Agradecemos a Anthony J. Koleske, PhD para fornecer os ratos YFP.

Parte 1: Optics Microprisma e Configuração do Microscópio <ol><li> O microprismas que usamos são de 1 mm, em ângulo reto prismas feitos de vidro comprado BK7 da Corporação Optosigma (Figura 1). Microprismas são tratadas usando uma pinça, sempre que possível. </li><li> A hipotenusa do microprisma é revestido com prata aperfeiçoado para fornecer uma refletividade de mais de 97% 4-200 nm. Isto proporciona reflexão interna, tanto de luz de excitação do laser e da fluorescência emitida. </li><li> Microprismas são sempre tratados com pinças, quando possível. As luvas são usadas em todos os momentos para manter o prisma limpo. </li><li> Nós usamos uma custom-built microscópio de dois fotões capaz de pequenos animais de imagem. O animal, ligado ao dispositivo de estereotaxia, é colocado em um palco de 3 eixos motorizados. </li><li> O microscópio está ligado a um PC Windows rodando software scanimage. Scanimage é um software de aquisição de imagem escritos em Matlab por Pologruto et al 1. </li><li> Para geração de imagens microprisma, coletamos imagens com uma resolução de 1024 x 1024 ou 512 x 512 pixels. Além disso, nós tipicamente scan em 2 ms / linha ou 4 ms / linha. </li><li> Dois tipos de objetivos são utilizados nos experimentos: <ul><li> A menor abertura numérica objetivo de ar (NA) para um largo campo de visão de imagem (veja a Tabela de Reagentes e Equipamentos). </li><li> Um objetivo maior de ar NA com um colar de correção de vidro capazes de minimizar aberrações esféricas induzida por 0-2 mm de vidro (ver Tabela de Reagentes e Equipamentos). </li></ul></li><li> Quando o animal está pronto para a gráfica em um objetivo baixa ampliação, alinhar o padrão raster quadrados de varredura do laser com o topo da microprisma. Isso ajudará a orientar a imagem e fazer uso eficiente da potência do laser. </li><li> Usando um objetivo maior NA com um colar de correção de vidro, coloque o colar de correção para corrigir a aproximadamente 1 mm de vidro. Uma vez que uma imagem fluorescente aparece durante o experimento, pode-se chegar com cuidado dentro do microscópio para otimizar o colar de correção para maximizar a resolução de imagem. </li></ol> Parte 2: Non-sobrevivência Cirurgia Animal e Inclusão Microprisma <ol><li> A duração do procedimento completo, desde a incisão inicial até a conclusão de imagem, depende dos objetivos do experimento. No entanto, a colocação não-sobrevivência procedimento cirúrgico e microprisma pode ser esperado para levar cerca de 30-40 minutos para ser concluído. </li><li> Camundongos (P30-P60) são pesados ​​e anestesiados adequadamente usando uma injeção IP de cetamina (100 mg / kg) e xilazina (10 mg / kg) para um plano adequado para procedimentos cirúrgicos. </li><li> Ao longo de todo o experimento, o animal deve ser verificado a cada 15-20 minutos para avaliar o seu nível de anestesia. Se o animal reage a uma empresa toe pinch, uma dose suplementar de cetamina / xilazina é necessária. Tipicamente, uma dose suplementar é um terço da dose inicial e pode ser preparado com antecedência, em uma seringa para injeção IP imediato, se necessário. </li><li> Uma vez que o mouse esteja devidamente anestesiado, a maioria dos pêlos sobre a cabeça do animal é raspado com um aparador com uma lâmina # 40. </li><li> Nair ® é aplicado para os cabelos restantes na cabeça para ajudar a criar uma superfície livre de fios que podem ficar no caminho do microprisma durante o exame. Após 5 minutos, o ® é Nair limpos usando um cotonete. </li><li> O animal anestesiado está devidamente colocada em um aparelho estereotáxico mouse. Durante os procedimentos cirúrgicos e sessões de imagem do animal é mantido em um cheio de água almofada de aquecimento fixado em 36 graus Celsius. </li><li> Com a cabeça do animal corretamente fixada no local, uma incisão limpa é feito usando um bisturi para baixo da linha medial do crânio para separar a pele. </li><li> Uma pequena quantidade de peróxido de hidrogênio 3% é colocado em um cotonete e aplicados ao crânio para limpar as membranas entre o crânio ea pele. </li><li> O peróxido de hidrogênio também ajuda a revelar bregma, um marco importante em saber onde realizar a craniotomia e insira o microprisma. </li><li> Antes da craniotomia é iniciado, as barras de