Nous remercions Anthony J. Koleske, PhD pour fournir les souris YFP.

Partie 1: Optique microprismes et de configuration de microscope <ol><li> Les microprismes que nous utilisons sont de 1 mm, angle droit prismes à base de verre BK7 achetés auprès de la Société Optosigma (figure 1). Microprismes sont traitées en utilisant une pince quand c&#39;est possible. </li><li> L&#39;hypoténuse du microprismes est revêtu d&#39;argent améliorée afin de fournir une réflectivité supérieure à 97% de 400 à 2000 nm. Ceci fournit une réflexion interne à la fois la lumière d&#39;excitation laser et la fluorescence émise. </li><li> Microprismes sont toujours traités en utilisant une pince quand c&#39;est possible. Les gants sont portés en tout temps pour garder le prisme propre. </li><li> Nous utilisons une coutume construit microscope à deux photons capables d&#39;imagerie du petit animal. L&#39;animal, attaché à l&#39;appareil de stéréotaxie, est placé sur une scène à 3 axes motorisés. </li><li> Le microscope est connecté à un PC Windows exécutant le logiciel scanimage. Scanimage est un logiciel d&#39;acquisition d&#39;image écrit en Matlab par Pologruto et al 1. </li><li> Pour l&#39;imagerie microprismes, nous recueillons des images à une résolution de 1024 x 1024 ou 512 x 512 pixels. De plus, nous avons généralement scan à 2 ms / ligne ou 4 ms / ligne. </li><li> Deux types d&#39;objectifs sont utilisés dans les expériences: <ul><li> Inférieure un objectif chiffré de l&#39;air d&#39;ouverture (NA) pour grand champ de vision d&#39;imagerie (voir tableau des réactifs et des équipements). </li><li> Un objectif plus élevé de l&#39;air NA avec un collier de verre de correction susceptible de minimiser les aberrations sphériques induites par 0 - 2 mm de verre (voir le tableau des réactifs et des équipements). </li></ul></li><li> Lorsque l&#39;animal est prêt pour l&#39;imagerie sous un objectif à faible grossissement, d&#39;aligner le modèle raster carrés de balayage du laser avec le haut de la microprismes. Cela aidera à orienter l&#39;image et de faire une utilisation efficace de la puissance du laser. </li><li> En utilisant un objectif plus élevé NA avec un collier de verre de correction, régler la bague de correction pour corriger environ 1 mm de verre. Une fois une image fluorescente apparaît pendant l&#39;expérience, on peut bien accéder à l&#39;intérieur du microscope pour optimiser la bague de correction afin de maximiser la résolution d&#39;imagerie. </li></ol> Partie 2: La chirurgie des animaux non-survie et d&#39;insertion microprismes <ol><li> La longueur entière procédure, à partir de l&#39;incision initiale jusqu&#39;à l&#39;achèvement de l&#39;imagerie, dépend des objectifs de l&#39;expérience. Cependant, le placement non-intervention chirurgicale et la survie de microprismes peuvent être devrait prendre environ 30 - 40 minutes à compléter. </li><li> Souris (P30-P60) sont pesés et anesthésiés appropriée en utilisant une injection IP de kétamine (100 mg / kg) et de xylazine (10 mg / kg) à un plan approprié pour les procédures chirurgicales. </li><li> Tout au long de toute l&#39;expérience, l&#39;animal doit être vérifié