Molte grazie a Dr. Jennifer Gutzman che per primo ha sviluppato questa tecnica in laboratorio Sive. Grazie anche a JB Verde presso il Dana-Farber Cancer Institute di Boston, MA che ha gentilmente fornito la codifica plasmide CAAX GFP-mRNA. L&#39;imaging confocale è stato condotto presso il WM Keck Foundation Imaging Fondo Biologiche presso l&#39;Whitehead. HS è supportato da NIHMH70926. EG è supportato da un pre-dottorato NSF.

1. Preparazione del mRNA per iniezione <ol><li> L&#39;mRNA utilizzati in questa procedura è trascritto da un plasmide codifica CAAX-eGFP (memGFP) mRNA. In primo luogo linearizzare il plasmide secondo Gutzman et al, 2008. </li><li> Poi trascrivere mRNA memGFP utilizzando il mMessage mMachine kit. </li><li> Diluire l&#39;mRNA risultante 1μg/μl, aliquota, e conservare a -80 ° C. </li><li> Preparare uno stampo ad iniezione di 1% agarosio con corsie della larghezza di embrioni allo stadio di una cellula. </li><li> Il giorno prima dell&#39;iniezione, istituito gabbie accoppiamento che separa il maschio dalla femmina. </li><li> Il giorno di iniezione, tirare aghi capillare con un estrattore micropipetta per preparare l&#39;iniezione. </li></ol> 2. Iniezione del mRNA <ol><li> Il giorno di iniezione, disgelo un&#39;aliquota di mRNA memGFP 1μg/μl sul ghiaccio. </li><li> Diluire il mRNA 1:5 in acqua per una concentrazione finale di 200ng/μl, e tenere in ghiaccio. </li><li> Caricare l&#39;ago capillare con 1ml di mRNA memGFP preparati a 200ng/μl. </li><li> Luogo carico ago in un micromanipolatore collegato a un alimentati a gas microinjector. </li><li> Regolare il volume di iniezione di 1nl. </li><li> Tirare i divisori di pesce di tipo selvatico in gabbia accoppiamento del giorno precedente. </li><li> Raccogliere gli embrioni non appena sono previste. Orientarle nello stampo ad iniezione agarosio coperto da media embrione (Westerfield, 1995) con la singola cella sul lato opposto del micromanipolatore. </li><li> Iniettare attraverso il corion e tuorlo in modo tale che l&#39;mRNA viene depositato direttamente nella cella singola. Iniettare circa 50 embrioni per sperimentare. Non iniettare se la cella ha diviso. </li><li> Embrioni incubare a 28 ° C per una notte in media embrione </li></ol> 3. Montaggio e embrioni Imaging <ol><li> Per montare l&#39;immagine e gli embrioni, rimuovere il corion con una pinza da embrioni sotto uno stereomicroscopio un&#39;ora prima del punto di tempo di interesse. </li><li> Preparare un vetrino per il montaggio fino a 4 embrioni. <ul><li> Usare l&#39;aspiratore grasso al silicone per sigillare un coprioggetto sulla parte inferiore di un apposito 2mm scivolo di plastica con un foro di circa 1 cm di diametro. </li><li> Riempire il buco nella diapositiva con il 0,7% di agarosio, e lasciate riposare circa 20 minuti o fino a quando indurito </li><li> Una volta che l&#39;agarosio si è solidificato, rimuovere una piccola spina con 200μl puntali per formare fori cilindrici in cui montare ogni embrione. </li></ul></li><li> Mettere gli embrioni in agarosio pieno vetrino sotto un microscopio da dissezione. Aggiungere 50μl di tricaine (Westerfield, 1995) per anestetizzare gli embrioni. </li><li> Usare pinze per posizionare gli embrioni nei fori cilindrici in agarosio con il cervello o la regione di interesse contro il coprioggetto sottostante. </li><li> Coprire gli embrioni in agarosio con un altro vetrino fissato con grasso al silicone vuoto. </li><li> Immagine l&#39;embrione utilizzando un invertito, fluorescente, laser-scanning o spinning microscopio confocale disco. Immagine a 63x o superiore per raccogliere immagini ad alta risoluzione di singole cellule all&#39;interno del neuroepitelio. </li><li> Le immagini vengono esportate come file TIFF dal software LSM e analizzati utilizzando Photoshop. </li></ol> 4. Risultati rappresentante / Esito Mostrato nella Figura 1 è un&#39;immagine rappresentativa confocale di un neuroepitelio 24 zebrafish hpf tra il mesencefalo e romboencefalo ventricoli. Ogni cella è etichettato con membrana GFP. <img src="/files/ftp_upload/1217/Graeden JoVE figure 1.png" alt="" width="450" height="449" /> Figura 1. Vivere l&#39;imaging confocale di un memGFP a iniezione 24 embrione zebrafish HPF. (A) neuroepitelio di un embrione di 24 HPF con ogni cella delineato da GFP. Regione ripreso separa il mesencefalo e romboencefalo ventricoli (M, H rispettivamente), che formano il mesencefalo-rombencefalo confine.

