Bu çalışma Ulusal Sağlık Enstitüleri tarafından kısmen desteklenen Sağlık Hibeler HL-085.887, HL-092.992, S10RR027535 ve MOP-93.676.

1.. Gemi Diseksiyon ve Görüntüleme <ol><li> Martens ve diğ 27 de tarif edildiği gibi iç çocuk domuz Hasat doku. Bir polidimetilsiloksanın (PDMS) HEPES fizyolojik tuzlu su solüsyonu (PSS) tamponlu içeren alt diseksiyon çanak içine hasat domuz sağ ventrikülleri yerleştirin. <ol><li> Bir stereomikroskopta yardımıyla, incelemek ve çevre kardiyak doku katmanlarından damar segmentini kaldırarak forseps ve yaylı makas kullanılarak çevreleyen dokudan koroner arter (~ 8 mm uzunluk, 0.5 mm çap) sol ön inen bir bölümünü kaldırın. NOT: damar duvarı delmek değil dikkat edin. </li><li> Diseksiyon çanak içine PDMS bloğu (1 x 0.5 x 0.5 cm) yerleştirin, ve alt iğnelemekle bir iğne kullanın. Mikro iğnelerin şekillendirme, oniki ~ 0.3 cm uzunluk kesimleri içine yaklaşık 40 mikron çaplı tungsten tel kesme ve çanak bu yerleştirin. Forseps kullanılarak, bir micropi ile blok damar segmentinin bir ucunu güvenlin. </li><li> Dikkatle damar lümenine küçük yaylı makas yerleştirin ve tamamen açmak için geminin bir tarafında aşağı uzunlamasına kesilir. Endotel yukarıya açılan damar segmentini yönlendirin. </li><li> Damar düz bir dikdörtgen oluşturur, öyle ki engellemek için açılan damar segmentinin sınırları güvence altına almak için, geri kalan mikro iğnelerin kullanın. NOT: Micropin üstleri 90 ° eğildi ve blok yüzeyi ile aynı hizada kadar takılmalıdır. Care gerilmeden olmadan damar hazırlık uzatmak için alınmalıdır; Nihai genişliği ~ başlangıç ​​esnetilmemiş genişliğini 1.5x edilmelidir. </li><li> 134 NaCl, KCl 6: a HEPES içinde pluronik (% 0.03) ile DMSO içinde çözülmüş Fluo-04:00 (10 uM) karıştırılarak Fluo-04:00 yükleme çözeltisi küçük bir hacmi (~ 1 ml) hazırlayın mM ihtiva eden PSS (tamponlu , 1 MgCİ, 10 HEPES, 10 glukoz) ve karanlıkta oda sıcaklığında yaklaşık 40 dakika boyunca yükleme çözeltisi içine bütün bir blok yerleştirin. </li><li> Yükleme işleminden sonra, HEPES tamponlu blok yıkayın5-10 dakika için PSS. </li><li> HEPES PSS&#39;ye tamponlu içeren bir coverglass alt odasına 50-100 mikron kalınlığında ayırıcılar üzerine bloğunu monte edin. NOT: Metal pimler ayırıcılar olarak kullanılan ve damar segment aşağı bakacak ve aralıklarına değmiyor emin olabilir. </li><li> Konfokal görüntüleme için donatılmış bir inverted mikroskop