Damos las gracias a Anthony J. Koleske, PhD de proporcionar a los ratones YFP.

Parte 1: Óptica microprismas y configuración del microscopio <ol><li> El microprismas que utilizamos son de 1 mm, en ángulo recto a partir de prismas de cristal BK7 comprados a la Corporación Optosigma (Figura 1). Microprismas se manejan con las pinzas siempre que sea posible. </li><li> La hipotenusa del microprism está recubierta con plata mejorado para proporcionar una reflectividad superior a 97% 400 a 2000 nm. Esto proporciona la reflexión interna de los dos la luz de excitación láser y la fluorescencia emitida. </li><li> Microprismas siempre son manejados con pinzas si es posible. Se usan guantes en todo momento para mantener el prisma limpio. </li><li> Utilizamos una costumbre-construido de dos fotones microscopio capaz de pequeños animales de imagen. El animal, que se adjunta al dispositivo de estereotaxia, se coloca en un escenario de 3 ejes motorizados. </li><li> El microscopio está conectado a un PC con Windows con software scanimage. Scanimage es un software de adquisición de imágenes en Matlab por Pologruto et al 1. </li><li> Para obtener imágenes de microprismas, recopilamos las imágenes con una resolución de 1024 x 1024 o 512 x 512 píxeles. Además, típicamente escanean a los 2 ms / línea y 4 ms / línea. </li><li> Hay dos tipos de objetivos son utilizados en los experimentos: <ul><li> A la baja apertura numérica (NA) de aire para todo el objetivo de imágenes de campo de visión (ver la tabla de reactivos y equipos). </li><li> Un alto NA objetivo de aire con un collar de corrección de vidrio capaz de minimizar las aberraciones esféricas inducidas por 0-2 mm de vidrio (véase la tabla de reactivos y equipos). </li></ul></li><li> Cuando el animal está listo para la imagen en un objetivo de bajo aumento, alinear el patrón de trama cuadrada de escaneo de láser con la parte superior de la microprism. Esto le ayudará a orientar a la imagen y hacer un uso eficiente de la potencia del láser. </li><li> El uso de un mayor objetivo de NA con un collar de cristal de corrección, ajuste el collar de corrección para corregir aproximadamente 1 mm de vidrio. Una vez que una imagen fluorescente aparece durante el experimento, un cuidado puede alcanzar en el interior del microscopio con el fin de optimizar el collar de corrección para maximizar la resolución de imagen. </li></ol> Parte 2: la supervivencia Cirugía Animal e Inserción microprisma <ol><li> La duración del procedimiento, desde la incisión inicial hasta la finalización de la imagen, depende de los objetivos del experimento. Sin embargo, la colocación no la supervivencia de procedimiento quirúrgico y microprism se puede esperar que tomará aproximadamente 30 a 40 minutos para completar. </li><li> Ratones (P30-P60) se pesan y se anestesiaron con una inyección adecuada de propiedad intelectual de la ketamina (100 mg / kg) y xilazina (10 mg / kg) a un plano adecuado para procedimientos quirúrgicos. </li><li> A lo largo de todo el experimento, el animal debe ser revisado cada 15-20 minutos para evaluar su nivel de anestesia. Si el animal reacciona a una empresa puntera de sujetador, una dosis suplementaria de ketamina / xilazina es necesario. Normalmente, una dosis suplementaria es de un tercio de la dosis inicial y se puede preparar de antemano en una jeringa para inyección IP inmediata si es necesario. </li><li> Una vez que el ratón está bien anestesiado, la mayoría de los pelos de la cabeza del animal se afeita con un condensador de ajuste con una cuchilla # 40. </li><li> Nair ® se aplica a los pelos que queda en la cabeza para ayudar a crear una superficie libre de pelos que se pueden obtener en el camino de la microprism durante la exploración. Después de 5 minutos, el Nair ® se limpia con un hisopo de algodón. </li><li> El animal anestesiado se coloca correctamente en un dispositivo de estereotaxia ratón. Durante los procedimientos quirúrgicos y sesiones de formación de imágenes del animal se mantiene en un agua llena de almohadilla térmica a 36 grados Celsius. </li><li> Con la cabeza del animal bien colocado en su lugar, una incisión limpia se realiza con un bisturí en la línea media del cráneo para separar la piel. </li><li> Una pequeña cantidad de peróxido de hidrógeno al 3% se coloca en un bastoncillo de algodón y se aplica en el cráneo para eliminar las membranas entre el cráneo y la piel. </li><li> El peróxido de hidrógeno también ayuda a revelar bregma, un hito importante en saber dónde realizar la craneotomía y la inserción microprism. </li><li> Antes de la craneotomía se inicia, las barras