Questo lavoro è stato finanziato da un Dipartimento di Era Difesa del Premio Speranza Scholar (n. W81XWH-05-0396) e un Scholar Pew al Premio Scienze Biomediche a Edward B. Brown III.

1. Allineare l&#39;ottica. L&#39;attrezzatura fondamentale per un MP-FRAP esperimento comprendono: un modo bloccato sorgente laser, Cell Pockels (per la modulazione del fascio), generatore di impulsi, dicroici, lenti dell&#39;obiettivo, emissione di fluorescenza filtro, tubo fotomoltiplicatore recintato, e un sistema di registrazione dei dati (fotoni contatore e multicanale scaler). 2. Determinare i poteri del monitor di sicurezza. <ol><li> Attenuare il laser ad un basso, ma ragionevole, il potere di generare fluorescenza all&#39;interno del campione. </li><li> Messa a fuoco all&#39;interno del campione. </li><li> Impostare un contatore di fotoni per integrare su un arco temporale molto più lungo del previsto il recupero di fluorescenza. Con il volume focale tenuto fermo all&#39;interno del campione (il nostro software di questo microscopio è ottenuto da una scansione punto), registrare un numero ragionevole di dati di conteggio di fotoni. </li><li> Aumentare la potenza e prendere un&#39;altra serie di dati di conteggio di fotoni. Ripetere fino a quando l&#39;aumento del numero dei fotoni sembra diminuire in modo significativo rispetto alle aumentare la potenza. </li><li> Plot log (conta fotone) in funzione di log (potenza). Una deviazione da un pendio di due indica sbiancamento durante il monitor. Scegli come alimentazione del monitor un potere che è inferiore a tale tagliata, ma che consente una ragionevole rapporto segnale rumore. </li></ol> 3. Determinare i parametri di ingresso al generatore di impulsi e multicanale scaler. <ol><li> Determinare back-of-the-envelope stime del coefficiente di diffusione e tempi di recupero basato sul / o letteratura e la equazione di Stokes-Einstein e l&#39;equazione di diffusione. </li><li> Impostare i valori dei parametri necessari per il generatore di impulsi e lo scaler. <ol class="lalpha"><li> Sul generatore di impulsi, impostare i tempi di pre-lavaggio e di lavaggio. Un buon set di regole pratiche è che la pre-candeggina dovrebbe essere 1-2 volte più grande del previsto il recupero del primo tempo, e il tempo di candeggina dovrebbe essere un decimo il tempo di recupero previsto metà. </li><li> Sul scaler, impostare la larghezza bin a circa la metà della durata di candeggina, e impostare il numero di cassonetti per registrare in modo tale che il prodotto della vostra larghezza bin selezionato e contenitori per ogni record è superiore o pari a circa il recupero completo più attesi pre -candeggina e tempi di candeggina. </li><li> Ritorno al generatore di impulsi e impostare la frequenza della sequenza pre-bleach/bleach/monitor pari a un valore appena inferiore l&#39;inverso del prodotto di volte la larghezza del bin il numero di cassonetti per record. * </li><li> Infine, impostare il numero di record / scansione sul scaler in base all&#39;intensità del segnale di alimentazione del monitor a vostra scelta. * E &#39;molto importante che il tempo per una sequenza pre-bleach/bleach/monitor completa (1/frequenza) impostata sul generatore di impulsi è maggiore del tempo di raccolta dati (bin larghezza x cassonetti / registrazione) impostato sul scaler. Se questi criteri non è soddisfatto, fa scattare candeggina multiple possono apparire all&#39;interno di un singolo record sul scaler. </li></ol></li><li> Impostare l&#39;alimentazione del monitor ad un livello accettabile, determinato in precedenza. Impostare la potenza a 200 mW candeggina o giù di sopra del valore soglia sbiancamento determinata durante la prova di monitorare sbiancamento. Questi poteri sono entrambi impostati come differenti tensioni in tutto il cristallo PC. </li><li> Sigillare il microscopio, spegnere le luci, accendere la PMT, iniziare una scansione punto sul software microscopio, quindi avviare il generatore di impulsi e scaler. </li><li> Analizzare la curva di recupero conseguente tracciando la curva e marcatura tre punti importanti: 1) la pre-candeggina fluorescenza, 2) la fluorescenza immediatamente