Name: 3d orbital tracking in a modified two-photon microscope: an application to the tracking of intracellular vesicles
Uploaded: 2014-10-01T13:00:00.000+00:00
Duration: 11 min 28 s
Description: Watch this Scientific Journal Video about 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles at JoVE.com

This project was supported by grants NIH NIGMS 8P41 GM103540-28 and P50-GM076516

Preparazione del campione 1 <ol><li> Mantenere le cellule CHOK1 in tessuto fiasche di coltura utilizzando DMEM supplementato con 10% di siero fetale bovino e 100 UI / ml di penicillina 50 mg / ml di streptomicina. Incubare le cellule in un 5% di CO 2 incubatore umidificato a 37 ° C. </li><li> Raccolta e poi plate cellule CHOK1 su un diametro di micro-pozzetto 14 mm e uno spessore superficie di 0,16 mm. Cellule seme per densità ottimale per l&#39;imaging, intorno al 60-70% di confluenza. </li><li> Incubare le cellule per una notte a 37 ° C, 5% di CO 2. Lavare le cellule tre volte in HBSS (Hank Buffered Saline Solution), e incubare le cellule in una soluzione contenente 50 nM di Lysotracker DND26 verde e 150 Nm di tubulina inseguitore verde. Incubare le cellule per 1 ora a 37 ° C. Passaggio facoltativo: Prima di incubazione, eseguire una macchia supplementare con un colorante colorazione matrice mitocondriale (25 Nm). </li><li> Lavare le cellule tre volte in HBSS per rimuovere il colorante non legato. Aggiungere fresco terreno di coltura DMEM prima imagzione le cellule. </li></ol> 2. Configurazioni Microscopio Il microscopio per il tracciamento di particelle descritto nel protocollo video viene montato sul telaio di un microscopio invertito disponibile in commercio (Figura 4). Tuttavia moduli commerciali per 3D tracciamento di particelle orbitali sono ora disponibili. <ol><li> Utilizzare un coerente Chameleon-Ultra II Ti: Sa a femtosecondi sorgente di luce di eccitazione laser, con una gamma di lunghezze d&#39;onda di uscita regolabile tra 690 nm-1.040 nm. </li><li> Assicurarsi che il raggio laser è allineato nella porta posteriore del microscopio utilizzando specchi IR-rivestito. Il raggio laser viene attenuato mediante una piastra semionda rotante seguito da un polarizzatore lineare calcite. Attenuazione del fascio in modo che la potenza media al campione è compreso tra 0,5 e 2 mW. NOTA: Il raggio laser viene riflesso da una coppia di specchi a comando galvanometro motori che consentono il controllo della posizione e traiettoria del fascio focalizzato nel piano campione. Inconfigurazione tipica del fascio laser collimato uscire degli specchi galvanometro viene espanso (10X), che passa attraverso un espansore del fascio, prima di entrare nel retro dell&#39;obiettivo microscopio dopo la riflessione nel passo corto specchio dicroico. </li><li> Raccogliere la luce di fluorescenza dal campione ponendo un obiettivo numerico ad elevata dell&#39;acqua di apertura (60X, NA 1.2) nel percorso ottico. Scegli un cubo filtro di fluorescenza in funzione delle lunghezze d&#39;onda di emissione desiderati in configurazione sia uno o due canali. Impiegare filtri passa-banda di emissione per selezionare ulteriormente la gamma spettrale della fluorescenza emessa. </li><li> Porre il campione su un palco motorizzato, e regolare la multa movimento dell&#39;obiettivo del microscopio con un piezo-controllore obiettivo. </li><li> Dirigere la luce dal cubo del filtro in tubi fotomoltiplicatori dove il segnale viene discriminati e inviato a una scheda di acquisizione dati I / O digitale. Generato dal computer forme d&#39;onda sono l&#39;uscita analogica della scheda I / O e sono provid: NOTAed agli scanner di controllo elettronica. L&#39;ingresso di conteggio di fotoni dai fotomoltiplicatori e il segnale di uscita per gli scanner sono misurati e controllati tramite la scheda I / O dal Laboratorio per il software Fluorescence Dynamics SimFCS. </li></ol> 3. Imaging <ol><li> Posizionare le cellule sul palco e mettere a fuoco utilizzando l&#39;illuminazione a luce trasmessa. </li><li> Passa alla scansione di immagini raster per identificare le cellule che hanno incorporato il colorante e di visualizzare i microtubuli sottostanti. Identificare l&#39;area iniziale in cui il movimento delle vescicole è presente. </li><li> Una volta che una vescicola isolato è identificato, impostare i seguenti parametri per l&#39;inseguimento orbitale: <ol><li> Selezionare il raggio dell&#39;orbita per definire le dimensioni della scansione circolare secondo la dimensione della particella monitorata. Per un emettitore punto, impostare il raggio dell&#39;orbita pari alla vita della Point Spread Function (PSF) del fascio di eccitazione al massimo la sensibilità e la risposta. </li><li> Impostare il assialedistanza per definire la distanza tra le due orbite che vengono eseguite per localizzare la posizione della particella lungo la direzione assiale. Impostare la distanza assiale a 1,5-3 volte la vita PSF. </li><li> Definire il tempo di sosta in base alla luminosità della particella per impostare il tempo speso su ogni punto dell&#39;orbita, che determinerà anche il tasso-photo sbiancamento. Utilizzare un tempo di sosta tra 10-100 msec. </li><li> Impostare il numero di punti in ciascuna orbita di 64 o 128 per produrre periodi orbita nell&#39;ordine di 4-32 msec e fornire un&#39;elevata risoluzione temporale per la determinazione della posizione della particella. </li></ol></li><li> Modificare il segnale di offset DC inviata agli specchi in modo da centrare il fascio sulla particella attraverso l&#39;interfaccia utente grafica mediante un cursore di selezione nell&#39;immagine raster-digitalizzata. </li><li> Inizia il monitoraggio commutando la modalità microscopio da raster-imaging per la scansione orbitale. </li><li> Attivare la raccolta di feedback e dati. </li></ol> 4. TrajectAnalisi ory <ol><li> Utilizzare il software per visualizzare la fluorescenza raccolte in ogni punto lungo l&#39;orbita e ad ogni tempo sotto forma di un &#39;tappeto intensità&#39;. L&#39;intensità raccolto lungo il tappeto fornisce informazioni sulle interazioni della particella con altri oggetti luminosi. Utilizzare il software per visualizzare sia le informazioni traiettoria (cioè lo spostamento CC nel tempo di x, y scanner e del -piezo z) e l&#39;intensità di fluorescenza raccolti dal tubo fotomoltiplicatore nel tempo sotto forma di serie temporali. </li><li> Utilizzare la rappresentazione delle serie storiche e le informazioni &#39;tappeto&#39; intensità &#39;di scegliere solo una regione di interesse nella traiettoria. </li><li> Utilizzare il software per visualizzare una proiezione 2D della porzione selezionata della traiettoria della particella. Selezionare i controlli appropriati per codice colore traiettoria secondo l&#39;intensità di fluorescenza delle particelle. </li><li> Selezionare l&#39;opzione per visualizzare la particolo traiettoria in 3D, e codice-colore che secondo l&#39;intensità di fluorescenza. Ruotare la traiettoria in 3D utilizzando i comandi per visualizzare le caratteristiche del moto lisosomi lungo il microtubulo. </li></ol>