de bar morder la oreja y estén debidamente ajustados a la inclinación del cráneo expuesto de tal manera que es, básicamente, al nivel de la mesa. Esto proporciona una mejor plataforma para la craneotomía. Además, esto hace que la superficie superior de la perpendicular de prisma con la luz de excitación de entrada. Esto ayudará a minimizar las aberraciones ópticas, mientras que la imagen. </li><li> Una pequeña fresa dental (~ 0,8 mm tamaño de la cabeza) está conectado a una herramienta Dremel ® utilizando un brazo de extensión flexible. Para craneotomías, que establece la velocidad a aproximadamente entre 7.000 y 10.000 revoluciones por minuto. </li><li> La fresa dental es desnatada a lo largo del cráneo hasta una ranura cuadrada se establece en el hueso. Los lados de la plaza es de aproximadamente 2-3 mm de longitud y caudal centrada 1,5 mm y 1 mm lateral a bregma. </li><li> Continuando con el adelgazamiento del hueso hasta una pequeña abertura se realiza a través del cráneo. Un par de pinzas de can levantar el colgajo óseo fuera del cráneo. </li><li> La duración debe ser eliminado antes de la microprism puede ser insertado en la corteza. El sangrado se controla mejor al permitir que la sangre coagule en la superficie durante varios minutos. Después, la sangre coagulada se retira con una esponja quirúrgica antes de insertar el microprism. </li><li> Para ayudar a ayudar a la alineación del prisma con la corteza, la cara vertical de la microprism es alineadas en paralelo con las asas de las pinzas. </li><li> Con el prisma de considerar adecuadamente, el microprism todo se inserta hasta la parte superior del prisma esté al ras con la superficie neocortical. </li><li> Cuando se inserta correctamente, el microprism debe permanecer en la posición correcta. Cualquier sangrado que se produce es mínima y puede ser absorbido con una esponja quirúrgica pequeña. Una vez que el prisma está en el lugar que el animal está listo para su imagen. </li><li> Después de un microprism se ha utilizado en un animal, se puede volver a utilizar varias veces. Sin embargo, tiene que ser limpiado para eliminar cualquier resto de material biológico. Esto se puede lograr mediante la inmersión del prisma en un pequeño volumen de HCl ácido, seguido de un baño en metanol. El microprism limpieza puede ser envuelto en papel de la lente hasta su uso futuro. </li></ol> Parte 3: Resultados de Representante <img src="/files/ftp_upload/1509/1509fig1.jpg" alt="Figura 1" /> Figura 1: Imágenes de la microprism en ángulo recto. Prismas utilizados en este experimento son de un milímetro, prismas de vidrio BK7 con una capa de plata mejora de la hipotenusa para que la reflexión interna de la excitación y de emisión de luz. <img src="/files/ftp_upload/1509/1509fig2.jpg" alt="Figura 2" /> Figura 2: Imagen de la capa de neuronas piramidales V YFP. Amplio campo de visión de imágenes con la microprism revela una gran población de cuerpos de las células piramidales ~ 800 a 900 m de profundidad desde la superficie cortical. Además, las dendritas apical se extiende hacia la capa I también se puede resolver. Barra de escala = 200 micras. <img src="/files/ftp_upload/1509/1509fig3.jpg" alt="Figura 3" /> Figura 3: Un objetivo mayor apertura numérica que se utiliza en conjunción con el microprismas ofrece una resolución espacial suficiente para resolver las espinas dendríticas en las dendritas apicales en la capa V neuronas piramidales. Barra de escala = 10 micras. <img src="/files/ftp_upload/1509/1509fig4.jpg" alt="Figura 4" /> Figura 4: Una imagen obtenida con el microprism y una inyección de cola vena de un colorante fluorescente. La imagen muestra los vasos vertical, de gran calibre que se extiende desde las regiones profundas del cerebro y pequeños capilares se desvía a través del volumen cortical. Barra de escala = 200 micras. <img src="/files/ftp_upload/1509/1509fig5.jpg" alt="Figura 5" /> Figura 5: El uso de un objetivo de mayor apertura numérica, la red de capilares en la neocorteza se pueden crear imágenes con mayor detalle. Barra de escala = 50 micras. <img src="/files/ftp_upload/1509/1509fig6.jpg" alt="Figura 6" /> Figura 6: microprismas también permite que las células de imágenes rojos (GR) que fluye por los vasos. Los glóbulos rojos no absorben el colorante fluorescente y por lo tanto se ven como rayas oscuras en todo el buque. Por línea de exploración a través de la nave, se pueden obtener mediciones de flujo de glóbulos rojos y la velocidad. Barra de escala = 10 micras (barra horizontal en la imagen superior e inferior) y 250 ms (barra vertical en la imagen inferior).

