 ميكرون على الفواصل سميكة في غرفة أسفل ساترة تحتوي على HEPES مخزنة PSS. ملاحظة: دبابيس معدنية يمكن أن تستخدم الفواصل وضمان تواجه قطاع السفينة إلى أسفل وعدم لمس الفواصل. </li><li style=";text-align:right;direction:rtl"> وضع الغرفة على مرحلة مجهر مقلوب مجهزة للتصوير متحد البؤر، والتركيز على طبقة الخلايا البطانية. </li><li style=";text-align:right;direction:rtl"> تسلسل التقاط صورة مرور الوقت مضان النسبي القاعدية ل~ 3 دقائق في 20X التكبير ومعدل الإطار ل~ 8 إطارات في الثانية باستخدام صورة متحد البؤر البرمجيات aquisition التسلسل. </li><li style=";text-align:right;direction:rtl"> بعد ~ 3 دقائق من تسجيل مضان القاعدية، استبدال HEPES مخزنة حل PSS مع نفس الحجم (~ 1 مل) من مادة P (100 م) المذاب في HEPES مخزنة جهاز الأمن الوقائي، وسجل لالإضافيون 3 دقائق. </li></ol></li></ol> 2. التحليل الآلي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تقديم تسلسل الصور (ق) من النشاط الكالسيوم الفلورسنت من برنامج الحصول متحد البؤر إلى 8 بت، زrayscale &#39;. TIF&#39; شكل ملفات مع عدم وجود المعلومات على نطاق و. </li><li style=";text-align:right;direction:rtl"> يماغيج مفتوحة، انقر فوق &#39;فتح&#39; في القائمة ملف، وحدد تسلسل الصور المناسبة في نافذة المستكشف لعرض تسلسل الصور (ق) في ImageJ. </li><li style=";text-align:right;direction:rtl"> تحديد العائد على الاستثمار يبلغ قطرها المناسب باستخدام أداة مستطيلة العائد على الاستثمار لتقدير حدود العلوي والسفلي من انتشار المكاني للنشاط ضمن تسلسل الصور (ق). ملاحظة: قطر مستطيل متوسط ​​هو اختيار مناسب لقطر العائد على الاستثمار. </li><li style=";text-align:right;direction:rtl"> إنشاء مجلد جديد على القرص الصلب لجهاز الكمبيوتر، وإضافة تسلسل الصور (ق) إلى الدليل المجلد. </li><li style=";text-align:right;direction:rtl"> في ImageJ، انقر فوق نافذة &#39;الإضافات&#39;، ثم انقر &#39;LC_Pro&#39; لبدء التحليل. </li><li style=";text-align:right;direction:rtl"> أدخل القيمة قطر العائد على الاستثمار وتحديد قيمة العتبة تصفية (إما 0.05 أو 0.01). </li><li style=";text-align:right;direction:rtl"> انقر على &#39;العلاج من تعاطي المخدرات&#39; مربع الاختيار وأدخل القيم نقطة زمنية على الفور قبل وبعد وأضيف المخدرات (بالثواني). </li><li style=";text-align:right;direction:rtl"> انقر &#39;موافق&#39;، وأدخل الدليل تسلسل الصور في إطار ملف اكسبلورر. </li></ol> 3. إخراج الرسومية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحميل النسخة 3.0.2 من R <a href="http://www.r-project.org/">http://www.r-project.org/</a> . </li><li style=";text-align:right;direction:rtl"> فتح إصدار 32 بت من ر. </li><li style=";text-align:right;direction:rtl"> انقر على زر &quot;ملف&quot;، ثم &quot;النصي فتح &#39;وحدد&#39; traceplot.R &#39;R النصي لتوليد التقارير الرسومية التجريبية. </li><li style=";text-align:right;direction:rtl"> تثبيت معايرة، وgplots الحزم عن طريق تحديد &quot;حزم&quot;، &quot;تثبيت حزم&quot;، واختيار حزم appropirate. </li><li style=";text-align:right;direction:rtl"> انقر &#39;ملف&#39;، ثم &#39;تغيير الدليل&#39; الأوامر، واستخدام نافذة المستكشف لتحديد الدليل الذي يتوافق مع الإخراج تحليل LC_Pro. </li><li style=";text-align:right;direction:rtl"> انقر على إطار البرنامج النصي ثم &#39;تشغيل جميع&#39; الأوامر لتشغيل البرنامج النصي. </li></ol>

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والكتاب ليس لديهم ما يكشف.

