Bu çalışma, Umut Scholar Ödülü Savunma Era Bölümü (No W81XWH-05-0396) ve Edward B. Brown III Biyomedikal Bilimler Ödülü Pew Scholar tarafından finanse edildi.

1. Optik hizalayın. MP-sıkı bağlamak deney için önemli ekipman şunlardır: mod-kilitli bir lazer kaynağı, Pockel Hücre (ışın modülasyon), nabız jeneratör, renk değişikliği, objektif lens, floresan emisyon filtresi, kapılı photomultiplier tüp, ve bir veri kayıt sistemi (foton sayacı ve çok kanallı ölçekleyici). 2. Güvenli monitör yetkilerini belirler. <ol><li> Örnek içinde floresan üretmek için düşük, ama makul, güç lazer zayıflatır. </li><li> Örnek içinde odaklanın. </li><li> Beklenen floresan kurtarma daha çok uzun bir zaman ölçeği üzerinde entegre bir foton sayacı ayarlayın. Mikroskop yazılım odak hacmi (bu bir nokta tarama ile elde edilir) numune içinde sabit tutulan makul bir sayıda foton sayımı verileri kaydeder. </li><li> Gücünü artırmak ve foton sayımı verileri başka bir dizi çekmek. Foton sayılarında artış gücünü artırmak açısından önemli ölçüde azaltmak için görünene kadar tekrarlayın. </li><li> Arsa log (güç) bir fonksiyonu olarak log (foton sayısı). Monitör sırasında beyazlatma iki yamacında bir sapma gösterir. Monitörün güç olarak bu cut off bir güç seçin, ancak makul bir sinyal gürültü oranı sağlar. </li></ol> 3. Pulse jeneratör ve çok kanallı ölçekleyici giriş parametreleri belirleyin. <ol><li> Belirleyin geri-of-the-zarf edebiyat ve / veya Stokes-Einstein denklemi, difüzyon denklemi dayalı difüzyon katsayısı ve iyileşme süresi tahminleri. </li><li> Darbe jeneratörü ve ölçekleyici gerekli parametre değerleri ayarlayın. <ol class="lalpha"><li> Darbe jeneratörü, önceden çamaşır suyu ve ağartıcı kez ayarlayın. Başparmak kuralları iyi bir ön-beyazlatıcı beklenen yarısında iyileşme süresi daha 1-2 kat daha büyük olması gerektiğini ve çamaşır suyu beklenen yarısında iyileşme süresi onda biri olmalıdır. </li><li> Ölçekleyici, yaklaşık bir yarım çamaşır suyu süresi bin genişliği ve kayıt başına kutularının sayısını ayarlamak seçtiğiniz bin genişliği ve kayıt başına kutuları ürün beklenen tam iyileşme artı ön yaklaşık eşit veya daha büyük olduğu gibi -Çamaşır suyu ve ağartıcı kez. </li><li> Darbe jeneratörü dönün ve sadece bin genişliği kez kayıt başına kutuların sayısı ürünü ters daha küçük bir değere eşit pre-bleach/bleach/monitor dizisi frekansını ayarlamak. * </li><li> Son olarak, kayıtların sayısını ayarlamak / seçtiğiniz monitör güçte sinyal yoğunluğuna göre ölçekleyici tarama. * Darbe jeneratör tam bir pre-bleach/bleach/monitor dizisi (1/frequency) için zaman ölçekleyici veri toplama süresi (bin genişlik x kutuları / kayıt) daha fazla olduğu çok önemlidir. Bu kriterleri yerine değilse, birden fazla ağartıcı tetikler ölçekleyici tek bir kayıt içinde görünebilir. </li></ol></li><li> Monitörün güç, önceden belirlenmiş kabul edilebilir bir seviyeye ayarlayın. Monitör beyazlatma test sırasında belirlenen beyazlatma kesim üzerinde 200 mW veya çamaşır suyu gücünü ayarlayın. Bu güçler PC kristal boyunca farklı gerilim olarak ayarlanır, her ikisi de. </li><li> Mikroskop Seal, ışıkları kapatmak PMT açmak, mikroskop yazılımı üzerinde bir nokta tarama başlamak, sonra darbe jeneratörü ve ölçekleyici başlar. </li><li> 1) ön-bleach floresan, 2) floresan hemen post-çamaşır suyu, ve 3) veri setinin sonunda floresan: eğrisinin çizilmesi ve üç önemli nokta işaretleme çıkan kurtarma eğrisi analiz edin. Floresans değerleri öncesi ve hemen sonrası yarı-iyileşme süresi daha iyi tahmin etmek için çamaşır