toutes les 15-20 minutes pour évaluer son niveau de l&#39;anesthésie. Si l&#39;animal réagit à une entreprise toe-pincement, une dose supplémentaire de kétamine / xylazine est nécessaire. Généralement, une dose supplémentaire est d&#39;un tiers de la dose initiale et peut être préparé à l&#39;avance dans une seringue pour injection IP immédiat si nécessaire. </li><li> Une fois que la souris est correctement anesthésié, la majorité des cheveux sur la tête de l&#39;animal est rasé en utilisant un potentiomètre avec une lame # 40. </li><li> Nair ® est appliqué aux poils restant sur la tête pour aider à créer une surface libre de poils qui peuvent obtenir de la manière de l&#39;microprismes pendant l&#39;imagerie. Après 5 minutes, le Nair ® est nettoyée en utilisant un coton-tige. </li><li> L&#39;animal anesthésié est correctement placé dans un appareil stéréotaxique de la souris. Pendant les procédures chirurgicales et des séances d&#39;imagerie est gardé l&#39;animal sur un rempli d&#39;eau coussin chauffant réglé à 36 degrés Celsius. </li><li> Avec tête de l&#39;animal soit correctement fixé, une incision propre est faite en utilisant un scalpel sur la ligne médiane du crâne de séparer la peau. </li><li> Une petite quantité de peroxyde d&#39;hydrogène à 3% est placée sur un coton-tige et appliqué sur le crâne pour déblayer les membranes entre le crâne et la peau. </li><li> Le peroxyde d&#39;hydrogène permet également de révéler bregma, un jalon important de savoir où effectuer la craniotomie et insérez le microprismes. </li><li> Avant la craniotomie est démarré, les barres d&#39;oreille bouchée et un bar sont correctement ajustés à l&#39;inclinaison de la boîte crânienne exposée telle qu&#39;elle est essentiellement au niveau du tableau. Cela fournit une meilleure plateforme pour la craniotomie. De plus, ce qui rend la surface supérieure de la perpendiculaire prisme à la lumière d&#39;excitation entrants. Cela permettra de minimiser les aberrations optiques tout en imagerie. </li><li> Une petite bavure dentaires (~ 0,8 mm taille de la tête) est connecté à un outil de Dremel ® à l&#39;aide d&#39;un bras flexible de prolongation. Pour craniotomies, nous avons mis la vitesse à environ 7.000 à 10.000 RPM. </li><li> La fraise dentaire est écrémé le long du crâne jusqu&#39;à une rainure carrée est établie dans l&#39;os. Les côtés du carré sont environ 2-3 mm de longueur et 1,5 centré caudale et 1 mm latéral bregma. </li><li> Poursuivant l&#39;amincissement de l&#39;os jusqu&#39;à ce qu&#39;une petite ouverture se fait à travers le crâne. Une paire de pinces à can soulever le volet osseux loin du crâne. </li><li> La durée doit être enlevé avant que le microprismes peuvent être insérés dans le cortex. Le saignement est mieux contrôlée en laissant le sang coagule à la surface pendant plusieurs minutes. Ensuite, le sang coagulé est enlevée à l&#39;aide d&#39;une éponge chirurgicale avant d&#39;insérer la microprismes. </li><li> Pour aider à faciliter l&#39;alignement du prisme avec le cortex, la face verticale de la microprismes est aligné parallèlement à la poignée sur la pince. </li><li> Avec le prisme bien tenu, les microprismes