Diese Arbeit wurde durch ein Department of Defense Era of Hope Scholar Award (Nr. W81XWH-05 bis 0396) und eine Pew Scholar in der Biomedical Sciences Award Edward B. Brown III finanziert.

1. Richten Sie die Optik. Die wichtigsten Geräte für einen MP-FRAP-Experiment sind: eines modengekoppelten Laserquelle, Pockels-Zelle (für Strahlmodulation), Impulsgeber, dichroitische, Objektiv, Fluoreszenzemission Filter, gated Photomultiplier und einem Datenerfassungssystem (Photonen-Zähler und Mehrkanal-Scaler). 2. Bestimmen Sie sicher überwachen Kräfte. <ol><li> Dämpfen Sie den Laser auf einen niedrigen, aber vernünftig, Energie zur Erzeugung von Fluoreszenz in der Probe. </li><li> Fokus in der Probe. </li><li> Set ein Photon Zähler zu integrieren über eine Zeitskala viel länger als die erwartete Fluoreszenz-Recovery. Mit dem Fokalvolumen stationär gehalten in der Probe (auf unserem Mikroskop-Software ist dies durch einen Punkt erreicht Scan), Aufnahme einer angemessenen Anzahl von Photonen Zähldaten. </li><li> Steigern Sie die Leistung und nehmen eine andere Reihe von Photonenzahl Daten. Wiederholen, bis die Erhöhung der Photonen offenbar deutlich verringert in Bezug auf die Leistung zu erhöhen. </li><li> Plot log (Photonen) als Funktion von log (Leistung). Eine Abweichung von einem Hang aus zwei zeigt Bleichen während des Monitors. Wählen Sie als Ihren Monitor Macht eine Macht, die unter diesem abgeschnitten, aber die einen vernünftigen Signal-Rausch-Verhältnis ermöglicht. </li></ol> 3. Bestimmen Sie Input-Parameter mit dem Impulsgenerator und Multichannel-Scaler. <ol><li> Bestimmen back-of-the-envelope Schätzungen der Diffusionskoeffizient und Recovery-Zeit auf die Literatur und / oder die Stokes-Einstein-Gleichung und der Diffusionsgleichung basiert. </li><li> Stellen Sie die erforderlichen Parameter-Werte auf der Impulsgeber und der Scaler. <ol class="lalpha"><li> Auf der Impulsgeber, stellen Sie den Pre-Bleichmittel und Bleich-Zeiten. Ein guter Satz von Regeln besagt, dass der Pre-Bleiche sollte 1-2 mal größer als die erwartete Erholung ein halb Mal, und das Bleichmittel Zeit sollte ein Zehntel des erwarteten ein halb Recovery-Zeit. </li><li> Auf der Scaler, stellen Sie den Bin-Breite auf etwa die Hälfte der Bleiche Dauer und die Anzahl der Behälter pro Datensatz, so dass das Produkt von Ihnen gewählten Bin-Breite und Behälter pro Datensatz größer als oder ungefähr gleich Ihre voraussichtliche vollständige Genesung sowie Pre ist -Bleichmittel und Bleich-Zeiten. </li><li> Zurück zum Impulsgeber und stellen Sie die Frequenz des pre-bleach/bleach/monitor Sequenz gleich auf einen Wert knapp kleiner als der Kehrwert des Produkts der Bin-Breite mal der Anzahl der