<ol>
	<li>Gutzman, J. H., Graeden, E., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+zebrafish+midbrain-hindbrain+boundary+constriction+requires+laminin-dependent+basal+constriction.">Formation of the zebrafish midbrain-hindbrain boundary constriction requires laminin-dependent basal constriction.</a> Mech Dev. 125 (974), 11-12 (2008).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book: a Guide for the Laboratory Use of Zebrafish. , (1995).</li></ol>

In questo video, si dimostra un metodo per iniezione di mRNA in embrioni di zebrafish singola cellula. Qui, usiamo mRNA codifica una membrana mirati GFP di etichettare ogni cella. Abbiamo poi dimostrare come montare e immagine lo sviluppo del cervello a risoluzione singola cellula. Questa tecnica ci ha permesso di studiare un nuovo tipo di cella di cambiare forma, costrizione basale, necessari per la formazione del mesencefalo-romboencefalo confine (Gutzman et al, 2008). Un&#39;analisi simile di altri fenomeni ha il pot...

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		<th>Comment</th>
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<tr>
<td>SeaKem GTG agarose</td>
<td> Reagent</td>
<td>Lonza</td>
<td>50027</td>
<td>&nbsp;</td>
<tr>
<td>3-aminobenzoic acid ethyl ester (Tricaine)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-5040</td>
<td>&nbsp;</td>
<tr>
<td>Capillary tubing</td>
<td>Tool</td>
<td>Frederick Haer and Co.</td>
<td>30-30-1</td>
<td>Borosil 1.0mmOD x 0.5mm ID/fiber 100mm</td>
</tr>
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<td>Silicone vacuum grease</td>
<td>Reagent</td>
<td>VWR</td>
<td>W0S717</td>
<td>&nbsp;</td>
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<td>Forceps</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11232-20</td>
<td>Dumont #5 Bio Inox</td>
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<td>1-200 &#x03BC;l Pipette tips</td>
<td>Tool</td>
<td>Tip One, US Scientific</td>
<td>111-0806</td>
<td>These are the correct size for 24 hpf embryos. </td>
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<td>mMessage mMachine transcription kit</td>
<td>Reagent</td>
<td>Ambion</td>
<td>AM1340</td>
<td>SP6 RNA polymerase</td>
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<td>Zeiss LSM 510 scanning confocal </td>
<td>Microscope</td>
<td>Zeiss</td>
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<td>Micromanipulator</td>
<td>Tool</td>
<td>Narishige</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Microinjector</td>
<td>Tool</td>
<td>Medical Systems Research Products Harvard Apparatus</td>
<td>PLI-100</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette puller</td>
<td>Tool</td>
<td>Sutter Instruments</td>
<td>&nbsp;</td>
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live imaging of the zebrafish embryonic brain by confocal microscopy