ensemble est inséré jusqu&#39;au sommet du prisme est au ras de la surface du néocortex. </li><li> Lorsqu&#39;elle est insérée correctement, les microprismes doit rester dans la bonne position. Tout saignement qui en résulte est minime et peut être absorbé avec une petite éponge chirurgicale. Une fois le prisme est en place l&#39;animal est prêt pour l&#39;imagerie. </li><li> Après un microprisme a été utilisé dans un animal, il peut être réutilisé plusieurs fois. Toutefois, il ne doivent être correctement nettoyées pour éliminer tout résidu biologique. Ceci peut être accompli en trempant le prisme dans un petit volume d&#39;acide HCl, suivie par un plongeon dans le méthanol. Les microprismes peuvent ensuite être nettoyé enveloppés dans du papier lentille jusqu&#39;à une utilisation future. </li></ol> Partie 3: Résultats Représentant <img src="/files/ftp_upload/1509/1509fig1.jpg" alt="Figure 1" /> Figure 1: Images de l&#39;microprismes à angle droit. Prismes utilisés dans cette expérience sont d&#39;un millimètre, des prismes de verre BK7 avec un revêtement en argent renforcée sur l&#39;hypoténuse pour permettre une réflexion interne de l&#39;excitation et l&#39;émission de lumière. <img src="/files/ftp_upload/1509/1509fig2.jpg" alt="Figure 2" /> Figure 2: Image de la couche de neurones pyramidaux V YFP. Large champ de vision avec l&#39;imagerie microprismes révèle une grande population de corps cellulaires pyramidaux ~ 800 à 900 um de profondeur de la surface corticale. En outre, les dendrites apicales s&#39;étendant jusqu&#39;à vers la couche I peuvent également être résolus. Barre d&#39;échelle = 200 pm. <img src="/files/ftp_upload/1509/1509fig3.jpg" alt="Figure 3" /> Figure 3: Un objectif d&#39;ouverture numérique élevée utilisés en conjonction avec les microprismes fournit une résolution spatiale suffisante pour résoudre les épines dendritiques sur les dendrites apicales des neurones pyramidaux couche V. Barre d&#39;échelle = 10 um. <img src="/files/ftp_upload/1509/1509fig4.jpg" alt="Figure 4" /> Figure 4: Une image obtenue en utilisant les microprismes et une injection de queue veine d&#39;un colorant fluorescent. L&#39;image montre verticales, les navires de gros calibre qui s&#39;étend de régions profondes du cerveau et les petits capillaires bifurquant à travers le volume cortical. Barre d&#39;échelle = 200 pm. <img src="/files/ftp_upload/1509/1509fig5.jpg" alt="Figure 5" /> Figure 5: Utilisation d&#39;un objectif plus élevé ouverture numérique, le réseau de capillaires dans le néocortex peuvent être imagées avec plus de détails. Barre d&#39;échelle = 50 microns. <img src="/files/ftp_upload/1509/1509fig6.jpg" alt="Figure 6" /> Figure 6: microprismes permet aussi pour les cellules sanguines d&#39;imagerie rouges (hématies) circulant dans les vaisseaux. Globules rouges n&#39;absorbent pas le colorant fluorescent et sont donc considérés comme des rayures foncées sur le navire. En ligne de balayage dans le récipient, on peut obtenir des mesures de flux de RBC et de vitesse. Barre d&#39;échelle = 10 microns (barre horizontale à l&#39;image de haut et bas) et 250 ms (barre verticale à l&#39;image de fond).

