Nous tenons à remercier Olga Garaschuk pour des discussions utiles sur l&#39;optimisation du chargement en vrac des indicateurs de calcium.

<ol><li> Préparer la solution de calcium indicateur fluorescent. Nous utilisons des colorants ester acétoxyméthyle, colorants ou AM pour faire court. Ce sont des colorants qui peuvent être prises par les cellules lorsqu&#39;elles sont injectées extracellulaire. Nous utilisons généralement l&#39;Oregon-Vert BAPTA-1 AM ou Fluo-quatre heures à une concentration de 1 mM. AM colorants peuvent être obtenus auprès de Invitrogen dans 50 flacons de microgramme. Nous avons d&#39;abord préparer une solution à 20% de Pluronic F-127 dans le DMSO. Nous chauffer la solution avant de l&#39;utiliser chaque jour jusqu&#39;à son clair. Nous dissoudre l&#39;indicateur de calcium du F-127/DMSO Pluronic 20% pour les 15-20 minutes à une concentration de 10 mM. Nous avons ensuite diluer la solution à 1 mM dans une solution contenant, en mm: 150 NaCl, 2,5 KCl, 10 HEPES, pH 7,4. En outre, 100 101 uM Sulforhodamine est habituellement inclus aux astrocytes étiquettes et de visualiser la pipette lors de l&#39;injection. Nous filtrer la solution avant l&#39;injection d&#39;un filtre 0,49 micron centrifuge (Stosiek et al, 2003;.. Garaschuk et al, 2006). </li><li> Implant une fenêtre crânienne sur la zone à imager. Nous avons <a href="http://jove.com/index/details.stp?ID=680" target="_blank">décrit l&#39;approche générale de JoVE</a> , mais les modifications suivantes sont importantes pour l&#39;injection de colorant de calcium: Faites une petite craniotomie de 2 mm de diamètre sur la surface corticale d&#39;intérêt, prenant un soin extrême à ne pas endommager le sous-tendent-mère. Fixez une lamelle en place au cours de la craniotomie, mais ne couvrent que partiellement la craniotomie, laissant un petit espace entre le bord de la craniotomie et la lamelle de verre pour laisser place à une pipette pour entrer dans le cortex oblique. Avant l&#39;injection, faire un puits peu profond avec du ciment dentaire autour de la craniotomie. Le bien doit s&#39;étendre en direction rostrale et être assez peu profond pour permettre l&#39;insertion de la micropipette pour l&#39;injection. </li><li> Transfert de la souris transféré à l&#39;étape du microscope et d&#39;immobiliser la tête, comme le montre notre <a href="http://jove.com/index/details.stp?ID=678" target="_blank">vidéo sur l&#39;imagerie du flux sanguin</a> . </li><li> Tirez pipettes microélectrode de verre à un diamètre de l&#39;extrémité céder 2-4 Ohms Mega dans la résistance, en utilisant un extracteur de pipette. Chargez le mélange de teinture de calcium indicateur sur la pipette de verre à l&#39;aide d&#39;une microseringue. L&#39;utilisation d&#39;un micromanipulateur, abaissez doucement la micropipette sur la surface du cerveau en utilisant un objectif 4X, qui est ensuite finement placé sous un objectif 40X. Choisissez un emplacement approprié pour l&#39;injection, de sorte qu&#39;il ya peu ou pas de vaisseaux sanguins recouvrant à obscurcir l&#39;image. Utiliser microscopie à deux photons, la pointe de la pipette est visualisée et s&#39;avança lentement dans le cortex, à une profondeur de 200 micromètres en dessous de la dure-mère. Ensuite, la pression d&#39;injecter le mélange de teinture à 10 psi pendant 1 minute en utilisant un Picospritzer. En général, nous livrer deux à quatre injections du mélange de colorants AM calcium, séparés dans l&#39;espace d&#39;environ 200-300 microns. Cette approche de chargement en vrac des injections qui se chevauchent légèrement assure une région aussi grande que 600 x 600 microns est suffisamment colorées pour l&#39;imagerie calcique. Commencez d&#39;imagerie 1 heure après l&#39;injection du mélange de colorant AM, afin de permettre au colorant de diffuser et être repris par les neurones. </li><li> Effectuer in vivo à deux photons avec une imagerie personnalisée faite microscope à deux photons en utilisant un caméléon Ti-Sapphire Laser (800-880 accordé à nm) et des miroirs de Cambridge Technology galvanomètre. Nous acquérir des images à l&#39;aide du logiciel développé dans le laboratoire scanimage Karel Svoboda (Pologruto et al., 2003). Puissance du laser est généralement bien en dessous de 70 mW à l&#39;échantillon. Ensemble du champ des images sont acquises soit en utilisant un 20X (0,95 NA) ou 40X (0,8 NA) objectifs Olympus, à 1.95 à 15.63 images par seconde (512 x 256 pixels à 256 x 64 pixels). Ligne scans sont acquises à 500 Hz. Lors d&#39;imagerie, les animaux sont maintenus sous anesthésie à l&#39;isoflurane léger (0,6-0,9%), et sont également maintenus à 37 ° C en utilisant un appareil Harvard température de l&#39;appareil de contrôle. Il est pris soin de garder le rythme respiratoire des animaux au plus près à 100 respirations / minute que possible. Ce niveau d&#39;utilisation d&#39;anesthésie est le niveau le plus bas qui immobilise l&#39;animal, mais permet encore l&#39;activité spontanée d&#39;être enregistré. </li></ol>

