Vorremmo riconoscere Dionna Gamble, Allison Larson, e Katia Torres per l&#39;assistenza con generazione di modelli e misure, Monica Mahre per assistenza manoscritto, Gary Williams per l&#39;assistenza tecnica, Jerrald Spencer Jr. per l&#39;assistenza con le figure e le Fairview Imaging servizi al Università del Minnesota. Il finanziamento è stato ricevuto presso l&#39;Istituto di Ingegneria in Medicina e Chirurgia (Università del Minnesota) e in parte da un contratto di ricerca con Medtronic Inc.

Procedura La tabella 1 riassume i materiali utilizzati durante il processo. Figura 1 fornisce una panoramica del processo. 1. Campioni e scansione di preparazione <ol><li> Ottenere i isolati cuori umani freschi e successivamente perfusione fissarli in 10% formalina tamponata nel loro stato telediastolico. </li><li> Sciacquare i cuori da acquisire in acqua il giorno prima della scansione al fine di rimuovere la maggior parte della formalina. </li><li> Prima di dirigersi verso lo scanner, incannulare il seno (CS) vena coronaria all&#39;interno di ogni cuore con un catetere a palloncino venogramma. Ottenere l&#39;accesso al CS attraverso sia la vena cava superiore o inferiore con visualizzazione diretta o l&#39;uso di videoendoscopi. </li><li> Una volta in posizione, gonfiare il palloncino del catetere presente venogram per ancorare il catetere in CS. </li><li> Posizionare ogni cuore all&#39;interno di un contenitore di polimero sigillabile in cima a una spugna che è stato progettato in modo cheil cuore può sedersi nella sua posizione anatomica attitudinally corretta. </li></ol> 2. Tomografia computerizzata <ol><li> Posizionare un dato di cuore sul piano dello scanner CT come se un paziente era sdraiato supino e la testa in primo luogo sullo scanner. </li><li> Collegare l&#39;estremità prossimale del catetere venogram ad un iniettore che contiene due siringhe per iniezione: uno per contrasto e una per salina. </li><li> Iniettare automaticamente 40 ml di contrasto nel sistema venoso cardiaca a 5 ml / sec. </li><li> TC cuore 8 secondi dopo l&#39;inizio della iniezione di contrasto. Impostare la TAC a 512 x 512 pixel di risoluzione da 0,6 mm di spessore fetta. </li><li> Iniettare automaticamente 40 ml di soluzione fisiologica nel sistema venoso cardiaca a 5 ml / sec per irrigare il contrasto. </li><li> Esportare le immagini TC dicom su un disco rigido esterno. </li></ol> 3. Ricostruzione e Misure <ol><li> Carica immagini TC DICOM in Mimics Software. </li><li> Generare una maschera per il CT images che contiene solo i pixel con alta unità Hounsfield per evidenziare solo il contrasto presente nel cuore. </li><li> Rimuovere contrasto che è fuoriuscito nelle camere o diffusi nel tessuto in modo che la maschera contiene solo il contrasto all&#39;interno dei principali vene cardiache. </li><li> Compilare manualmente in sacche d&#39;aria all&#39;interno di un determinato filone fotogramma per fotogramma. </li><li> Generare un oggetto 3D dalla maschera risultante. </li><li> Liscia e avvolgere l&#39;oggetto da eliminare geometrie approssimative. Video 1 presenta uno di questi modelli 3D rotante nello spazio. </li><li> Generare linee centrali per ogni modello 3D creato. </li><li> Utilizzando queste linee centrali, misurare la lunghezza d&#39;arco, angolo di ramificazione, tortuousity (lunghezza dell&#39;arco / distanza lineare) e diametri per ogni peschereccio importante in ogni cuore. Nostra nomenclatura anatomica impiegata viene visualizzato in Figura 2. </li></ol>

<ol>
	<li>Burkhardt, J. D., Wilkoff, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interventional+electrophysiology+and+cardiac+resynchronization+therapy:+delivering+electrical+therapies+for+heart+failure.">Interventional electrophysiology and cardiac resynchronization therapy: delivering electrical therapies for heart failure.</a> Circ. 115, 2208-2220 (2007).</li><li>Lu, F., Iaizzo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+resynchronization+therapy.">Cardiac resynchronization therapy.</a> Handbook of cardiac physiology and anatomy. , 475-497 (2009).</li><li>Eggen, M. D., Swingen, C. M., Iaizzo, P. 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Nessun conflitto di interessi dichiarati.

Il nostro laboratorio sta sviluppando una libreria di perfusione-fissi campioni di cuore per i vari studi anatomici. Ad oggi, abbiamo oltre 240 esemplari conservati. I metodi specifici che abbiamo usato per preparare questi campioni sono stati precedentemente descritti 3. Il presente studio descrive una nuova metodologia per la mappatura del sistema venoso cardiaco umano e per lo sviluppo di un database anatomico, che potrebbe essere utilizzato per la progettazione di dispositivi cardiaci impiegate all&#39;in...