<ol>
	<li>Pologruto, T. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.">ScanImage: flexible software for operating laser scanning microscopes.</a> Biomed Eng Online. 2, 13-13 (2003).</li><li>Feng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28 (1), 41-41 (2000).</li><li>Kleinfeld, D., Mitra, P. P., Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+and+stimulus-induced+changes+in+blood+flow+observed+in+individual+capillaries+in+layers+2+through+4+of+rat+neocortex.">Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex.</a> Proc Natl Acad Sci U S A. 95 (26), 15741-15741 (1998).</li></ol>

Expériences sur les animaux ont été effectuées en conformité avec les directives et règlements établis par l&#39;Université de Yale IACUC.

Utiliser un microprisme dans une expérience d&#39;imagerie est relativement simple et offre de nombreux avantages pour les études in vivo sur le néocortex. Des exemples représentatifs en utilisant cette technique comprennent des images prises à partir des souris transgéniques exprimant une protéine fluorescente jaune dans les neurones V couche corticale. Utiliser microprismes, on peut voir une collection de grands corps des cellules pyramidales dans la couche V, près de 1 mm en dessous de la surface corticale. O...

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<td>With enhanced silver reflective coating-(R77)</td>
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<td>0.28 NA, 4x air objective</td>
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in vivo imaging of deep cortical layers using a microprism

Nous présentons un protocole pour l&#39;imagerie in vivo du tissu cortical en utilisant une sonde d&#39;imagerie cérébrale profonde dans la forme d&#39;un microprisme. Microprismes sont de 1 mm en taille et ont un revêtement réfléchissant sur l&#39;hypoténuse pour permettre une réflexion interne de l&#39;excitation et l&#39;émission de lumière. La sonde microprismes simultanément plusieurs images couches corticales avec une perspective typiquement observée uniquement dans les préparations tranche. Les images sont recueillies avec un grand champ de vue (~ 900 um). En outre, nous fournissons des détails sur la procédure de non-chirurgical et la survie de configuration de microscope. Les résultats représentatifs comprennent des images de neurones pyramidaux couche V de Thy-1-H YFP souris montrant leurs dendrites apicales qui s&#39;étend à travers la couche superficielle du cortex et s&#39;étendant dans des touffes. Résolution a été suffisante pour épines dendritiques image proche du soma des neurones V couche. Une injection de queue veine de colorant fluorescent révèle le réseau complexe de vaisseaux sanguins dans le cortex. À balayage linéaire de globules rouges (hématies) qui coule à travers les capillaires révèle la vitesse de RBC et les taux de flux peuvent être obtenues. Cette sonde microprismes roman est un élégant mais puissant méthode nouvelle de visualiser en profondeur les structures cellulaires et la fonction corticale in vivo.

Watch this Scientific Journal Video about In vivo Imaging of Deep Cortical Layers using a Microprism at JoVE.com

In vivo Imaging of Deep Cortical Layers using a Microprism

À angle droit microprismes inséré dans le néocortex de souris permet d&#39;imagerie profonde de plusieurs couches corticales avec un point de vue on trouve généralement dans les tranches. Un millimètre microprismes offrent un champ de vision large (~ 900 um) et des résolutions spatiales suffisantes pour résoudre les épines dendritiques. Nous démontrons la couche V imagerie neuronale et néocorticale imagerie vasculaire en utilisant microprismes.

L&#39;imagerie in vivo de Deep couches corticales utilisant un microprisme

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

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Biology

In vivo Imaging of Deep Cortical Layers using a Microprism

11.3K vues.  Yale University. À angle droit microprismes inséré dans le néocortex de souris permet d'imagerie profonde de plusieurs couches corticales avec un point de vue on trouve généralement dans les tranches. Un millimètre microprismes offrent un champ de vision large (~ 900 um) et des résolutions spatiales suffisantes pour résoudre les épines dendritiques. Nous démontrons la couche V imagerie neuronale et néocorticale imagerie vasculaire en utilisant microprismes.

Vidéo: In vivo Imaging of Deep Cortical Layers using a Microprism

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

Préparation de cultures souris Dissocié neurones corticaux

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

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Biologie

Calcium Imaging of Cortical Neurons using Fura-2 AM

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths.  The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding.  Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.

Imagerie calcique des neurones corticaux utilisant Fura-2 heures

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium ...

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

In vivo Bioluminescent imagerie des tumeurs mammaires qui utilisent des fréquences IVIS

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Mésoscopique fluorescence tomographie pour In vivo L&#39;imagerie du développement Drosophile</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

In vivo d&#39;embryons de drosophile Centrifugation

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Imagerie interactions protéine-protéine In vivo</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

Une seule molécule d&#39;imagerie du règlement Gene<em&gt; In vivo</em&gt; Utilisation de l&#39;activation par clivage cotraductionnelle (COTRAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

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 suivi 4-Dimensional de hématopoïétiques cellules souches et progénitrices adultes dans la voûte du crâne de souris de moelle osseuse

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

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Quantification de l&#39;expression protéique et co-localisation utilisant multiplexé Immuno-histochimie Coloration et multispectrale Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Mesure de la rigidité de<em&gt; Ex Vivo</em&gt; Souris aortes Utiliser Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...