Nos gustaría agradecer Dionna Gamble, Allison Larson y Katia Torres para obtener ayuda con la generación de modelos y medidas, Monica mahre de asistencia manuscrito, Gary Williams para la asistencia técnica, Jerrald Spencer Jr. para obtener ayuda con las cifras y las imágenes de los servicios de Fairview en el Universidad de Minnesota. Se ha recibido financiación del Instituto de Ingeniería en Medicina (Universidad de Minnesota) y en parte de un contrato de investigación con Medtronic Inc.

Procedimiento La Tabla 1 resume los materiales utilizados durante el proceso. Figura 1 ofrece una visión general del proceso. 1. Preparación de muestras y análisis <ol><li> Obtener los corazones aislados de humanos frescos y, posteriormente, la perfusión fijarlos en formalina tamponada al 10% en su estado final de la diástole. </li><li> Enjuague los corazones que se exploró en agua el día antes de explorar con el fin de eliminar la mayoría de la formalina. </li><li> Antes de dirigirse al escáner, canular el seno vena coronaria (CS) dentro de cada corazón con un catéter de balón venografía. Obtener acceso a la CS ya sea a través del superior o inferior de la vena cava bajo visualización directa o el uso de videoscopios. </li><li> Una vez en su lugar, inflar el globo de este catéter venograma para anclar el catéter en el CS. </li><li> Coloque cada corazón dentro de un contenedor de polímero sellable en la parte superior de una esponja que ha sido diseñado de manera queel corazón puede sentarse en su posición anatómica correcta attitudinally. </li></ol> 2. Tomografía computarizada <ol><li> Coloque un corazón dado en el escáner CT como si el paciente estaba acostado en posición supina y la cabeza por primera vez en el escáner. </li><li> Conectar el extremo proximal del catéter venograma a un inyector que contiene dos jeringas de inyección: uno para el contraste y uno para la solución salina. </li><li> Inyectar automáticamente 40 ml de contraste en el sistema venoso cardíaco en 5 ml / seg. </li><li> CT scan del corazón 8 segundos después de que se inició la inyección de contraste. Establezca la tomografía computarizada para x 512 píxeles de resolución 512 con 0,6 mm de grosor de corte. </li><li> Inyectar automáticamente 40 ml de solución salina en el sistema venoso cardíaco en 5 ml / seg para eliminar el contraste. </li><li> Exportar las imágenes de TC DICOM en un disco duro externo. </li></ol> 3. Reconstrucción y Medidas <ol><li> Subir CT imágenes DICOM en Imita Software. </li><li> Generar una máscara para la CT images que sólo contiene píxeles con unidades de alta Hounsfield para resaltar sólo el contraste presente en el corazón. </li><li> Retire contraste que se ha filtrado en las cámaras o difunde en el tejido de manera que la máscara sólo contiene el contraste dentro de las grandes venas cardiacas. </li><li> Llenar manualmente en bolsas de aire dentro de un marco vena dada por cuadro. </li><li> Generar un objeto 3D de la máscara resultante. </li><li> Suave y envolver el objeto de eliminar geometrías irregulares. Vídeo 1 presenta uno de estos modelos 3D que gira en el espacio. </li><li> Generar líneas centrales de cada modelo 3D creado. </li><li> El uso de estas centrales, mida la longitud del arco, ángulo de ramificación, tortuosidad (longitud de arco / distancia lineal) y diámetros para cada vaso principal en cada corazón. Nuestra nomenclatura anatómica empleado se muestra en la Figura 2. </li></ol>