ouvido e bar mordida estão adequadamente ajustadas para inclinar o crânio exposto de tal forma que é, basicamente, ao nível da mesa. Isso proporciona uma melhor plataforma para a craniotomia. Além disso, o que torna a superfície superior do prisma perpendicular com a luz de excitação de entrada. Isso ajudará a minimizar aberrações ópticas, enquanto imagem. </li><li> A pequena rebarba dental (~ 0,8 tamanho da cabeça mm) é conectado a uma ferramenta Dremel ® usando um braço de extensão flexível. Para craniotomias, vamos definir a velocidade para cerca de 7.000 a 10.000 rpms. </li><li> A broca dental é desnatado ao longo do crânio, até um groove praça é estabelecida no osso. Os lados do quadrado são aproximadamente 2-3 mm de comprimento e 1,5 centrada caudal mm e 1 mm lateral ao bregma. </li><li> Continuar afinando o osso até uma pequena abertura é feita através do crânio. Um par de fórceps can levantar o retalho ósseo longe do crânio. </li><li> A dura precisa ser removido antes da microprisma pode ser inserido no córtex. Sangramento é melhor controlada, permitindo que o sangue coagule na superfície por alguns minutos. Posteriormente, o sangue coagulado é removido com uma esponja cirúrgica antes de inserir o microprisma. </li><li> Para ajudar a ajudar o alinhamento do prisma com o córtex, a face vertical do microprisma está alinhado paralelo com as alças do fórceps. </li><li> Com o prisma corretamente realizada, o microprisma inteiro é inserido até o topo do prisma é nivelada com a superfície neocortical. </li><li> Quando inserido corretamente, a microprisma deve permanecer na posição adequada. Qualquer hemorragia que resulta é mínima e pode ser absorvido com uma pequena esponja cirúrgica. Uma vez que o prisma está em vigor o animal está pronto para geração de imagens. </li><li> Depois de um microprisma tem sido usado em um animal, ele pode ser reutilizado várias vezes. No entanto, ele não precisa ser devidamente limpo para remover qualquer material restante biológica. Isso pode ser feito por imersão do prisma em um pequeno volume de HCl ácido, seguida de um mergulho em metanol. O microprisma limpa pode então ser embrulhado em papel de lente até o uso futuro. </li></ol> Parte 3: Resultados Representante <img src="/files/ftp_upload/1509/1509fig1.jpg" alt="Figura 1" /> Figura 1: Imagens do microprisma em ângulo reto. Prismas usados ​​neste experimento são um milímetro, prismas de vidro BK7 com um revestimento de prata reforçada com a hipotenusa para permitir reflexão interna de excitação e emissão de luz. <img src="/files/ftp_upload/1509/1509fig2.jpg" alt="Figura 2" /> Figura 2: Imagem da camada V neurônios piramidais YFP. Imagem de campo de visão ampla com a microprisma revela uma grande população de corpos celulares piramidal ~ 800-900 mM de profundidade da superfície cortical. Além disso, dendrites apical estendendo-se até para a camada I também pode ser resolvido. Barra de escala = 200 mM. <img src="/files/ftp_upload/1509/1509fig3.jpg" alt="Figura 3" /> Figura 3: Um objetivo maior abertura numérica usada em conjunto com o microprisma fornece resolução espacial suficiente para resolver as espinhas dendríticas nos dendritos apicais nos neurônios piramidais da camada V. Barra de escala = 10 mM. <img src="/files/ftp_upload/1509/1509fig4.jpg" alt="Figura 4" /> Figura 4: Imagem obtida pela microprisma e uma cauda de injeção venosa de corante fluorescente. A imagem mostra vertical, vasos de grosso calibre, que se estende desde as regiões cerebrais profundas e capilares menores ramificando-se através do volume cortical. Barra de escala = 200 mM. <img src="/files/ftp_upload/1509/1509fig5.jpg" alt="Figura 5" /> Figura 5: Usando um objetivo maior abertura numérica, a rede de capilares no neocórtex pode ser trabalhada com maior detalhe. Barra de escala = 50 mm. <img src="/files/ftp_upload/1509/1509fig6.jpg" alt="Figura 6" /> Figura 6: microprismas também permite que as células vermelhas do sangue de imagem (hemácias) que flui através dos vasos. Hemácias não absorvem o corante fluorescente e, portanto, são vistos como listras escuras em todo o navio. Por linha de varredura em toda a embarcação, pode-se obter medições de fluxo e velocidade de RBC. Barra de escala = 10 mM (barra horizontal na imagem superior e inferior) e 250 ms (barra vertical na imagem em baixo).

