Gostaríamos de agradecer Olga Garaschuk para discussões úteis sobre como otimizar o carregamento a granel de indicadores de cálcio.

<ol><li> Prepare a solução indicador fluorescente de cálcio. Nós usamos corantes éster acetoxymethyl, ou AM corantes para breve. Estes são corantes que podem ser absorvidas pelas células quando injetado extracelularmente. Normalmente usamos Oregon-Green-BAPTA 01:00 ou 04:00 Fluo-a uma concentração de 1 mM. Corantes AM podem ser obtidas Invitrogen em frascos de 50 microgramas. Primeiro, preparar uma solução de 20% dos PLURONIC F-127 em DMSO. Nós aquecer a solução antes de usar todos os dias até a sua clara. Nós dissolver o indicador de cálcio F-127/DMSO PLURONIC 20% por 15-20 minutos a uma concentração de 10 mM. Em seguida, diluir a solução de 1mM em uma solução contendo, em mM: NaCl 150, KCl 2,5, 10 Hepes, pH 7.4. Além disso, 100 Sulforhodamine M 101 é normalmente incluído para astrócitos rótulo e visualize a pipeta durante a injecção. Nós filtrar a solução antes da injeção com um filtro de 0,49 micron centrífuga (Stosiek et al, 2003;.. Garaschuk et al, 2006). </li><li> Implante uma janela cranial sobre a área a ser trabalhada. Temos <a href="http://jove.com/index/details.stp?ID=680" target="_blank">descreveu a abordagem geral em JOVE</a> , mas as seguintes modificações são importantes para a injeção de corante de cálcio: Fazer uma pequena craniotomia de 2 mm de diâmetro sobre a área cortical de interesse, tomando extremo cuidado para não danificar a dura-máter subjacente. Garantir uma lamela no lugar sobre a craniotomia, mas apenas parcialmente cobrir a craniotomia, deixando um pequeno espaço entre a borda da craniotomia e da lamela de vidro para permitir espaço para uma pipeta para entrar no córtex obliquamente. Antes da injeção, faça um buraco raso com cimento dental ao redor do craniotomia. O bem deve estender-se em direção rostral e ser rasa o suficiente para permitir a inserção da micropipeta de injeção. </li><li> Transferir o mouse transferido para o estágio do microscópio e imobilizar a cabeça, como mostra a nossa <a href="http://jove.com/index/details.stp?ID=678" target="_blank">imagem de vídeo no fluxo sanguíneo</a> . </li><li> Puxe pipetas de vidro de microeletrodos para um diâmetro da ponta rendendo 2-4 Ohms mega na resistência, usando um extrator pipeta. Carregar o mix de cálcio corante indicador para a pipeta de vidro, utilizando uma micro. Usando um micromanipulador, delicadamente abaixar a micropipeta sobre a superfície do cérebro usando uma objetiva de 4X, que é então finamente posicionado sob objetiva de 40X. Escolha um local adequado para injeção, de tal forma que existem vasos sobrejacente poucos ou nenhum sangue para obscurecer a imagem. Utilizando dois fótons de microscopia, a ponta da pipeta é visualizado e avançado lentamente no córtex, a uma profundidade de 200 micrômetros abaixo da dura-máter. Então, a pressão de injetar a mistura de corante em 10 PSI por 1 minuto usando um Picospritzer. Costumamos oferecer 2-4 injeções da mistura de corante de cálcio AM, separados no espaço por cerca de 200-300 microns. Esta abordagem carregamento a granel de injeções ligeiramente sobrepostos garante que uma área tão grande como 600 x 600 mícrons é adequadamente manchada para imagens de cálcio. Começar uma hora de imagens após a injeção da mistura de corante AM, para permitir o corante para difundir e ser absorvido pelos neurônios. </li><li> Realizar in-vivo de dois fótons de imagem com um custom made microscópio de dois fotões utilizando um Chameleon Ti-Safira Laser (ajustado para 800-880 nm) e espelhos galvanômetro Cambridge Technology. Nós adquirir imagens utilizando Software scanimage desenvolvido em laboratório Karel Svoboda do (Pologruto et al., 2003). Potência do laser é tipicamente bem abaixo de 70 mW na amostra. Imagens campo inteiro são adquiridos utilizando uma 20X (0,95 NA) ou 40x (0.8 NA) objetivos Olympus, no 1,95-15,63 frames por segundo (512 x 256 pixels para 256 x 64 pixels). Varreduras de linha são adquiridas a 500 Hz. Durante a gravação, os animais são mantidos sob anestesia com isoflurano luz (0,6-0,9%), e também são mantidos a 37 ° C usando um aparelho de Harvard dispositivo de controle de temperatura. Cuidado é tomado para manter a freqüência respiratória dos animais, enquanto cerca de 100 respirações / minuto possível. Este nível de uso de anestesia é o nível mais baixo que imobiliza o animal, mas ainda permite a atividade espontânea a ser gravado. </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>