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	<li>So, P. T., Dong, C. Y., Masters, B. R., Berland, K. M. <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+excitation+fluorescence+microscopy.">Two photon excitation fluorescence microscopy.</a> Annu Rev Biomed Eng. 2, 399-429 (2000).</li><li>Dupont, A., Lamb, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+three+dimensional+single+particle+tracking.">Nanoscale three dimensional single particle tracking.</a> Nanoscale. 3, 4532-4541 (2011).</li><li>Estrada, L. C., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+nanometer+images+of+biological+fibers+by+directed+motion+of+gold+nanoparticles.">3D nanometer images of biological fibers by directed motion of gold nanoparticles.</a> Nano Lett. 11, 4656-4660 (2011).</li><li>Kis-Petikova, K., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distance+measurement+by+circular+scanning+of+the+excitation+beam+in+the+two+photon+microscope.">Distance measurement by circular scanning of the excitation beam in the two photon microscope.</a> Microscopy Research and Technique. 63, 34-49 (2004).</li><li>Levi, V., Ruan, Q., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3+D+particle+tracking+in+a+two+photon+microscope+application+to+the+study+of+molecular+dynamics+in+cells.">3 D particle tracking in a two photon microscope application to the study of molecular dynamics in cells.</a> Biophys J. 88, 2919-2928 (2005).</li><li>Lund, F. W., Wustner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+single+particle+tracking+and+temporal+image+correlation+spectroscopy+for+quantitative+analysis+of+endosome+motility.">A comparison of single particle tracking and temporal image correlation spectroscopy for quantitative analysis of endosome motility.</a> Journal of Microscopy. 252, 169-188 (2013).</li><li>Nath, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spreading+of+Neurodegenerative+Pathology+via+Neuron+to+Neuron+Transmission+of+beta+Amyloid.">Spreading of Neurodegenerative Pathology via Neuron to Neuron Transmission of beta Amyloid.</a> Journal of Neuroscience. 32, 8767-8777 (2012).</li><li>Balint, S., Vilanova, I. V., Alvarez, A. 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S., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+particle+tracking+on+the+two+photon+microscope.">3D particle tracking on the two photon microscope.</a> Multiphoton Microscopy in the Biomedical Sciences. 2, 247-258 (2001).</li><li>Konig, K., So, P. T. C., Mantulin, W. W., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+response+to+near-infrared+femtosecond+laser+pulses+in+two+photon+microscopes.">Cellular response to near-infrared femtosecond laser pulses in two photon microscopes.</a> Optics Letters. 22, 135-136 (1997).</li><li>Caviston, J. P., Holzbaur, E. 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The authors have nothing to disclose.