<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>

Experimentos em animais foram realizados de acordo com as diretrizes e regulamentos estabelecidos pela IACUC da Universidade de Yale.

Usando um microprisma em um experimento de imagens é relativamente simples e oferece muitas vantagens para os estudos in vivo sobre o neocórtex. Exemplos representativos usando esta técnica incluem imagens tiradas a partir de camundongos transgênicos expressando a proteína fluorescente nos neurônios da camada amarela V cortical. Usando microprismas pode-se ver uma coleção de grandes corpos celulares piramidais da camada V, cerca de 1 mm abaixo da superfície cortical. Visto também são os dendritos apicais que ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>With enhanced silver reflective coating-(R77)</td>
<td>&nbsp;</td>
<td>OptoSigma Corp. (Santa Ana, CA)</td>
<td>055-0010</td>
<td>With enhanced silver reflective coating-(R77)</td>
</tr>
<tr>
<td>Fluorescein-dextran</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>FD70</td>
<td>70 kDa </td>
</tr>
<tr>
<td>0.28 NA, 4x air objective</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>XLFLUOR 4x/340</td>
<td>&nbsp;</td>
<tr>
<td>0.60 NA, 40x air objective</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>LUCPLFLN</td>
<td>With 0-2 mm coverglass correction</td>
</tr>		</tbody>
		</table>
		</div>

in vivo imaging of deep cortical layers using a microprism

Nós apresentamos um protocolo para a imagem in vivo do tecido cortical usando uma sonda de imagem cerebral profunda na forma de um microprisma. Microprismas estão 1 mm em tamanho e têm um revestimento reflexivo sobre a hipotenusa para permitir reflexão interna de excitação e emissão de luz. A sonda microprisma simultaneamente imagens múltiplas camadas cortical com uma perspectiva tipicamente vistos apenas em preparações fatia. As imagens são coletados com um grande campo de visão (~ 900 mm). Além disso, nós fornecemos informações sobre o procedimento não-cirúrgico de sobrevivência e configuração microscópio. Resultados representativos incluem imagens de neurônios piramidais da camada V de Thy-1-H YFP camundongos mostrando suas dendrites apical estendendo-se através da camada superficial cortical e estendendo-se tufos. Resolução foi suficiente para espinhas dendríticas imagem perto do soma dos neurônios da camada V. A injeção de cauda veia de corante fluorescente revela a intrincada rede de vasos sanguíneos no córtex. Linha de varredura das células vermelhas do sangue (hemácias) que flui através dos capilares revela RBC velocidade e taxas de fluxo pode ser obtida. Esta sonda microprisma romance é um elegante, o método ainda nova e poderosa de visualizar profundas estruturas celulares e da função cortical 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

Em ângulo reto microprismas inserido no neocórtex do mouse permite a imagem profunda de múltiplas camadas cortical com um ponto de vista tipicamente encontrados em fatia. Um milímetro microprismas oferecer um campo de visão ampla (~ 900 mm) e resolução espacial suficiente para resolver as espinhas dendríticas. Demonstramos camada de imagem V neuronal e de imagem vascular neocortical usando microprismas.

Em Imaging vivo do Deep camadas corticais usando um Microprisma

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 ...

in-vivo-imaging-of-deep-cortical-layers-using-a-microprism

Research

JoVE Journal

Biology

In vivo Imaging of Deep Cortical Layers using a Microprism

11.3K visualizações.  Yale University. Em ângulo reto microprismas inserido no neocórtex do mouse permite a imagem profunda de múltiplas camadas cortical com um ponto de vista tipicamente encontrados em fatia. Um milímetro microprismas oferecer um campo de visão ampla (~ 900 mm) e resolução espacial suficiente para resolver as espinhas dendríticas. Demonstramos camada de imagem V neuronal e de imagem vascular neocortical usando microprismas.

Vídeo: 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.

Preparação de Dissociated rato Culturas Neuron Cortical

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

Biologia

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.

Imagem de cálcio dos neurônios corticais usando Fura-02:00

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 imagem de tumores mamários Usando Spectrum 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 ...

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 Centrifugação de Embriões Drosophila

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.

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, ...

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.

Molécula única imagem da regulação gênica<em&gt; Em vivo</em&gt; Uso de ativação Cotranslational por clivagem (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 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) ...

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.

A quantificação da expressão da proteína e co-localização Usando multiplexada Imuno-histoquímica Coloração e Multispectral Imagiologia

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.

Medindo a rigidez<em&gt; Ex Vivo</em&gt; Mouse aortas Usando Microscopia de Força Atômica

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

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

Drosophila Preparação e imagem longitudinal do coração Função In Vivo Usando Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...