suyu. Kullanın. </li><li> Yarı-iyileşme süresi, bu tahmin, önceden çamaşır suyu, çamaşır suyu için yeni değerlerini belirlemek için daha önce belirtilen başparmak kuralları ve bin genişliği süreleri kullanın. Ayrıca, tam iyileşme elde etmek için gerekli süreyi daha iyi tahmin etmek için veri kümesinin sonunda öncesi ağartıcı floresan ve floresan kullanın. Genel bir kural olarak, veri, verilerin ikinci yarısı için rapor edilmesi tam iyileşme sağlar bir süre için alınmalıdır. </li><li> Eğrisi güçlü bir çamaşır suyu ve pürüzsüz bir iyileşme sergiliyor kadar darbe jeneratörü ve ölçekleyici parametrelerini ayarlayın, yeni bir eğri parametreleri almak ve ince ayar devam. Mümkünse, çamaşır suyu süresi göz önünde bulundurun ve başparmak kural altına düşürülmelidir. Çok sığ, ya da çok derin, çamaşır suyu elde edilir, çamaşır suyu gücü de ayarlanabilir. </li></ol> 4. Test uyarım doygunluk. <ol><li> Önceden belirlenmiş uygun monitör ve çamaşır suyu güçler, MP-sıkı bağlamak eğrilerini kullanarak, bir dizi alır. Uygun matematiksel formu ve doğrusal olmayan en küçük kareler uydurma algoritması (bkz. aşağıdaki &quot;Veri Analizi&quot;) kullanarak, ön ağartıcı floresan normalize her kurtarma, analiz edin. Çamaşır suyu derinliği parametre monte değeri kaydedin.</li><li> MP-sıkı bağlamak eğrileri birbirini izleyen seriler alma ve analiz çamaşır suyu güç değerlerini artırarak devam edin. </li><li> Arsa log (güç) bir fonksiyonu olarak log (çamaşır suyu derinliği parametresi). Herhangi bir sapma, bir yamaç iki uyarma doygunluk gösterir. Güçlü bir çamaşır suyu verimi çamaşır suyu güç seçin, ancak doygunluk neden olmaz. </li></ol> 5. MP-sıkı bağlamak eğrileri çekmeye devam edin. <ol><li> Yukarıdaki gibi, bütün parametreler düzgün koyacaktır, eğriler çekmeye devam edin. </li></ol> 6. Veri Analizi. <ol><li> Floresan veri öncesi, çamaşır suyu ve ağartıcı veri, bu veri seti ve ayarlamak kaldırın t = 0 hemen sonrası-ağartıcı floresan değeri ile karşılık gelir. </li><li> -Bleach öncesi ortalama floresan açısından ortaya çıkan floresan kurtarma Normale. </li><li> Uygun bir matematiksel model ve MATLAB lsqcurvefit fonksiyonu olarak doğrusal olmayan, en küçük kareler uydurma algoritması kullanılarak normalize eğrisi sığdır. Burada sunulan model, difüzyon ve konvektif akışı hem de etkilemiş floresan kurtarma hesapları: <img src="/files/ftp_upload/1636/1636eq.jpg" alt="Denklem" /> F o ön ağartıcı ortalama floresan nerede çamaşır suyu derinliği parametresi, β, τ D karakteristik uzadıya iyileşme süresi, R eksenel radyal (w r) genişlikleri karesine oranı (w z) iki foton odak hacmi, τ vx = w r / v x, τ Vy = w r / v y τ vz = w, z / v z ve v 2 = v x 2 + v y 2 + v, z 2- konvektif akış hızı. Görüntü düzlemi ile sınırlı akışı için, τ vz → ∞ ve 1 / τ vx + 1 / τ Vy 1 / τ vr ile değiştirilebilir. Konvektif akışının olmaması, pay hem de üsteller 1 gidip ifade sadece iki uydurma parametreleri, β ve τ D büyük ölçüde azaltılmış oluyor . Difüzyon katsayısı D = w r 2 / 8τ D olarak kolayca hesaplanır. </li></ol> 7. Temsilcisi Sonuçlar. <img src="/files/ftp_upload/1636/1636fig1.jpg" alt="Şekil 1" /> Şekil 1 Temsilcisi çamaşır suyu ve FITC-BSA için kurtarma eğrisi. Iyi bir iyileşme eğrisi verilerin ikinci yarısında tam bir iyileşme floresan ile, güçlü bir çamaşır suyu ve pürüzsüz bir iyileşme sergiliyor. <img src="/files/ftp_upload/1636/1636fig2.jpg" alt="Şekil 2" /> Şekil 2 FITC-BSA Normalize kurtarma eğrisi (kırmızı) ve en küçük kareler ilgili uyum (siyah). Bu uyum, literatür ile tutarlı, difüzyon katsayısı, 52.9 um2 / s verir.