Nonostante gli enormi progressi delle tecniche di microscopia a fluorescenza e strumentazioni nel corso degli ultimi anni, raggiungendo traiettorie delle particelle fluorescenti in tre dimensioni con una elevata risoluzione temporale è rimasta una sfida nel campo. Se alta risoluzione temporale è stato raggiunto particelle di inseguimento in due dimensioni, estensione alla direzione assiale comporta tipicamente una drastica riduzione della risposta in frequenza del sistema 10. In...

Un gran numero di approcci sono stati sviluppati fino ad oggi per monitorare le particelle fluorescenti in tre dimensioni utilizzando un microscopio. La maggior parte approcci si basano sull&#39;impiego di telecamere veloci, ideale per monitorare in due dimensioni, di solito in combinazione con modifiche personalizzate dell&#39;ottica emissione del microscopio per ottenere inseguimento in direzione assiale. Microscopi a scansione laser confocale (uno o due fotoni) convenzionalmente in grado di monitorare una particella fluorescente eseguendo una sequenza temporale di z-stack, anche se questo processo è in genere richiede tempo, e produce la risoluzione di tempo ragionevole (10 Hz) solo se la particella monitorata è conservato nel centro di una piccola regione di imaging raster da un meccanismo di feedback attivo 2. L&#39;idea di bloccaggio-in per la particella è alla base del metodo di monitoraggio orbitale. Invece di un raster scan un&#39;orbita circolare viene eseguita intorno alla particella fluorescente. L&#39;intensità della fluorescenza lungol&#39;orbita localizza con precisione la posizione delle particelle 4. La posizione localizzato della particella può quindi essere utilizzato per azionare gli scanner microscopio e ri-centro orbita sulla posizione delle particelle. Gli scanner galvanometro del microscopio sono guidate da una tensione analogica. La componente alternata della tensione permette di eseguire un&#39;orbita con il fascio laser focalizzato, cioè un sinusoidale e un&#39;onda coseno applicata agli specchi X e Y scansione consentirà di eseguire un&#39;orbita circolare. Il DC offset del segnale facilita la modifica della posizione del centro orbita. Dopo un periodo di orbita è determinata e la forma d&#39;onda per il segnale AC è configurato, il sistema di retroazione deve aggiornare solo la componente continua del segnale. Un software in grado di leggere il segnale fluorescente raccolti dai rivelatori è necessario per controllare gli scanner e rivelatori, per calcolare la posizione della particella e aggiornare il DC offset. Per una valutazione di successo di imaging un&#39;accurata scelta dei parametri orbitali (dimensioni e della tempistica) è necessario e questi parametri devono essere regolati in base alle caratteristiche delle particelle fluorescenti che è necessario tenere traccia. I fondamenti fisici e matematici della tecnica sono stati descritti in passato 4,5 per entrambe le applicazioni 2D e 3D. In questo protocollo si illustrano brevemente le principali componenti hardware del setup, e più in dettaglio la scelta dei parametri e la preparazione del campione richiesto per un tipico esperimento che ci permette in seguito lo spostamento di un lisosoma con una risoluzione temporale elevata all&#39;interno di una cellula vivente.