Behälter pro Datensatz. * </li><li> Schließlich stellen Sie die Anzahl der Datensätze / Scan auf dem Scaler basierend auf der Signalintensität am gewünschten Monitor mit Strom versorgt. * Es ist sehr wichtig, dass die Zeit für eine vollständige pre-bleach/bleach/monitor Sequenz (1/frequency) am Puls Generator größer als die Datensammlung Zeit (bin Breite x bins / record) auf der Scaler eingestellt ist. Wenn diese Kriterien nicht erfüllt ist, können mehrere Bleichmittel Trigger innerhalb eines einzelnen Datensatzes auf dem Scaler erscheinen. </li></ol></li><li> Stellen Sie den Monitor mit Strom versorgt, um ein akzeptables Niveau, ermittelt zuvor. Stellen Sie die Bleiche Leistung bis 200 mW oder so über das Bleichen Cutoff während der Monitor Bleichen ermittelt. Diese Befugnisse gesetzt sind, wie unterschiedliche Spannungen über den PC-Kristall. </li><li> Seal das Mikroskop, löschte das Licht, schalten Sie den PMT, beginnen einen Punkt Scan auf dem Mikroskop-Software, starten Sie dann den Impulsgeber und Scaler. </li><li> Analysieren Sie die resultierende Erholung durch Auftragen der Kurve und Kennzeichnung drei wichtige Punkte: 1) die Pre-Bleiche Fluoreszenz, 2) die Fluoreszenz sofort post-Bleichmittel, und 3) die Fluoreszenz am Ende des Datensatzes. Verwenden Sie die Fluoreszenz-Werte vor und unmittelbar nach der Bleiche zur besseren Abschätzung der Halb-Recovery-Zeit. </li><li> Mit dieser Einschätzung der Halb-Recovery-Zeit, die Faustregeln zuvor, um neue Werte für die Pre-Bleichmittel, Bleichmittel bestimmen skizziert und bin Breite Dauer. Auch die Vor-Bleiche Fluoreszenz-und Fluoreszenz am Ende des Datensatzes zu einer besseren Abschätzung der Zeitaufwand für die vollständige Genesung zu erreichen. Als Faustregel gilt, sollten die Daten für einen Zeitraum, der für die vollständige Wiederherstellung für die zweite Hälfte der gemeldeten Daten ermöglicht gesammelt werden. </li><li> Passen Sie die Parameter auf der Impulsgeber und Scaler, nehmen Sie eine neue Kurve, und weiterhin Feinabstimmung der Parameter, bis Ihr Kurve zeigt eine starke Bleichmittel und reibungslose Genesung. Beachten Sie, dass das Bleichmittel Dauer kann und sollte, unter der Faustregel verringert werden, wenn möglich. Das Bleichmittel Macht kann auch eingestellt werden, wenn zu flach oder zu tief, ein Bleichmittel erreicht werden. </li></ol> 4. Test für die Anregung Sättigung. <ol><li> Mit entsprechenden Monitor und Bleichmittel Kräfte bestimmt vorher, nehmen eine Reihe von MP-FRAP Kurven. Analysieren Sie jede Erholung, normiert auf die Pre-Bleiche Fluoreszenz, mit den entsprechenden mathematischen Form und eine nicht-lineare Least-Squares Fitting-Algorithmus (siehe &quot;Data Analysis&quot; weiter unten). Notieren Sie den angepassten Wert der Bleiche Tiefe Parameter.