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

Una comprensione dettagliata della complessità e della variabilità relativa all&#39;interno del sistema venoso cardiaco umano è cruciale per lo sviluppo di dispositivi cardiaci che richiedono l&#39;accesso a questi vasi. Per esempio, anatomia venosa cardiaca è conosciuto per essere una delle limitazioni chiave per la corretta erogazione della terapia di resincronizzazione cardiaca (CRT) 1 Pertanto, lo sviluppo di un database di parametri anatomici per sistemi venosi cardiache umane può aiutare nella progettazione di consegna CRT dispositivi per superare tale limitazione. In questo progetto di ricerca, i parametri anatomici sono stati ottenuti da ricostruzioni 3D del sistema venoso mediante tomografia a contrasto computerizzata (TC), l&#39;imaging e software di modellazione (Materialise, Leuven, Belgio). I seguenti parametri sono stati valutati per ogni vena: lunghezza d&#39;arco, tortuousity, angolo di ramificazione, distanza in seno ostio coronarico, e diametro del vaso. CRT è un potenziale trattamento per patigenitori con dissincronia elettromeccanica. Circa il 10-20% dei pazienti con insufficienza cardiaca possono beneficiare di 2 monitor CRT. Dissincronia elettromeccanica implica che le parti del contratto di attivare miocardio e prima o dopo il normale percorso di conduzione del cuore. In CRT, aree dyssynchronous del miocardio sono trattati con stimolazione elettrica. Stimolazione CRT in genere comporta elettrocateteri di stimolazione che stimolano l&#39;atrio destro (RA), ventricolo destro (RV), e il ventricolo sinistro (LV) per produrre ritmi più risincronizzata. L&#39;elettrocatetere VS è tipicamente impiantato all&#39;interno di una vena cardiaca, con l&#39;obiettivo di sovrapporre all&#39;interno del sito di attivazione ultime miocardica. Noi crediamo che i modelli ottenuti e le analisi dello stesso promuoveranno l&#39;educazione anatomico per i pazienti, studenti, medici, e progettisti di dispositivi medici. Le metodologie impiegate qui possono anche essere utilizzati per studiare altre caratteristiche anatomiche dei nostri campioni del cuore umano, come ad esempiole arterie coronarie. Per incoraggiare ulteriormente il valore educativo di questa ricerca, abbiamo condiviso i modelli venose sul nostro sito web libero accesso: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu / atlante</a> .

La tabella 2 presenta i parametri anatomici mediani per le principali vene cardiache per 42 campioni cuore umano. Tutti i campioni di cuore contenevano una vena interventricolare posteriore (PIV) e vena interventricolare anteriore (AIV). Alcuni esemplari conteneva più di una vena posteriore del ventricolo sinistro (PVLV), vena postero-laterale (PLV), sinistra vena laterale (LLV), e / o vena antero-laterale (ALV), mentre altri cuori non possono aver avuto una o due di queste vene specifiche presenti. </...

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

L&#39;obiettivo di questa ricerca è quello di ricreare e quindi accedere alla anatomia del sistema venoso cardiaco umano con ricostruzioni 3D generate dal contrasto-tomografia computerizzata.

Ricostruzioni anatomiche del sistema umano venoso cardiaco utilizzando Contrasto-tomografia computerizzata di campioni di perfusione-fissi

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 Views.  University of Minnesota. L'obiettivo di questa ricerca è quello di ricreare e quindi accedere alla anatomia del sistema venoso cardiaco umano con ricostruzioni 3D generate dal contrasto-tomografia computerizzata.

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.

Rilevamento e identificazione batterica Utilizzando Sensori elettrochimici

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

Ricerca

Bioingegneria

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.

Coltura mouse cardiaca Valvole in miniatura Tissue Culture Sistema

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.

Studiando la funzione delle proteine ​​e il ruolo di Altered espressione della proteina da anticorpo interferenza e ricostruzioni tridimensionali

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.

Costruzione di Engineered Definito umani cardiache tessuti per studiare i meccanismi della terapia cellulare 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.

In Vivo quantitativa valutazione della struttura del miocardio, la funzione, la perfusione e sostenibilità Utilizzando cardiaca micro-tomografia computerizzata

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).

Utilizzando la trasmissione ottica straordinaria per quantificare i biomarcatori cardiaci nel siero umano

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.

Efficiente generazione e la modifica di IPSCs privo di alimentatore da cellule pancreatiche umane utilizzando il sistema di 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 metodo basato su stampo netto della creazione senza impalcatura tridimensionale del tessuto 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.

Rigida incorporamento di fisso e macchiato, complesso, scala millimetrata esemplari per l'istologia privo di sezione 3D da Micro-tomografia computerizzata

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.

Catetere leggero intra-cardiaco per il monitoraggio del metabolismo cellulare utilizzando la spettroscopia transmurale di riambente di cuori mammiferi perfusi

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