<table><tbody><tr><td>Lysotracker DND 26</td><td>Life Technologies</td><td>L-7526</td><td></td></tr><tr><td>Tubuline tracker Green</td><td>Life Technologies</td><td>T34075</td><td></td></tr><tr><td>Mitotracker RED</td><td>Life Technologies</td><td>M7512</td><td></td></tr><tr><td>Coherent Chamelon- ultra II TI</td><td>Coherent</td><td></td><td></td></tr><tr><td>Glan Taylor Calcite Polarizer</td><td>Melles Griot</td><td>03PTA001</td><td></td></tr><tr><td>Galvanometer-motor mirror</td><td>Cambridge Technologies</td><td>M 6350</td><td></td></tr><tr><td>Dichroic mirror</td><td>Chroma Technologies</td><td>700 DCSPXR</td><td></td></tr><tr><td>Motorized stage</td><td>ASI</td><td>MS2000</td><td></td></tr><tr><td>Piezo&amp;nbsp;</td><td>PI</td><td>P721-LLQ</td><td></td></tr><tr><td>Photomultiplier tube</td><td>Hamamatsu</td><td>H7422P-40</td><td></td></tr><tr><td>Data acquisition card</td><td>IO tech</td><td>PCI 1128-4000</td><td></td></tr><tr><td>Imaging software</td><td>LFD</td><td>Global for images-SimFCS</td><td></td></tr></tbody></table>

3d orbital tracking in a modified two-photon microscope: an application to the tracking of intracellular vesicles

L&#39;obiettivo di questo protocollo video è quello di discutere come eseguire e analizzare un esperimento tridimensionale fluorescente orbitale particle tracking utilizzando una versione modificata del microscopio a due fotoni 1. A differenza di approcci convenzionali (scansione raster o largo campo basato su una pila di frame), il tracciamento orbitale 3D permette di localizzare e seguire con un spaziale elevata (10 precisione nm) e risoluzione temporale (risposta in frequenza 50 Hz) lo spostamento 3D di una particella fluorescente passare lunghezza-scale di centinaia di micron 2. Il metodo si basa su un algoritmo di feedback che controlla l&#39;hardware di un microscopio a scansione laser a due fotoni per eseguire un&#39;orbita circolare intorno all&#39;oggetto da tracciare: il meccanismo di feedback manterrà dell&#39;oggetto fluorescente al centro da comandare lo spostamento dei il raggio di scansione 3-5. Per dimostrare i vantaggi di questa tecnica, abbiamo seguito un organello in rapido movimento, il lisosoma, dentro alcellule iving 6,7. Le cellule sono state piastrate secondo protocolli standard, e colorate con un colorante commercialmente lisosoma. Discutiamo brevemente la configurazione hardware e più in dettaglio il software di controllo, effettuare un esperimento di tracciamento orbitale 3D all&#39;interno delle cellule viventi. Si parlerà in dettaglio i parametri richiesti per controllare il microscopio a scansione e consentire il movimento del fascio in un&#39;orbita chiusa attorno alla particella. Concludiamo dimostrando come questo metodo può essere efficacemente utilizzato per tracciare il movimento veloce di un lisosoma marcato lungo i microtubuli in 3D all&#39;interno di una cella dal vivo. Lisosomi possono muoversi con velocità nell&#39;intervallo 0,4-0,5 micron / sec, tipicamente visualizzando un moto diretto lungo la rete microtubuli 8.

Secondo questo protocollo veloce inseguimento singola particella 3D può essere eseguita all&#39;interno di cellule viventi utilizzando un microscopio a due fotoni modificato per monitorare lo spostamento dei lisosomi fluorescente. L&#39;esperimento eseguito consiste di tracciare un lisosoma isolato muoversi all&#39;interno della cella dopo il processo di maturazione endosome 9. I lisosomi sono stati colorati con un colorante verde fluorescente ed eccitato a 930 nm che sfruttano 2-fotone di eccitazione. I nos...

Watch this Scientific Journal Video about 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles at JoVE.com

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3D Orbital Inseguimento in una Modified microscopio a due fotoni: un&#39;applicazione per il monitoraggio di intracellulare vescicole

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

3d-orbital-tracking-modified-two-photon-microscope-an-application-to

Research

JoVE Journal

Bioengineering

10.2K Views.  University of California, Irvine. In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope. 