sahnede odasına yerleştirin ve endotel hücre tabakası üzerine odaklanır. </li><li> ~ 3 20X büyütme dakika ve konfokal görüntü sırası aquisition yazılımını kullanarak saniyede ~ 8 kare hızı için bazal göreceli floresan yakalama zaman atlamalı görüntü dizileri. </li><li> Bazal floresans kayıt ~ 3 dakika sonra, bir HEPES addtional 3 dakika için aynı tampon HEPES içinde PSS çözündürüldü, Madde P (100 pM) hacimle (~ 1 ml), ve bir kayıt ile PSS tamponlu çözelti değiştirin. </li></ol></li></ol> 2.. Otomatik Analizi <ol><li> 8 bit, g konfokal toplama yazılımı floresan kalsiyum aktivitesinin görüntü dizisi (ler) RenderHiçbir ölçekli bilgi rayscale &#39;. tif&#39; formatındaki dosyalar. </li><li> Açık ImageJ, dosya menüsünde &#39;açık&#39; tıklayın ve ImageJ görüntü dizisi (ler) görüntülemek için explorer penceresinde uygun görüntü dizisini seçin. </li><li> Görüntü sekans (lar) içinde faaliyet mekansal yayılma üst ve alt sınırları tahmin etmek için dikdörtgen ROI aracını kullanarak, uygun bir ROI çapı belirler. NOT: ortalama dikdörtgen çapı ROI çapı için uygun bir seçimdir. </li><li> Bilgisayarın sabit sürücüsünde yeni bir klasör oluşturun ve klasörü dizine görüntü dizisi (s) ekleyebilirsiniz. </li><li> ImageJ, &#39;Eklentiler&#39; penceresini tıklayın, ardından analizi başlatmak için &#39;LC_Pro tıklayın. </li><li> ROI çap değerini girin ve filtre eşik değerini (0.05 veya 0.01) seçin. </li><li> &#39;Ilaç tedavisi&#39; onay kutusunu tıklayın ve hemen önce ve sonra ilaç ilave edildi (saniye) zaman noktası değerleri girin. </li><li> &#39;Tamam&#39; düğmesini tıklayın vedosya gezgini penceresine görüntü dizisi dizini girin. </li></ol> 3.. Grafik Çıktı <ol><li> Den R sürüm 3.0.2 indir <a href="http://www.r-project.org/">http://www.r-project.org/</a> . </li><li> R. 32 bit sürümünü açın </li><li> &quot;Dosya&quot;, daha sonra &#39;açık komut tıklayın ve grafiksel deney raporları oluşturmak için&#39; traceplot.R &#39;R komut seçin. </li><li> &#39;Paketler&#39; seçerek kalibre ve gplots paketleri yüklemek, &#39;paketleri yüklemek&#39;, ve uygun sistemlerin paketleri seçerek. </li><li> &#39;Change directory&#39; komutu ardından, &#39;dosya&#39; tıklayın ve LC_Pro analiz çıkışına karşılık dizini seçmek için kaşif penceresini kullanın. </li><li> Komut penceresinde tıklatın ve sonra da &#39;çalışan tüm&#39; komut dosyasını çalıştırmak için komut. </li></ol>