In questo video, dimostriamo il metodo nostro laboratorio ha sviluppato per analizzare i cambiamenti delle cellule forma e riarrangiamenti necessari per piegare e ripiegare il cervello pesce zebra di sviluppo (Gutzman et al, 2008). Tale analisi offre una nuova comprensione della biologia cellulare di base necessari per lo sviluppo della struttura 3D del cervello dei vertebrati, e aumenta notevolmente la nostra capacità di studiare la morfogenesi del tubo neurale. Il cervello ha la forma embrionale di zebrafish con inizio alle 18 ore dopo la fecondazione (HPF), come i ventricoli all&#39;interno del neuroepitelio gonfiare. Da 24 HPF, i passi iniziali della morfogenesi del tubo neurale sono completi. Utilizzando il metodo descritto qui, gli embrioni allo stadio di una cellula sono iniettati con mRNA codifica membrana mirati proteina verde fluorescente (memGFP). Dopo l&#39;iniezione e l&#39;incubazione, l&#39;embrione, ormai tra i 18 ei 24 HPF, è montato, capovolta, in agarosio e fotografato al microscopio confocale. In particolare, l&#39;embrione di zebrafish è trasparente che lo rende un sistema ideale per l&#39;imaging a fluorescenza. Mentre le nostre analisi si sono concentrate sul mesencefalo e il romboencefalo confine romboencefalo, questo metodo potrebbe essere esteso per l&#39;analisi di qualsiasi altra regione del zebrafish ad una profondità di 80-100 micron.

Watch this Scientific Journal Video about Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy at JoVE.com

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In questo video, si dimostra un metodo con cui analizzare il cervello dei vertebrati sviluppo in embrioni di zebrafish dal vivo a una risoluzione singola cellula al microscopio confocale. Questo include il metodo con cui iniettare il unicellulare zebrafish e, successivamente, montare e immagine lo sviluppo del cervello.

Vivere Imaging of the Brain Zebrafish embrionali da microscopia confocale

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

live-imaging-of-the-zebrafish-embryonic-brain-by-confocal-microscopy

Research

JoVE Journal

Biology

18.5K Views.  MIT - Massachusetts Institute of Technology. In questo video, si dimostra un metodo con cui analizzare il cervello dei vertebrati sviluppo in embrioni di zebrafish dal vivo a una risoluzione singola cellula al microscopio confocale. Questo include il metodo con cui iniettare il unicellulare zebrafish e, successivamente, montare e immagine lo sviluppo del cervello.

Video: Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

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

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Immagini dal vivo di Drosophila melanogaster Migrazioni Hemocyte embrionali

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

Immagini dal vivo di estrusione in cella dal Epidermide di Zebrafish in via di sviluppo

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Basato sulla fluorescenza Misura Store-Entry operato di calcio in cellule vive: dal Cancer Cell coltura per fibra muscolare scheletrica

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

Imaging 3D e ricostruzione di strutture cerebrovascolari in Zebrafish embrionali

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Analizzando craniofacciale morfogenesi in Zebrafish Uso 4D Microscopia confocale

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

Imaging cellulare dal vivo di primaria ratto neonato Cardiomyocytes seguito Adenoviral e lentivirali trasduzione Uso confocale Spinning Disk Microscopia

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Here we present a protocol to image cells expressing green fluorescent protein-tagged angiotensin type 1a receptors during endocytosis initiated by angiotensin II treatment. This technique includes labeling lysosomes with a second fluorescent marker, and then utilizing software to analyze the co-localization of receptor and lysosome in three dimensions over time.

Imaging cellulare dal vivo e analisi 3D del recettore dell&#39;angiotensina di tipo 1a tratta degli transfettati embrionali umane cellule renali Utilizzando Microscopia confocale

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Preparazione del campione e la formazione immagine di esosomi da microscopia elettronica di trasmissione

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...