</li><li> Setzen Sie die Einnahme und Analyse von aufeinander folgenden Reihe von MP-FRAP Kurven bei steigenden Werten der Bleiche Macht. </li><li> Plot log (Bleiche Tiefe Parameter) als Funktion von log (Leistung). Jede Abweichung von einem Hang aus zwei gibt Anregung Sättigung. Wählen Sie ein Bleichmittel Macht, die ein starkes Bleichmittel ergibt, bewirkt aber keine Sättigung. </li></ol> 5. Weiter unter MP-FRAP Kurven. <ol><li> Setzen Sie die Einnahme Kurven, wie oben beschrieben, werden alle Parameter richtig eingestellt sind. </li></ol> 6. Data Analysis. <ol><li> Entfernen Sie die Pre-Bleichmittel und Bleich-Daten aus der Fluoreszenz-Daten einstellen und die Zeit-Daten, so dass t = 0 entspricht der Fluoreszenz-Wert sofort nach der Bleiche. </li><li> Normalisieren der resultierenden Fluoreszenz Erholung in Bezug auf die durchschnittliche pre-Bleiche Fluoreszenz. </li><li> Fit der normierten Kurve mit den entsprechenden mathematischen Modell und eine nicht-lineare, Least Squares Fitting-Algorithmus, wie die lsqcurvefit Funktion in MATLAB. Das hier vorgestellte Modell-Konten für die Fluoreszenz-Rückforderungen von beiden Diffusion und konvektive Strömung beeinflusst: <img src="/files/ftp_upload/1636/1636eq.jpg" alt="Gleichung" /> wo F o der Pre-Bleiche durchschnittliche Fluoreszenz, β der Bleiche Tiefe Parameter ist, τ D die charakteristische diffusive Recovery-Zeit ist, ist R das Verhältnis des Quadrats der axialen (w z) radial (w r) Breiten der Zwei-Photonen-Fokalvolumen, τ vx = w r / v x, τ vy = w r / v y, τ vz = w z / v z und v 2 = v x 2 + v y 2 + v z 2 ist die Geschwindigkeit des konvektiven Strömung. Für flow beschränkt sich auf die Bildebene, τ vz → ∞ und 1 / τ vx + 1 / τ vy kann mit 1 / τ vr ersetzt werden. In Abwesenheit von Konvektionsströmung, beide Exponenten im Zähler auf 1 zu gehen, und der Ausdruck ist stark reduziert, um nur zwei Anpassungsparameter, β und τ D. Der Diffusionskoeffizient ist leicht als D = w r 2 / 8τ D berechnet. </li></ol> 7. Repräsentative Ergebnisse. <img src="/files/ftp_upload/1636/1636fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Representative Bleich-und Recovery-Kurve für FITC-BSA. Ein gutes Recovery-Kurve zeigt eine starke Bleichmittel und glatte Erholung, mit der Fluoreszenz bei voller Erholung wird für die zweite Hälfte der gemeldeten Daten. <img src="/files/ftp_upload/1636/1636fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Normalized Recovery-Kurve für FITC-BSA (rot) und die damit verbundenen kleinsten Quadrate (schwarz). Diese passen ergibt sich ein Diffusionskoeffizient von 52,9 um2 / s, im Einklang mit der Literatur.