post-candeggina, e 3) la fluorescenza alla fine del set di dati. Utilizzare i valori di fluorescenza pre e immediatamente post-candeggina per valutare meglio il mezzo tempo di recupero. </li><li> Con questa stima di metà tempo di recupero, utilizzare le regole pratiche descritte in precedenza per determinare i nuovi valori per la pre-candeggina, candeggina, e durate larghezza bin. Inoltre, utilizzare il pre-candeggina fluorescenza e fluorescenza, alla fine del set di dati per stimare meglio il tempo necessario per raggiungere il pieno recupero. Come regola generale, i dati devono essere raccolti per un periodo di tempo che consente il pieno recupero da segnalare per la seconda metà dei dati. </li><li> Regolare i parametri del generatore di impulsi e scaler, prendere una nuova curva, e continuare il ritocco dei parametri fino a quando la curva mostra un forte candeggina e recupero liscia. Tenete a mente che la durata della candeggina, e dovrebbe, essere ridotto sotto la regola del pollice, se possibile. La potenza candeggina può anche essere regolato se troppo bassa o troppo profonda, una candeggina è raggiunto. </li></ol> 4. Test per la saturazione di eccitazione. <ol><li> Utilizzando monitor appropriato e poteri candeggina, determinato in precedenza, prendere una serie di MP-FRAP curve. Analizzare ogni recupero, normalizzato per il pre-candeggina fluorescenza, utilizzando l&#39;apposito modulo matematico e un non-lineare dei minimi quadrati algoritmo di montaggio (si veda &quot;Analisi dei dati&quot; di seguito). Registrare il valore stimato del parametro profondità candeggina.</li><li> Continuare a prendere e analizzare serie successiva di MP-FRAP curve ad aumentare i valori di potenza candeggina. </li><li> Plot log (profondità parametro candeggina) in funzione di log (potenza). Qualsiasi deviazione da un pendio di due indica saturazione eccitazione. Scegli una potenza candeggina che produce un forte candeggina, ma non causa la saturazione. </li></ol> 5. Continuare ad assumere MP-FRAP curve. <ol><li> Continuare a prendere le curve, come sopra, saranno tutti i parametri impostati correttamente. </li></ol> 6. Analisi dei dati. <ol><li> Rimuovere i dati di pre-lavaggio e dai dati di fluorescenza impostare e regolare i dati al momento in cui t = 0 corrisponde al valore di fluorescenza immediatamente post-candeggina. </li><li> Normalizzare il recupero di fluorescenza risultante rispetto alla media pre-candeggina fluorescenza. </li><li> Montare la curva normalizzata utilizzando il modello appropriato matematico e un algoritmo non lineare, minimi quadrati, come ad esempio la funzione lsqcurvefit in MATLAB. Il modello presentato qui rappresenta recuperi fluorescenza influenzata sia la diffusione e il flusso convettivo: <img src="/files/ftp_upload/1636/1636eq.jpg" alt="Equazione" /> dove F o è la pre-candeggina fluorescenza media, β è il parametro profondità candeggina, τ D è il tempo caratteristico di recupero diffusivo, R è il rapporto tra il quadrato della assiale (w z) a radiale (w r) larghezze della a due fotoni del volume focale, τ vx w = r / v x, τ vy = w r / v y, τ vz = w z / v z e v 2 = v x 2 + v y 2 + v z 2 è il velocità del flusso convettivo. Per il flusso confinato al piano di imaging, vz τ → ∞ e 1 / τ vx + 1 / τ vy può essere sostituito con 1 / τ vr. In assenza di flusso convettivo, esponenziali sia al numeratore va a 1 e l&#39;espressione è notevolmente ridotto, a solo due parametri di montaggio, β e τ D. Il coefficiente di diffusione può essere facilmente calcolato come D = w r 2 / D 8τ. </li></ol> 7. Rappresentante dei risultati. <img src="/files/ftp_upload/1636/1636fig1.jpg" alt="Figura 1" /> Figura 1. Candeggina Rappresentante e la curva di recupero per FITC-BSA. Una curva mostra un buon recupero candeggina forte e recupero graduale, con la fluorescenza in pieno recupero sono stati segnalati per la seconda metà dei dati. <img src="/files/ftp_upload/1636/1636fig2.jpg" alt="Figura 2" /> Figura 2. Curva recupero normalizzato per FITC-BSA (rosso) e il relativo ai minimi quadrati (nero). Questa misura produce un coefficiente di diffusione del 52,9 UM2 / s, coerente con la letteratura.