A vantagem deste método sobre gravações eletrofisiológicas é que é menos invasiva e permite que as gravações da atividade neuronal em densas redes neuronais. Ao fazer este procedimento é importante lembrar de tomar extremo cuidado ao realizar a craniotomia para não danificar a dura. Mesmo uma pequena quantidade de sangramento subdural com muito obscura a imagem.

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<td>Oregon Green BAPTA1-AM</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>O-6807</td>
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<td>Fluo 4- AM</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>F-14201</td>
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<td>Sulforhodamine 101</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>S-359</td>
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<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>
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in vivo 2-photon calcium imaging in layer 2/3 of mice

Para entender a dinâmica da rede de microcircuitos no neocórtex, é essencial para gravar simultaneamente a atividade de um grande número de neurônios. In-vivo de dois fótons de imagens de cálcio é o único método que permite registrar a atividade de uma densa população neuronal com uma única célula de resolução. O método consiste na implantação de uma janela de imagem do crânio, a injeção de um corante indicador fluorescente de cálcio que podem ser tomadas por um grande número de neurônios e, finalmente, gravar a atividade dos neurônios com o tempo de imagem usando um lapso de cálcio in-vivo duas microscópio de fótons. Co-injeção de astrócitos específicos corantes permite diferenciar os neurônios de astrócitos. A técnica pode ser realizada em camundongos expressando moléculas fluorescentes em subpopulações específicas de neurônios para entender melhor as interações de rede de diferentes grupos de células.

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

Para entender a dinâmica da rede de microcircuitos no neocórtex, é essencial para gravar simultaneamente a atividade de um grande número de neurônios. In-vivo de dois fótons de imagens de cálcio é o único método que permite registrar a atividade de uma densa população neuronal com uma única célula de resolução.

Vivo em 2-Photon Imagens de cálcio na camada 2 / 3 dos Ratos

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 visualizações.  University of California, Los Angeles. Para entender a dinâmica da rede de microcircuitos no neocórtex, é essencial para gravar simultaneamente a atividade de um grande número de neurônios. In-vivo de dois fótons de imagens de cálcio é o único método que permite registrar a atividade de uma densa população neuronal com uma única célula de resolução.

Vídeo: 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.

Dissecção e 2-Photon Imagens de linfonodos periféricos em ratos

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

Biologia

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.

Na imagem situ da Thymus Mouse Usando 2-Photon Microscopia

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.

Um método para 2-Photon Imagens de Fluxo de Sangue no neocórtex através de uma janela Cranial

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.

Geração de Medula Óssea Derivado Células Dendríticas murino para uso em dois fótons de imagem

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.

Imagem interações proteína-proteína 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.

Imagem Funcional de gordura marrom em ratos com 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 Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

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.

Face Detection En de óxido nítrico e superóxido na camada endotelial das artérias intactas

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.

Micropunção do espaço de Bowman em camundongos facilitada pela microscopia de fóton 2

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.

Dinâmica de cálcio de imagem em subpopulações de células de ilhotas pancreáticas do rato

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