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Anat. 22, 35-39 (2000).</li><li>Tada, H., Kurosaki, K., Naito, S., Koyama, K., Itoi, K., Ito, S., Ueda, M., Shinbo, G., Hoshizaki, H., Nogami, A., Oshima, S., Taniguchi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+visualization+of+the+coronary+venous+system+using+multidetector+row+computed+tomography.">Three-dimensional visualization of the coronary venous system using multidetector row computed tomography.</a> Circ. J. 69, 165-170 (2005).</li><li>Van de Veire, N. R., Schuijf, J. D., Sutter, J. D., Devos, D., Bleeker, G. B., de Roos, A., vander Wall, E. E., Schalij, M. J., Bax, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+visualization+of+the+cardiac+venous+system+in+coronary+artery+disease+patients+using+64-slice+computed+tomography.">Non-invasive visualization of the cardiac venous system in coronary artery disease patients using 64-slice computed tomography.</a> J. Am. Coll. Cardiol. 48, 1832-1838 (2006).</li><li>de Jong, P. A., Mayo, J. R., Golmohammadi, K., Nakano, Y., Lequin, M. H., Tiddens, H. A., Aldrich, J., Coxson, H. O., Sin, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+cancer+mortality+associated+with+repetitive+computed+tomography+scanning.">Estimation of cancer mortality associated with repetitive computed tomography scanning.</a> Am. J. Respir. Crit. Care Med. 173, 199-203 (2006).</li><li>Martin, D. R., Semelka, R. C., Chapman, A., Peters, H., Finn, P. J., Kalb, B., Thomsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephrogenic+systemic+fibrosis+versus+contrast-induced+nephropathy:+risks+and+benefits+of+contrast-enhanced+MR+and+CT+in+renally+impaired+patients.">Nephrogenic systemic fibrosis versus contrast-induced nephropathy: risks and benefits of contrast-enhanced MR and CT in renally impaired patients.</a> J. Magn. Reson. Imaging. 30, 1350-1356 (2009).</li></ol>

No hay conflictos de interés declarado.

Nuestro laboratorio está desarrollando una biblioteca de perfusión fijos especímenes de corazón para diversas investigaciones anatómicas. Hasta la fecha, contamos con más de 240 especímenes conservados. Los métodos específicos que hemos usado para preparar estos especímenes se han descrito anteriormente 3. El presente estudio describe una nueva metodología para el mapeo del sistema venoso cardíaco humana y para el desarrollo de una base de datos anatómica, que podría ser utilizado para el diseñ...

anatomical reconstructions of the human cardiac venous system using contrast-computed tomography of perfusion-fixed specimens

Una comprensión detallada de la complejidad y variabilidad relativa dentro del sistema venoso cardíaco humano es crucial para el desarrollo de dispositivos cardiacos que requieren acceso a estos vasos. Por ejemplo, la anatomía venosa cardíaca es conocido por ser una de las limitaciones principales para la entrega apropiada de la terapia de resincronización cardiaca (CRT) 1 Por lo tanto, el desarrollo de una base de datos de parámetros anatómicos para sistemas venosos cardíacos humanos puede ayudar en el diseño de la administración de TRC dispositivos para superar esta limitación. En este proyecto de investigación, se obtuvieron los parámetros anatómicos de las reconstrucciones 3D del sistema venoso mediante tomografía computarizada con contraste (TC) y el software de modelado (Materialise, Lovaina, Bélgica). Los siguientes parámetros fueron evaluados para cada sentido: longitud de arco, tortuosidad, el ángulo de ramificación, distancia al ostium del seno coronario, y el diámetro de los vasos. CRT es un tratamiento potencial para patipadres con asincronía electromecánica. Aproximadamente el 10-20% de los pacientes con insuficiencia cardíaca puede beneficiarse de la TRC 2. Asincronía electromecánica implica que las partes de la activación del miocardio y el contrato antes o después de la vía de conducción normal del corazón. En CRT, dyssynchronous áreas del miocardio son tratados con estimulación eléctrica. Marcapasos CRT suele implicar cables de estimulación que estimulan la aurícula derecha (AD), ventrículo derecho (VD) y ventrículo izquierdo (LV) para producir ritmos más vuelven a sincronizar. El cable de VI se implanta normalmente dentro de una vena cardíaca, con el objetivo de superponer dentro del sitio de la última activación del miocardio. Creemos que los modelos obtenidos y el análisis de los mismos promoverán la formación anatómica de los pacientes, estudiantes, médicos, y los diseñadores de dispositivos médicos. Las metodologías empleadas aquí también pueden ser utilizados para estudiar otras características anatómicas de los especímenes de corazón humano, tales comolas arterias coronarias. Para fomentar aún más el valor educativo de esta investigación, hemos compartido los modelos venosas en nuestro sitio web de acceso libre: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu / atlas</a> .

La Tabla 2 presenta los parámetros anatómicos mediana para los principales venas cardiacas para 42 muestras de corazón humano. Todas las muestras contenían corazón una vena interventricular posterior (PIV) y la vena interventricular anterior (VIA). Algunas muestras contenían más de una vena posterior del (PVLV), vena LV postero-lateral (PLV), vena lateral izquierdo (LLV), y / o vena antero-lateral (ALV), mientras que otros corazones no han tenido uno o dos de estas vetas específicas presentes. <...