<ol>
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Yazarlar ifşa hiçbir şey yok.

Hücresel ve çok hücreli düzeyde karmaşık Ca 2 + sinyalleri çözme titiz deneysel ve analitik yaklaşımlar gerektirir. Burada, bir yaklaşım Ca 2 + bağımlı floresans çözülmesi konfokal resim dizileri sunulan spesifik durumda sağlam hücre alanları içinde istatistiksel olarak ilgili Ca 2 + sinyalleri tespit eder ve miktarını belirler otomatik analizine tabi tutulur ve bu süre de açıklanan, bir arter kademeli bir izole edildi domuz kalbinden, tutturulmuş konfokal gö...

Ca 2 + molekülü ve sitozolik Ca 2 + düzeyleri yüksek regüle edilen sinyalizasyon bir yerde ikinci bir elçisidir. Hücre içi Ca 2 + sinyalleri karmaşık ve izole geçişlerini, salınımlar ve dalgalar 1 çoğaltım dahil - 4. Ca 2 + mekansal ve zamansal kontrol Fizyolojik sinyal özgüllüğünü altında yatan düşünce, ve bu nedenle Ca 2 + sinyal şekillerinin analizi birden fazla alanlarda 5 araştırmacılara büyük ilgi olduğunu. Bu tür Flu-4 ve Fura-2 ile Ca 2 + göstergesi boyalar genel olarak 5-12 floresan mikroskobu ile hücre içi Ca2 + sinyalleri ölçmek için kullanılır. 16 - Genellikle, geçici Ca 2 + sinyalleri zamana bağımlı kullanıcı tanımlı bir alanda ortalama floresan değişiklikler, ya da ilgi bölgesi (ROI) 5,6,13 olarak değerlendirilir. Şu anda, manuel ROI analizi zaman alıcı ve emek hem de bir19 - birçok İB&#39;leri belirlemek ve tekrarlayan hesaplamaları 17 gerçekleştirmek için kullanıcılar gerektirir çünkü da oldukça kapsamlıdır. Bu teknikler aynı zamanda yapay sinyal modları ve düşük genlikli veya yaygın sinyallerinin 18,20 dışlanma tanıtımı dahil olmak üzere, önemli kullanıcı hatası tabi olabilir. , Otomatik ROI algılama algoritmaları önceden optimum ROI yerleşimi belirlemek için istatistiksel yaklaşımlar çeşitli kullanarak uygulanan olmuştur, ama genellikle çizgi tarama ya da sözde-line zaman 17 tek bir mekansal boyut analizi kısıtlar tarama görüntüleri analiz sınırlı olmuştur 19-22. Ayrıca, birçok mevcut algoritmaları Ca periyodik, lokalize transientler dan yayılan dalgalar 23,24 aralığında 2 + serbest olaylar çeşitliliğini kapsayacak şekilde yeterli değildir. Fizyolojik Ca 2 + sinyalleri kapsamlı değerlendirme genellikle daha önemli görüntü artif varlığı ile komplikebirçok deneysel sistemlerde gürültü ayrımcılığa sinyalini karıştıran hareket. Daha önce, Ca otomatik ROI algılama algoritması çözüm 2 + (Sağlık, Bethesda, MD National Institutes), gelişmiş ve 25,26 valide NIH ImageJ yazılım için bir eklenti olarak uygulanan geçici algılama sinyal. LC_Pro denilen Bu algoritma, iki-boyutlu zaman atlamalı görüntü dizileri Ca 2 + sinyal geçişlerini kapsayan İB&#39;leri tanımlamak ve analiz etmek için tasarlanmıştır. İşte pratik bir deneysel protokol ve domuz koroner arter endotelyumda algoritmanın bir uygulama temsili gösteri kullanılabilir grafik çıktısı oluşturmak için açık kaynak istatistiksel işleme yazılım Ar kullanarak ek komutlar ile sağlanır.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
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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

Hücre içi Ca 2 + sinyalleri sık floresan Ca 2 + göstergesi boyalar ve mikroskopi teknikleri ile incelenmiştir. Bununla birlikte, Ca 2 + görüntüleme de niceliksel veri analizi, zaman alıcı ve önyargı tabidir. Ilgi (ROI) algılama bölgeye dayalı otomatik sinyal analizi algoritmaları tek boyutlu çizgi tarama ölçümleri için uygulanan olmuştur, ama iki-boyutlu görüntü dizilerinin optimize kimlik ve İB&#39;nin analizini entegre hiçbir geçerli algoritma yoktur. Burada, resim dizilerinin hızlı elde edilmesi ve ROI analizi için bir algoritma tarif edilmektedir. Bu elips optimum ROI yerleşimini belirlemek amacıyla gürültü filtre sinyallerine uygun kullanır, ve genlik, süre ve mekansal yayılma Ca 2 + sinyal parametrelerini hesaplar. Bu algoritma ImageJ (NIH) bir yazılım için serbestçe kullanılabilir eklenti olarak hayata geçirildi. Birlikte açık kaynak istatistiksel işleme yazılım Ar için yazılmış analizi komut ile,Bu yaklaşım, deneysel çıktı hızlı istatistiksel analiz yapmak için yüksek kapasiteli bir boru hattı sağlar. Yazarlar bu analiz protokolün kullanımı fizyolojik Ca 2 + sinyal bir daha tam ve tarafsız karakterizasyonu yol açacağını göstermektedir.

Özel bir algoritma, LC_Pro, gelişmiş ve Ca otomatik analizini gerçekleştirmek amacıyla hayata geçirildi konfokal görüntü dizileri üzerinde 2 + dinamikleri. Şekil 1 &#39;de gösterildiği gibi, algoritma A) algılar ve dinamik Ca parça siteleri ardışık işleme modülleri kullanır 2 + &lt;0.01), gürültü, B) aktif bölge merkezlerinde otomatik olarak (ROI) bölgeleri tanımlar ve istatistiksel (p yukarıda değiştirmek C) Belirli bir olay parametreleri belirlemek ...