<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>

Die Macht der Multi-Photonen-Fluoreszenz-recovery after photobleaching liegt in seiner Fähigkeit, dicken Proben mit 3D-Auflösung Sonde. Seit seiner Entwicklung in den 1990er Jahren hat MP-FRAP verwendet worden, um den Diffusionskoeffizienten (oder analoge Transport-Parameter) in Zellkörper, ex vivo dicken Gewebeschnitten und in vivo Gewebe und Interstitium bestimmen. In diesem Artikel haben wir die notwendige Ausrüstung, um ein MP-FRAP-Experiment, sowie das richtige Verfahren für die Ausrichtung d...

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<td>Ti:sapphire laser</td>
<td>&nbsp;</td>
<td>Spectra Physics</td>
<td>Mai Tai</td>
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<td>Pockels Cell</td>
<td>&nbsp;</td>
<td>Conoptics</td>
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<td>Olympus</td>
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<td>&nbsp;</td>
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<td>Hamamatsu</td>
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measuring diffusion coefficients via two-photon fluorescence recovery after photobleaching

Multi-Fluorescence Recovery After Photobleaching ist ein Mikroskopie-Technik verwendet, um den Diffusionskoeffizienten (oder analoge Transport-Parameter) von Makromolekülen zu messen, und kann sowohl angewendet werden<em&gt; In-vitro-</em&gt; Und<em&gt; In vivo</em&gt; Biologischen Systemen. Multi-Fluorescence Recovery After Photobleaching wird durch Bleichen einer Region von Interesse innerhalb einer fluoreszierenden Probe mit Hilfe eines intensiven Laser-Flash durchgeführt, dann Abschwächung des Strahls und die Überwachung der Fluoreszenz als noch fluoreszierende Moleküle von außerhalb der Region von Interesse diffuse in die gebleicht Moleküle ersetzen . Wir werden unsere Demonstration durch die Angleichung der Laserstrahl durch die Pockels-Zelle (Laser-Modulator) und entlang der optische Weg durch die Laser-Scan-Box und Objektiv auf die Probe beginnen. Der Einfachheit halber werden wir eine Stichprobe von wässrigen Fluoreszenzfarbstoff. Wir werden dann bestimmt die richtige experimentellen Parameter für unsere Stichprobe, einschließlich Monitor und Bleichen Mächte, Bleichmittel Dauer, bin Breiten (für Photon-Counting) und Fluoreszenz-Recovery-Zeit. Als nächstes werden wir das Verfahren für die Aufnahme Erholung Kurven, ein Prozess, der weitgehend über LabVIEW (National Instruments, Austin, TX) kann für verbesserten Durchsatz automatisiert. Schließlich ist der Diffusionskoeffizient durch Anpassung der Recovery-Daten an die entsprechenden mathematischen Modells mit Hilfe eines Least-Squares Fitting-Algorithmus, leicht programmierbar über Software wie MATLAB (The Mathworks, Natick, MA) ermittelt.

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

In diesem Artikel beschreiben wir das Verfahren zur Messung von Diffusionskoeffizienten mittels Multi-Photonen-Fluoreszenz-Erholung nach Ausbleichen. Wir werden durch die Angleichung der Laser entlang der optischen Pfad zu der Probe und Bestimmung der richtigen experimentellen Parameter beginnen, dann weiter generiert und schließlich passende Fluoreszenz Erholung Kurven.

Mess-Diffusionskoeffizienten über Zwei-Photonen-Fluorescence Recovery After Photobleaching

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

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

Research

JoVE Journal

Biology

11.1K Aufrufe.  University of Rochester. In diesem Artikel beschreiben wir das Verfahren zur Messung von Diffusionskoeffizienten mittels Multi-Photonen-Fluoreszenz-Erholung nach Ausbleichen. Wir werden durch die Angleichung der Laser entlang der optischen Pfad zu der Probe und Bestimmung der richtigen experimentellen Parameter beginnen, dann weiter generiert und schließlich passende Fluoreszenz Erholung Kurven.

Serie: 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.

Zwei-Photonen-Axotomie und Zeitraffer-konfokale Bildgebung in lebenden Zebrafisch-Embryonen

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

Forschung

Biologie

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.

Zeitraffer-Imaging der Mitose Nach siRNA-Transfektion

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.

Zwei-Photonen-Based Photoaktivierung in Live Zebrafischembryonen

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.

In vivo Quantifizierung der G Protein-gekoppelten Rezeptor-Wechselwirkungen mit spektral aufgelöste Zwei-Photonen-Mikroskopie

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

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

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

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

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

Photobleaching Assays (FRAP &amp; FLIP) zu Chromatin Protein Dynamics in Living embryonalen Stammzellen Measure

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.

Mapping molekulare Diffusion in der Plasmamembran durch 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 ...

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

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

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

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

Maus Sperma Kryokonservierung und Recovery mit der I · 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 ...

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.

Einfache Messung des Diffusionskoeffizienten von EGFP-markierten Plasmamembranproteine ​​Mit k-Raum-Bild-Korrelations-Spektroskopie

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.

Rückgewinnung von Adult Zebrafisch-Herzen für Hochdurchsatz-Anwendungen

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.

Zwei-Photonen-Imaging von intrazellulären Ca<sup&gt; 2+</sup&gt; Transport- und Stickoxid-Produktion in Endothelzellen und glatte Muskelzellen eines isolierten Rattenaorta

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