سوف فك معقدة كا 2 + إشارات على المستوى الخلوي وتتطلب نهجا متعددة الخلايا التجريبية والتحليلية الصارمة. هنا، يوصف هذا النهج في الوقت الذي تتعرض حل تسلسلات الصور متحد البؤر كا 2 + مضان تعتمد على تحليل الآلية التي تحدد والكمي كا 2 + إشارات ذات الصلة إحصائي...

كا 2 + هو رسول الثاني في كل مكان مما يشير جزيء وعصاري خلوي كا 2 + وينظم مستويات عالية. الكالسيوم داخل الخلايا 2 + إشارات معقدة وتشمل العابرين معزولة، التذبذبات، ونشر موجات 1-4. ويعتقد أن السيطرة المكانية والزمانية من كا 2 + لخصوصية تكمن وراء الفسيولوجية إشارة، وبالتالي فإن تحليل كا 2 + أنماط إشارة هو من مصلحة كبيرة للمحققين في حقول متعددة 5. الأصباغ كا 2 + المؤشر مثل فلوو-4-2 FURA وتستخدم عادة لقياس الكالسيوم داخل الخلايا 2 + إشارات مع مضان المجهري 5-12. عادة، يتم تقييم الزمانية كا 2 + إشارات عن التغيرات التي تعتمد على الوقت في متوسط ​​مضان داخل منطقة المعرفة من قبل المستخدم، أو المنطقة ذات الاهتمام (ROI) 5،6،13 - 16. حاليا، وتحليل العائد على الاستثمار دليل على حد سواء مضيعة للوقت والعمل فيتوتري لأنه يتطلب من المستخدمين لتحديد العديد من رويس وأداء العمليات الحسابية المتكررة 17-19. قد تكون هذه التقنيات أيضا عرضة للخطأ المستخدم كبيرة، بما في ذلك إدخال وسائط إشارة مصطنعة والاستبعاد من انخفاض السعة أو إشارات منتشر 18،20. وقد سبق أن نفذت خوارزميات الكشف الآلي العائد على الاستثمار باستخدام مجموعة متنوعة من النهج الإحصائية لتحديد العائد على الاستثمار الأمثل التنسيب، لكنها عموما اقتصرت على تحليل مسح سطر أو الزائفة خط مسح الصور، مما يحد من التحليل إلى البعد المكاني واحدة في الوقت المناسب 17، 19-22. بالإضافة إلى ذلك، العديد من الخوارزميات الموجودة ليست كافية لتشمل تنوع كا 2 + الأحداث الإفراج التي تتراوح من الدوري، العابرين المترجمة إلى نشر موجات 23،24. وغالبا ما تتعقد تقييم شامل للالفسيولوجية كا 2 + إشارات إضافية من خلال وجود صورة كبيرة artifالتصرف الذي يفند إشارة إلى التمييز الضوضاء في كثير من النظم التجريبية. سابقا، للكشف عن العائد على الاستثمار خوارزمية الحل الآلي لكا 2 + إشارة الكشف عابرة، كما تنفذ البرنامج المساعد لبرنامج NIH يماغيج (المعاهد الوطنية للصحة في بيثيسدا، MD)، تم تطويره والتحقق من صحتها 25،26. وقد تم تصميم هذه الخوارزمية، ودعا LC_Pro، لتحديد وتحليل رويس يشمل كا 2 + العابرين في إشارة ثنائية الأبعاد الوقت الفاصل بين متواليات الصورة. هنا يتم توفير بروتوكول التجريبية العملية ومظاهرة ممثل تطبيق الخوارزمية في الخنازير الشريان التاجي البطانة، مع تحليل نتائج إضافية باستخدام مفتوحة المصدر برامج معالجة الإحصائية R لتوليد الناتج رسومية قابلة للاستخدام.