Watch this Scientific Journal Video about Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens at JoVE.com

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

El objetivo de esta investigación es la de recrear y luego acceder a la anatomía del sistema venoso cardíaco humano utilizando reconstrucciones 3D generados por las exploraciones con contraste de tomografía computarizada.

Las reconstrucciones anatómicas del sistema venoso cardíaco humana usando tomografía computarizada con contraste de las muestras de perfusión fijos

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

anatomical-reconstructions-human-cardiac-venous-system-using-contrast

Research

JoVE Journal

Bioengineering

11.7K vistas.  University of Minnesota. El objetivo de esta investigación es la de recrear y luego acceder a la anatomía del sistema venoso cardíaco humano utilizando reconstrucciones 3D generados por las exploraciones con contraste de tomografía computarizada.

Video: Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Bacterial Detección e Identificación Usando sensores electroquímicos

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Investigación

Bioingeniería

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. 
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. 
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

El cultivo de ratón cardíaca válvulas en el sistema de cultivo tisular miniatura

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. Therefore, recent research invented different tools to target protein expression in mammalian cell lines or even animal models, including RNA and antibody interference. While the first strategy has gathered much attention during the past two decades, peptides &#160;mediating a translocation of antibody cargos across cellular membranes and into cells, obtained much less interest. In this publication, we provide a detailed protocol how to utilize a peptide carrier named Chariot in human embryonic kidney cells as well as in primary hippocampal neurons to perform antibody interference experiments and further illustrate the application of three-dimensional reconstructions in analyzing protein function. Our findings suggest that Chariot is, probably due to its nuclear localization signal, particularly well-suited to target proteins residing in the soma and the nucleus. Remarkably, when applying Chariot to primary hippocampal cultures, the reagent turned out to be surprisingly well accepted by dissociated neurons.

Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

El estudio de la función de proteínas y el Papel de la expresión de la proteína alterada por la interferencia de anticuerpos y reconstrucciones tridimensionales

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Construcción de Engineered Definido Humanos cardíacos tejidos para estudiar los mecanismos de la terapia celular cardiaca

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

En Vivo evaluación cuantitativa de la estructura miocárdica, la función, perfusión y viabilidad Usando computarizada micro-tomografía cardiaca

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor analytes against the background of human serum are crucial.
Nanoimprinting lithography (NIL) was used to fabricate, at a low cost, sensing areas as large as 1.5 mm x 1.5 mm. The sensing surface was made of high-fidelity arrays of nanoholes, each with an area of about 140 nm2. The great reproducibility of NIL made it possible to employ a one-chip, one-measurement strategy on 12 individually manufactured surfaces, with minimal chip-to-chip variation. These nanoimprinted localized surface plasmon resonance (LSPR) chips were extensively tested on their ability to reliably measure a bioanalyte at concentrations varying from 2.5 to 75 ng/mL amidst the background of a complex biofluid-in this case, human serum. The high fidelity of NIL enables the generation of large sensing areas, which in turn eliminates the need for a microscope, as this biosensor can be easily interfaced with a commonly available laboratory light source. These biosensors can detect cardiac troponin in serum with a high sensitivity, at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. They also show low chip-to-chip variance (due to the high quality of the fabrication process). The results are commensurable with widely used enzyme-linked immunosorbent assay (ELISA)-based assays, but the technique retains the advantages of an LSPR-based sensing platform (i.e., amenability to miniaturization and multiplexing, making it more feasible for POC applications).

This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff &#8764;10-400 pg/mL, depending on the assay).

Uso de transmisión óptica extraordinaria para cuantificar marcadores cardíacos en suero humano

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Eficiente generación y edición de IPSCs alimentador-libre de las células pancreáticas humanas utilizando el sistema CRISPR Cas9

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

Un método basado en el molde neto creación libre de andamio tridimensional de tejido cardiaco

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

Rígida fijación de fijo y manchados, todo, escala milimétrica muestras para histología sin sección 3D de tomografía Micro computada

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Catéter de luz intracardiaca para monitorear el metabolismo celular utilizando espectroscopia de absorbancia transmural de corazones de mamíferos perfundidos

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...