<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>

La potenza del multi-fotone recupero di fluorescenza dopo photobleaching risiede nella sua capacità di sondare i campioni di spessore con risoluzione 3D. Dal momento che il suo sviluppo nel 1990, MP-FRAP è stato utilizzato per determinare il coefficiente di diffusione (o parametri di trasporto analoghe) in corpi cellulari, ex fette di tessuto vivo di spessore, e nel tessuto vivo e nell&#39;interstizio. In questo articolo, abbiamo presentato l&#39;attrezzatura necessaria per eseguire...

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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>
<td>Fluoview</td>
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<td>Short pass dichroic mirror</td>
<td>&nbsp;</td>
<td>Chroma Technologies</td>
<td>670 DCSX-2P</td>
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measuring diffusion coefficients via two-photon fluorescence recovery after photobleaching

Multi-fluorescenza recupero dopo photobleaching è una tecnica di microscopia utilizzato per misurare il coefficiente di diffusione (o parametri di trasporto analoghe) delle macromolecole, e può essere applicato sia<em&gt; In vitro</em&gt; E<em&gt; In vivo</em&gt; Sistemi biologici. Multi-fluorescenza recupero dopo photobleaching viene eseguito da photobleaching una regione di interesse all&#39;interno di un campione fluorescente con un flash intenso laser, poi attenuare il fascio e il monitoraggio della fluorescenza come ancora molecole fluorescenti al di fuori della regione di diffuso interesse per sostituire le molecole fotodecolorate . Inizieremo la nostra dimostrazione, allineando il raggio laser attraverso il cellulare Pockels (modulatore laser) e lungo il cammino ottico attraverso la finestra di scansione laser e lenti dell&#39;obiettivo al campione. Per semplicità, useremo un campione di acquoso colorante fluorescente. Ci sarà quindi determinare i parametri corretti sperimentale per il nostro campione compreso, monitorare e poteri candeggio, durata candeggina, larghezze bin (per il conteggio di fotoni), e tempi di recupero di fluorescenza. Successivamente, verranno descritti la procedura di prelievo curve di recupero, un processo che può essere in gran parte automatizzato tramite LabVIEW (National Instruments, Austin, TX) per un throughput maggiore. Infine, il coefficiente di diffusione è determinato dal montaggio del recupero dei dati al modello matematico appropriato utilizzando un algoritmo dei minimi quadrati montaggio, facilmente programmabile tramite un software come MATLAB (The MathWorks, Natick, MA).

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

Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

In questo articolo andremo a descrivere il procedimento per determinare coefficienti di diffusione multi-fotone con il recupero di fluorescenza dopo photobleaching. Inizieremo allineando il laser lungo il percorso ottico al campione e determinare i parametri corretti sperimentale, poi continuare a generare ed infine montaggio curve recupero fluorescenza.

Misurare coefficienti di diffusione via a due fotoni di recupero fluorescenza Dopo 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 Views.  University of Rochester. In questo articolo andremo a descrivere il procedimento per determinare coefficienti di diffusione multi-fotone con il recupero di fluorescenza dopo photobleaching. Inizieremo allineando il laser lungo il percorso ottico al campione e determinare i parametri corretti sperimentale, poi continuare a generare ed infine montaggio curve recupero fluorescenza.

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.

Due fotoni assotomia e time-lapse confocale in embrioni di zebrafish vivo

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

Ricerca

Biologia

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.

Time-lapse imaging di mitosi dopo la trasfezione di siRNA

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.

Due-Photon-Based fotoattivazione in Live embrioni di zebrafish

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.

Quantificazione in vivo di G interazioni proteina recettore accoppiato con spettralmente risolte microscopia a due fotoni

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.

Fotodecolorazione saggi (FRAP e FLIP) per misurare la dinamica delle proteine ​​della cromatina in Living cellule staminali embrionali

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.

Mappatura diffusione molecolare nella membrana plasmatica da parte di più-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.

Crioconservazione del mouse Sperm and Recovery utilizzando il Kit · I Cryo

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.

Facile Misura di diffusione Coefficienti di EGFP-tagged plasma proteine ​​di membrana Uso k-spazio Spettroscopia di correlazione Immagine

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.

Recupero di Zebrafish adulti cuori per applicazioni ad alta velocità effettiva

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.

A due fotoni Imaging di Intracellulare Ca<sup&gt; 2+</sup&gt; Manipolazione e l&#39;ossido nitrico Produzione in endoteliale e della muscolatura liscia cellule di un isolato Rat Aorta

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