<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>

Photobleaching sonra çok foton floresan kurtarma gücü 3D çözünürlük ile kalın örnekleri prob yeteneği yatıyor. 1990 yılında gelişme bu yana, MP-sıkı bağlamak hücre gövdeleri, ex vivo kalın bir doku dilimleri ve in vivo doku ve interstisyumda difüzyon katsayısı (ya da analog ulaşım parametreleri) belirlemek için kullanılır olmuştur. Bu makalede, biz, deneysel parametreler ayar verileri alma ve kurtarma eğrileri analiz, MP-sıkı bağlamak gibi deney, ışın yolu hizalamak için uygun bir prosedür çalıştırmak için gerekli ekipman sundu. Uygun bir dalgaboyu ve emisyon filtresi seçimler iki foton kesitler ve emisyon spektrumları tarafından yönlendirilmelidir. Bu bilgiler genellikle, çeşitli boyalar için teknik veriler ile birlikte. Ayrıca, lazer ışınının hizalayarak, bu amaç arka lens uygun doldurulmasını elde edilmesi önemlidir. Bu genellikle bir ışın optik sistem genişletici ekleyerek gerçekleştirilir. Uygun doldurulmasını eksenel ve radyal yönlerde hem de alt çözünürlüklü sabit floresan boncuk tarama ve sonra komplo ve literatür değerleri ile karşılaştırılması için 1 / e 2 farklı genişlik bulmak için Gauss için floresan profilleri montajı tarafından kontrol edilebilir. Uygun izleme ve ağartma güçler seçmek için de önemlidir. Güç izlemek için kullanılan floresan öncesi ve sonrası çamaşır suyu iyi bir sinyal-gürültü oranı için izin yeterince yüksek ağartma kayda değer yol açmayacak kadar düşük olması, ama. Ağartma güç uyarma doygunluk kaçınmak gerekir. MP-sıkı bağlamak deneyde örnek güç olay karesi ile orantılı olan floresan uyarma oranı, bir üst limit vardır. Bu sınır, ikaz doygunluğu rejiminin başlangıç ​​işaretleri. Uyarma doygunluk rejimi faaliyet ağartıcı yetkilerini kullanarak üretilen Floresan kurtarma eğrileri yanlışlıkla düşük difüzyon katsayıları verecektir.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Ti:sapphire laser</td>
<td>&nbsp;</td>
<td>Spectra Physics</td>
<td>Mai Tai</td>
<td>&nbsp;</td>
<tr>
<td>Pockels Cell</td>
<td>&nbsp;</td>
<td>Conoptics</td>
<td>350-80</td>
<td>&nbsp;</td>
<tr>
<td>Laser Scanning Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>Fluoview</td>
<td>&nbsp;</td>
<tr>
<td>Short pass dichroic mirror</td>
<td>&nbsp;</td>
<td>Chroma Technologies</td>
<td>670 DCSX-2P</td>
<td>&nbsp;</td>
<tr>
<td>Photomultiplier tube</td>
<td>&nbsp;</td>
<td>Hamamatsu</td>
<td>HC125-02</td>
<td>&nbsp;</td>
<tr>
<td>Photon counter</td>
<td>&nbsp;</td>
<td>Stanford Research Systems</td>
<td>SR 400</td>
<td>&nbsp;</td>
<tr>
<td>Multi-channel scaler/averager</td>
<td>&nbsp;</td>
<td>Stanford Research Systems</td>
<td>SR 430</td>
<td>&nbsp;</td>
<tr>
<td>Pulse Generator</td>
<td>&nbsp;</td>
<td>Stanford Research Systems</td>
<td>DG 535</td>
<td>&nbsp;</td>
<tr>
<td>FITC-BSA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>--</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

measuring diffusion coefficients via two-photon fluorescence recovery after photobleaching