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			<td height="21" style="height:21px;width:216px;">dissection dish</td>
			<td style="width:71px;">Fisher Sci</td>
			<td style="width:119px;">#08-772-70</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polydimethylsiloxane (PDMS)</td>
			<td style="width:71px;">Fisher Sci</td>
			<td style="width:119px;">#NC9644388</td>
			<td>elastomer kit, must be molded into dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES-buffered PSS</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">#H3375-250G</td>
			<td>HEPES acid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">stereomicroscope</td>
			<td style="width:71px;">Nikon Inst.</td>
			<td style="width:119px;">#MNA42000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">forceps</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">#11223-20</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">spring scissors</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">15003-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">tungsten wire</td>
			<td style="width:71px;">Scientific Inst Svcs</td>
			<td style="width:119px;">#406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluo-4 AM</td>
			<td style="width:71px;">Life Tech.</td>
			<td style="width:119px;">#F-14201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pluronic F-127</td>
			<td style="width:71px;">Life Tech.</td>
			<td style="width:119px;">#P3000MP</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">metal pins</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">#26002-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cover-glass bottom chamber</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Custom designed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">spinning disc confocal microscope</td>
			<td style="width:71px;">Perkin Elmer</td>
			<td style="width:119px;">RS-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">imageJ software</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="http://rsbweb.nih.gov/ij/download.html">download at: http://rsbweb.nih.gov/ij/download.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LC_Pro plugin for imageJ</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="http://rsbweb.nih.gov/ij/plugins/lc-pro/index.html">download at: http://rsbweb.nih.gov/ij/plugins/lc-pro/index.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">R software</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="http://www.r-project.org/">download at: http://www.r-project.org/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">R traceplot script</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="https://docs.google.com/file/d/0B-PSp1D9e2fjV3NIcGppUzkxdEk/edit?usp=sharing">download at: https://docs.google.com/file/d/0B-PSp1D9e2fjV3NIcGppUzkxdEk/edit?usp=sharing</a></td>
		</tr>
	</tbody>
</table>

automated analysis of dynamic ca2+ signals in image sequences

يتم دراسة الخلايا كا 2 + إشارات عادة مع الأصباغ الفلورية كا 2 + المؤشر وتقنيات الفحص المجهري. ومع ذلك، والتحليل الكمي من كا 2 + بيانات التصوير هو مضيعة للوقت وتخضع للانحياز. وقد تم تنفيذ الآلية خوارزميات تحليل الإشارات يعتمد على المنطقة ذات الاهتمام (ROI) الكشف عن قياسات سطر واحد الابعاد المسح الضوئي، ولكن ليس هناك خوارزمية الحالي الذي يجمع بين تحديد الأمثل وتحليل رويس في تسلسلات الصور ثنائية الأبعاد. هنا يتم وصف خوارزمية لسرعة اقتناء وتحليل رويس في تسلسلات الصور. فإنه يستخدم الحذف يصلح لإشارات الضجيج تصفيتها من أجل تحديد العائد على الاستثمار الأمثل التنسيب، ويحسب كا 2 + المعلمات إشارة من السعة والمدة وانتشار المكاني. تم تنفيذ هذه الخوارزمية باعتباره المساعد متاحة بحرية لليماغيج (NIH) والبرمجيات. جنبا إلى جنب مع البرامج النصية تحليل مكتوب لمفتوحة المصدر برامج معالجة الإحصائية R،ويوفر هذا النهج خط أنابيب ذات قدرة عالية لأداء التحليل الإحصائي السريع للناتج التجريبية. كما يقترح الباحثون أن استخدام هذا البروتوكول التحليل سوف يؤدي إلى توصيف أكثر اكتمالا وغير منحازة من الفسيولوجية كا 2 + إشارات.