Video: 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

Visualizzazione del endosome Dynamics in Vivere terminazioni nervose con quattro-dimensionale imaging di fluorescenza

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Ricerca

Neuroscienze

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

Visualizzazione del traffico citoscheletro-dipendente di organelli contenenti lipidi in embrioni di Drosophila

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of ...

Biologia dello sviluppo

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 ...

Biologia

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Un protocollo per il monitoraggio in tempo reale 3D singola particella

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Bioingegneria

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

Quantificazione delle endosomi e dei motivi di lisosomi nei neuroni coltivati ​​utilizzando sonde fluorescenti

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

In Vivo Singola molecola Tracking al terminale presinaptico nervo motore Drosophila

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology. &#160;To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.&#160;

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

Microscopia 4D del lievito

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often require indirect methods due to their small diameter (40-250 nm), which is beneath the diffraction limit of typical light microscopy. We have developed a super-resolution microscopy-based visualization of EVs to bypass the diffraction limit in both two and three dimensions. Using this approach, we can resolve the three-dimensional shape of EVs to within +/- 20 nm resolution on the XY-axis and +/- 50 nm resolution along the Z-axis. In conclusion, we propose that super-resolution microscopy be considered as a characterization method of EVs, including exosomes, as well as enveloped viruses.

Direct stochastic optical reconstruction microscopy (dSTORM) is used to bypass the typical diffraction limit of light microscopy and to view exosomes at the nanometer scale. It can be employed in both two and three dimensions to characterize exosomes.

Microscopia a ricostruzione ottica stocastica diretta di vescicole extracellulari in tre dimensioni

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often ...

Visualization of Vesicle Motilities in Neurons Using Fluorescence Imaging

Source: Tsuruta, F., et al. <a href="https://app.jove.com/v/55488">Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes.</a> J. Vis. Exp. (2017).
This video demonstrates how neuronal vesicles are labeled with specific fluorescent proteins to visualize their movement. First, a plasmid coding for a fluorescent protein is incubated with a transfection agent and then introduced into a culture of neurons. Subsequently, images of the neurons are acquired and analyzed to track the vesicles&#39; movement.

Visualizzazione delle motilità delle vescicole nei neuroni mediante imaging a fluorescenza

1. Imaging Vesicle Motility

	Prepare the plasmids that will label each type of vesicle. For instance, use EGFP-Rab5 for early endosomes, EGFP-Rab7 for ...

3D Orbital Inseguimento in una Modified microscopio a due fotoni: un'applicazione per il monitoraggio di intracellulare vescicole

Riepilogo

Esplora Altri Video

Capitoli in questo video

Visualizzazione del endosome Dynamics in Vivere terminazioni nervose con quattro-dimensionale imaging di fluorescenza

Visualizzazione del traffico citoscheletro-dipendente di organelli contenenti lipidi in embrioni di Drosophila

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

Un protocollo per il monitoraggio in tempo reale 3D singola particella

Quantificazione delle endosomi e dei motivi di lisosomi nei neuroni coltivati utilizzando sonde fluorescenti

In Vivo Singola molecola Tracking al terminale presinaptico nervo motore Drosophila

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Microscopia 4D del lievito

Microscopia a ricostruzione ottica stocastica diretta di vescicole extracellulari in tre dimensioni

Visualizzazione delle motilità delle vescicole nei neuroni mediante imaging a fluorescenza

3D Orbital Inseguimento in una Modified microscopio a due fotoni: un'applicazione per il monitoraggio di intracellulare vescicole

Riepilogo

Esplora Altri Video

Capitoli in questo video

Visualizzazione del endosome Dynamics in Vivere terminazioni nervose con quattro-dimensionale imaging di fluorescenza

Visualizzazione del traffico citoscheletro-dipendente di organelli contenenti lipidi in embrioni di Drosophila

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

Un protocollo per il monitoraggio in tempo reale 3D singola particella

Quantificazione delle endosomi e dei motivi di lisosomi nei neuroni coltivati ​​utilizzando sonde fluorescenti

In Vivo Singola molecola Tracking al terminale presinaptico nervo motore Drosophila

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Microscopia 4D del lievito

Microscopia a ricostruzione ottica stocastica diretta di vescicole extracellulari in tre dimensioni

Visualizzazione delle motilità delle vescicole nei neuroni mediante imaging a fluorescenza

Quantificazione delle endosomi e dei motivi di lisosomi nei neuroni coltivati utilizzando sonde fluorescenti