<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>

Los experimentos con animales se realizaron de acuerdo con las directrices y normas establecidas por IACUC la Universidad de Yale.

El uso de un microprism en un experimento de imágenes es relativamente sencillo y ofrece muchas ventajas para los estudios in vivo en el neocórtex. Ejemplos representativos de esta técnica incluyen imágenes tomadas de ratones transgénicos que expresan la proteína amarilla fluorescente en la capa de neuronas corticales V. Usando microprismas se puede ver una colección de grandes cuerpos de las células piramidales de la capa V, cerca de 1 mm por debajo de la superficie cortical. También se ve son las dendritas ap...

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in vivo imaging of deep cortical layers using a microprism

Se presenta un protocolo de imágenes in vivo del tejido cortical mediante una sonda de imagen cerebral profunda en la forma de un microprism. Microprismas son de 1 mm de tamaño y tienen una capa reflectante de la hipotenusa para que la reflexión interna de la excitación y de emisión de luz. La sonda microprism simultáneamente múltiples imágenes de las capas corticales con una perspectiva general, visto sólo en los preparativos de división. Las imágenes se recogieron con un gran campo de visión (~ 900 m). Además, se proporcionan detalles sobre el procedimiento quirúrgico no la supervivencia y la configuración del microscopio. Los resultados representativos incluyen imágenes de la capa V neuronas piramidales de Thy-1-H YFP ratones que muestran sus dendritas apicales se extiende a través de la capa cortical superficial y se extiende a mechones. Resolución fue suficiente para las espinas dendríticas de imágenes cerca de la soma de la capa de neuronas V. Una inyección de cola vena de un colorante fluorescente revela la intrincada red de vasos sanguíneos en la corteza. De exploración lineal de los glóbulos rojos (GR) que fluye a través de los capilares revela la velocidad de glóbulos rojos y las tasas de flujo se pueden obtener. Esta sonda microprism novela es un método elegante, el nuevo y potente de visualizar profundidad las estructuras celulares y la función cortical en 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

En ángulo recto microprismas insertado en la corteza cerebral del ratón permite obtener imágenes profundas de múltiples capas corticales con un punto de vista típicamente se encuentran en el segmento. De un milímetro microprismas ofrecen un campo de visión amplio (~ 900 m) y resolución espacial suficiente para resolver las espinas dendríticas. Demostramos capa de imagen V neuronal y vascular mediante imágenes neocortical microprismas.

Imágenes in vivo de Deep capas corticales con un 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 vistas.  Yale University. En ángulo recto microprismas insertado en la corteza cerebral del ratón permite obtener imágenes profundas de múltiples capas corticales con un punto de vista típicamente se encuentran en el segmento. De un milímetro microprismas ofrecen un campo de visión amplio (~ 900 m) y resolución espacial suficiente para resolver las espinas dendríticas. Demostramos capa de imagen V neuronal y vascular mediante imágenes neocortical microprismas.

Video: 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.

Preparación de los cultivos disociados ratón neuronas corticales

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

Investigación

Biología

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.

Imagen de calcio de las neuronas corticales con Fura-2 a.m.

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.

En vivo bioluminiscente de imágenes de tumores de mama mediante el espectro de 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.

En vivo-La centrifugación de embriones de 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.

Imágenes de interacciones 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.

Una sola molécula de imágenes del Reglamento Gene<em&gt; En vivo</em&gt; Utilización de la activación cotraduccional por escisión (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.

En Vivo Tracking 4-Dimensional de madre hematopoyéticas y células progenitoras en ratones adultos de calota de Médula Ósea

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.

La cuantificación de la expresión de la proteína y Co-localización Uso de multiplexado inmunohistoquímica y tinción de imágenes multiespectrales

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

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

-Alta resolución de imagen de lapso de tiempo y análisis automatizado de los microtúbulos en la dinámica de estar umbilical humana Las células endoteliales de vena

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

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.

La medición de la rigidez de<em&gt; Ex Vivo</em&gt; Ratón aortas Usando microscopía de fuerza atómica

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