خوارزمية مخصصة، LC_Pro، تم وضعها وتنفيذها من أجل إجراء تحليل الآلي للكا 2 + ديناميات على تسلسلات الصور متحد البؤر. كما هو مبين في الشكل 1، ويستخدم خوارزمية وحدات معالجة متتابعة أن A) كشف وتعقب المواقع الديناميكية كا 2 + تغيير أعلاه إحصائية (ع &lt;0.01) الضو?...

Watch this Scientific Journal Video about Automated Analysis of Dynamic Ca2+ Signals in Image Sequences at JoVE.com

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

هنا يتجلى منطقة جديدة من بروتوكول تحليل الفائدة على أساس الفرز الحذف أفضل تناسب المخصصة لمناطق إشارة إيجابية خلال فترة زمنية ثنائي الأبعاد تسلسلات الصور الفاصل. هذه الخوارزمية قد تمكن المحققون على تحليل شامل الفسيولوجية كا 2 + إشارات مع الحد الأدنى من إدخال المستخدم والتحيز.

التحليل الآلي من ديناميك كا<sup&gt; 2 +</sup&gt; إشارات في تسلسلات الصور

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

automated-analysis-of-dynamic-ca2-signals-in-image-sequences

Research

JoVE Journal

Biology

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

17.0K Views.  University of South Alabama.  هنا يتجلى منطقة جديدة من بروتوكول تحليل الفائدة على أساس الفرز الحذف أفضل تناسب المخصصة لمناطق إشارة إيجابية خلال فترة زمنية ثنائي الأبعاد تسلسلات الصور الفاصل. هذه الخوارزمية قد تمكن المحققون على تحليل شامل الفسيولوجية كا 2 + إشارا?...

Video: Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

تنسيق كا 2 + الكشف العابر في العضلات الملساء

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

شبه الآلي التحليل البصري نبضات القلوب الصغيرة

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non coding sequences. One prominent function carried out by the non coding fraction of the genome is to regulate gene transcription; however, there are no effective methods to broadly predict cis-regulatory elements from primary DNA sequence. We have developed an efficient protocol to functionally evaluate potential cis-regulatory elements through zebrafish transgenesis. Our approach offers significant advantages over cell-culture based techniques for developmentally important genes, since it provides information on spatial and temporal gene regulation. Conversely, it is faster and less expensive than similar experiments in transgenic mice, and we routinely apply it to sequences isolated from the human genome. Here we demonstrate our approach to selecting elements for testing based on sequence conservation and our protocol for cloning sequences and microinjecting them into zebrafish embryos.

We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.

فسيفساء Transgenesis اسماك الزرد لتقييم متواليات محسن

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

الروبوتات والتحليل الديناميكي صورة للدراسات في التعبير الجيني في أنسجة النبات

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Development of automated imaging and analysis for zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

تطوير التصوير والتحليل الآلي للشاشات الكيميائية الزرد.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

تقنيات التصوير كاليفورنيا 2 + في اشارة الى الحيوانات المنوية البشرية

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

قياس المكانية والزمانية كاليفورنيا<sup&gt; 2+</sup&gt; إشارات في النباتات نبات الأرابيدوبسيس

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

التصوير المحلية الكالسيوم<sup&gt; 2+</sup&gt; الإشارات في خلايا الثدييات مثقف

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

عالية الدقة التصوير الوقت الفاصل بين وتحليل الآلي للأنيبيب حيوية في المعيشة السري الإنسان خلايا الوريد غشائي

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

واجهة المستخدم الرسومية لتتبع بمساعدة برنامج تركيز البروتين في النتوءات الخلوية الديناميكية

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...