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

Burada iki boyutlu zaman atlamalı görüntü dizileri içinde olumlu sinyal bölgelerine atanan en uygun elips sıralama dayanan faiz analiz protokolünün yeni bir bölge gösterilmiştir. Bu algoritma kapsamlı az kullanıcı girişi ve önyargı ile fizyolojik Ca 2 + sinyalleri analiz müfettişleri sağlayabilir.

Dinamik Ca Otomatik Analizi<sup&gt; 2 +</supGörüntü Dizilerinde&gt; Sinyaller

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 Görüntüleme.  University of South Alabama.  Burada iki boyutlu zaman atlamalı görüntü dizileri içinde olumlu sinyal bölgelerine atanan en uygun elips sıralama dayanan faiz analiz protokolünün yeni bir bölge gösterilmiştir. Bu algoritma kapsamlı az kullanıcı girişi ve önyargı ile fizyolojik Ca 2 + sinyalleri analiz müfettişleri sağlayabilir. 

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

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

Kronik Deneyleri Montaj Elektrotlar veya mikrodiyaliz Problar Işık tedavi Diş Yapıştırıcı Reçine Uygulama

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

Biyoloji

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.

Küçük Kalpler Yarı-otomatik Optik Heartbeat Analizi

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.

Mozaik Zebra balığı Transgenesis Enhancer Diziler değerlendirilmesi için

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.

Bitkisel Dokular Gen İfade Araştırmalar Robotik ve Dinamik Görüntü Analizi

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

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.

Görüntüleme Ca Teknikleri 2 + İnsan Sperm sinyal

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

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent 
proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest1, recent 
developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association 2, 3. One small 
tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH 
(green fluorescent), with high specificity even in live cells 2. The TC/biarsenical dye system offers far less steric constraints to the host protein than 
fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions 4-7. We recently developed 
a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington 
disease 7. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only 
binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer 
content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) 
with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).

The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.

ReAsH / Flash Etiketleme ve Hücreler Tetracysteine ​​Sensör Proteinlerin Görüntü Analizi

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells.  While fluorescent 
proteins ...

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.

Ölçme Mekansal ve zamansal Ca<sup&gt; 2+</supArabidopsis Bitkilerde&gt; Sinyalleri

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.

Görüntüleme Yerel Ca<sup&gt; 2+</sup&gt; Kültürlenmiş memeli hücrelerinde sinyaller

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.

Yüksek çözünürlüklü Time-lapse Görüntüleme ve Yaşayan İnsan Umbilikal Ven Endotelyal Hücrelerinde Mikrotubul Dinamiği Otomatik Analizi

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

Clock Scan Protocol for Image Analysis: ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a closed or segmented convex-shaped region of interest, leading to the generation of an averaged integral radial pixel-intensity profile. This protocol was originally developed in 2006, as a visual basic 6 script, but as such, it had limited distribution. To address this problem and to join similar recent efforts by others, we converted the original clock scan protocol code into two Java-based plugins compatible with NIH-sponsored and freely available image analysis programs like ImageJ or Fiji ImageJ. Furthermore, these plugins have several new functions, further expanding the range of capabilities of the original protocol, such as analysis of multiple regions of interest and image stacks. The latter feature of the program is especially useful in applications in which it is important to determine changes related to time and location. Thus, the clock scan analysis of stacks of biological images may potentially be applied to spreading of Na+ or Ca++ within a single cell, as well as to the analysis of spreading activity (e.g., Ca++ waves) in populations of synaptically-connected or gap junction-coupled cells. Here, we describe these new clock scan plugins and show some examples of their applications in image analysis.

This paper describes two novel ImageJ plugins for &#39;Clock Scan&#39; image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software package.

Görüntü Analizi için Saat Tarama Protokolü: ImageJ Eklentileri

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a ...