<ol>
	<li>Garaschuk, O., Milos, R. I., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+bulk-loading+of+fluorescent+indicators+for+two-photon+brain+imaging+in+vivo.">Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo.</a> Nat Protoc. 1, (2006).</li><li>Stosiek, C., Garaschuk, O., Holthoff, K., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vivo+two-photon+calcium+imaging+of+neuronal+networks.">In-vivo two-photon calcium imaging of neuronal networks.</a> Proc Natl Acad Sci USA. 100, 7319-7324 (2003).</li><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.&amp;nbsp;&amp;nbsp;+Biomed+Eng+Online+2:13.">ScanImage: flexible software for operating laser scanning microscopes.&amp;nbsp;&amp;nbsp; Biomed Eng Online 2:13.</a> Biomed Eng Online. 2, (2003).</li></ol>

L&#39;avantage de cette méthode sur des enregistrements électrophysiologiques, c&#39;est qu&#39;il est moins invasive et permet l&#39;enregistrement de l&#39;activité neuronale dans les denses réseaux de neurones. Lorsque vous effectuez cette procédure, il est important de se rappeler de prendre un soin extrême lors de l&#39;exécution de la craniotomie pour ne pas endommager la mère. Même une petite quantité de saignement sous-dural avec énormément obscurs de l&#39;imagerie.

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<td>Oregon Green BAPTA1-AM</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>O-6807</td>
<td>&nbsp;</td>
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<td>Fluo 4- AM</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>F-14201</td>
<td>&nbsp;</td>
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<td>Sulforhodamine 101</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>S-359</td>
<td>&nbsp;</td>
<tr>
<td>Pluronic F-127 in DMSO</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>P-3000MP</td>
<td>&nbsp;</td>
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<td>Centrifugal Filter (0.45 micron)</td>
<td>Reagent</td>
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in vivo 2-photon calcium imaging in layer 2/3 of mice

Pour comprendre la dynamique des réseaux de microcircuits du néocortex, il est indispensable d&#39;enregistrer simultanément l&#39;activité d&#39;un grand nombre de neurones. L&#39;imagerie in vivo de calcium à deux photons est la seule méthode qui permet d&#39;enregistrer l&#39;activité d&#39;une population dense de neurones avec une seule cellule de résolution. La méthode consiste à implanter une fenêtre d&#39;imagerie crânienne, l&#39;injection d&#39;un colorant fluorescent de calcium indicateur qui peut être repris par un grand nombre de neurones et finalement d&#39;enregistrer l&#39;activité des neurones à l&#39;imagerie calcique laps de temps en utilisant une in-vivo deux microscope photonique. La co-injection des astrocytes spécifiques colorants permet de différencier les neurones à partir des astrocytes. La technique peut être réalisée sur des souris exprimant des molécules fluorescentes dans les sous-populations spécifiques de neurones pour mieux comprendre les interactions en réseau des différents groupes de cellules.

Watch this Scientific Journal Video about In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice at JoVE.com

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

Pour comprendre la dynamique des réseaux de microcircuits du néocortex, il est indispensable d&#39;enregistrer simultanément l&#39;activité d&#39;un grand nombre de neurones. L&#39;imagerie in vivo de calcium à deux photons est la seule méthode qui permet d&#39;enregistrer l&#39;activité d&#39;une population dense de neurones avec une seule cellule de résolution.

In Vivo 2-Photon Imaging calcium dans la couche 2 / 3 de souris

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

in-vivo-2-photon-calcium-imaging-in-layer-23-of-mice

Research

JoVE Journal

Biology

28.0K vues.  University of California, Los Angeles. Pour comprendre la dynamique des réseaux de microcircuits du néocortex, il est indispensable d'enregistrer simultanément l'activité d'un grand nombre de neurones. L'imagerie in vivo de calcium à deux photons est la seule méthode qui permet d'enregistrer l'activité d'une population dense de neurones avec une seule cellule de résolution.

Vidéo: In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response 1. Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 2. Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37&deg;C.

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.

Dissection et 2-Photon Imaging des ganglions lymphatiques périphériques chez les souris

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the ...

Recherche

Biologie

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

Dans l&#39;imagerie in situ de la souris en utilisant deux Thymus microscopie biphotonique

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

Une méthode pour le 2-Photon Imaging du flux sanguin dans le néocortex travers une fenêtre crânienne

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

Génération de la moelle osseuse des cellules dendritiques dérivées murin pour l&#39;utilisation dans l&#39;imagerie 2-photons

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

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, ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Imagerie fonctionnelle de la graisse brune chez les souris avec FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

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) ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

En détection de visage de l&#39;oxyde nitrique et de superoxyde dans la couche endothéliale des artères intactes

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman&#39;s space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)-dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman&#39;s space and all segments of the nephron within the imaging depth limit, about 100 &#181;m below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman&#39;s space. We expect this technique to have wide applicability in renal physiologic investigation.

Micropuncture of Bowman&#039;s Space in Mice Facilitated by 2 Photon Microscopy

We present use of 2-photon microscopy to place a micropipette within Bowman&#39;s urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

Microponction de l’espace de Bowman chez les souris facilitée par 2 Photon Microscopy

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Imagerie de la dynamique du calcium dans les sous-populations de cellules d'islet pancréatique de souris

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...