Photobleaching sonra çok floresan kurtarma makromoleküllerin difüzyon katsayısı (ya da analog ulaşım parametreleri) ölçmek için kullanılan bir mikroskopi tekniği ve hem de uygulanabilir<em&gt; In vitro</em&gt; Ve<em&gt; In vivo</em&gt; Biyolojik sistemleri. Photobleaching sonra çok floresan kurtarma sonra ışın azaltıcı ve izleme photobleached moleküllerin yerine ilgi diffüz bölge dışından hala floresan moleküller olarak floresan yoğun bir lazer flaş kullanmadan floresan örnek bir bölge içinde ilgi photobleaching tarafından yapılır . Biz Pockel Hücre (lazer modülatör) ve örnek lazer tarama kutusu ve objektif lens ile optik yol boyunca lazer ışını hizalayarak gösteri başlayacak. Basitlik için, floresan boya, sulu bir örnek kullanabilirsiniz. Daha sonra uygun deneysel dahil olmak üzere bizim örnek parametreleri, monitör ve ağartma güçler, çamaşır suyu süresi, bin genişlikleri (foton sayma), ve floresan iyileşme süresi belirleyecek. Sonra, kurtarma eğrileri, büyük ölçüde gelişmiş akış için (National Instruments, Austin, Teksas) LabVIEW ile otomatik hale getirilebilir bir süreç ayırdığınız için prosedür anlatacağız. Son olarak, difüzyon katsayısı MATLAB (Mathworks, Natick, MA) gibi yazılımlar kullanarak kolayca programlanabilir en küçük kareler uydurma algoritması kullanılarak uygun bir matematiksel model kurtarma veri sıkıştırılmak suretiyle belirlenir.

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

Bu yazıda, photobleaching sonra çok foton floresan kurtarma kullanarak difüzyon katsayıları ölçmek için prosedür anlatacağız. Biz örnek optik yol boyunca lazer hizalayarak ve uygun deneysel parametrelerin belirlenmesi ile başlayacak, daha sonra devam üreten ve nihayet uydurma floresan kurtarma eğrileri.

İki foton Floresan Kurtarma üzerinden Photobleaching sonra Difüzyon Katsayıları Ölçüm

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

Nanyang Technological University

Research

JoVE Journal

Biology

11.1K Görüntüleme.  University of Rochester. Bu yazıda, photobleaching sonra çok foton floresan kurtarma kullanarak difüzyon katsayıları ölçmek için prosedür anlatacağız. Biz örnek optik yol boyunca lazer hizalayarak ve uygun deneysel parametrelerin belirlenmesi ile başlayacak, daha sonra devam üreten ve nihayet uydurma floresan kurtarma eğrileri.

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.

İki foton axotomy ve zaman atlamalı konfokal görüntüleme canlı zebrafish embriyolar

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

Biyoloji

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.

Zaman atlamalı Görüntüleme siRNA Transfeksiyon sonra Mitoz

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.

Canlı Zebra balığı Embriyolar iki-Foton-Tabanlı fotoaktivasyon

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.

Işıksal Çözülmüş İki foton Mikroskop kullanarak G Protein Coupled Reseptör Etkileşmeleri in vivo Niceleme

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 & 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.

Embriyonik Kök Hücreler Yaşayan Kromatin Protein Dynamics Tedbir Tahliller (sıkı bağlamak ve FLIP) Photobleaching

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.

Çoklu Hedef İzleme (MTT) tarafından Plazma Membran Moleküler Difüzyon Haritalama

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.

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.

I · Cryo Kit kullanarak Fare Sperm Kriyoprezervasyon ve Kurtarma

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.

K-Uzay Görüntü Korelasyon Spektroskopisi kullanarak EGFP-etiketli Plazma Membran Proteinleri Difüzyon Katsayılarının kolay Ölçümü

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.

Yüksek verim Uygulamaları Yetişkin Zebra Hearts Kurtarma

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.

Hücre içi Ca İki foton Görüntüleme<sup&gt; 2+</sup&gt; Bir İzole Sıçan Aort Kullanım ve Endotel Nitrik Oksit Üretimi ve düz kas hücreleri

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