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Beeswax as dental filling on a neolithic human tooth.
Evidence of prehistoric dentistry has been limited to a few cases, the most ancient dating back to the Neolithic. Here we report a 6500-year-old human mandible from Slovenia whose left canine crown bears the traces of a filling with beeswax. The use of different analytical techniques, including synchrotron radiation computed micro-tomography (micro-CT), Accelerator Mass Spectrometry (AMS) radiocarbon dating, Infrared (IR) Spectroscopy and Scanning Electron Microscopy (SEM), has shown that the exposed area of dentine resulting from occlusal wear and the upper part of a vertical crack affecting enamel and dentin tissues were filled with beeswax shortly before or after the individuals death. If the filling was done when the person was still alive, the intervention was likely aimed to relieve tooth sensitivity derived from either exposed dentine and/or the pain resulting from chewing on a cracked tooth: this would provide the earliest known direct evidence of therapeutic-palliative dental filling.
Authors: Miquella G. Chavez, Jimmy Hu, Kerstin Seidel, Chunying Li, Andrew Jheon, Adrien Naveau, Orapin Horst, Ophir D. Klein.
Published: 05-01-2014
Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal's life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.
16 Related JoVE Articles!
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Shrinkage of Dental Composite in Simulated Cavity Measured with Digital Image Correlation
Authors: Jianying Li, Preetanjali Thakur, Alex S. L. Fok.
Institutions: University of Minnesota.
Polymerization shrinkage of dental resin composites can lead to restoration debonding or cracked tooth tissues in composite-restored teeth. In order to understand where and how shrinkage strain and stress develop in such restored teeth, Digital Image Correlation (DIC) was used to provide a comprehensive view of the displacement and strain distributions within model restorations that had undergone polymerization shrinkage. Specimens with model cavities were made of cylindrical glass rods with both diameter and length being 10 mm. The dimensions of the mesial-occlusal-distal (MOD) cavity prepared in each specimen measured 3 mm and 2 mm in width and depth, respectively. After filling the cavity with resin composite, the surface under observation was sprayed with first a thin layer of white paint and then fine black charcoal powder to create high-contrast speckles. Pictures of that surface were then taken before curing and 5 min after. Finally, the two pictures were correlated using DIC software to calculate the displacement and strain distributions. The resin composite shrunk vertically towards the bottom of the cavity, with the top center portion of the restoration having the largest downward displacement. At the same time, it shrunk horizontally towards its vertical midline. Shrinkage of the composite stretched the material in the vicinity of the “tooth-restoration” interface, resulting in cuspal deflections and high tensile strains around the restoration. Material close to the cavity walls or floor had direct strains mostly in the directions perpendicular to the interfaces. Summation of the two direct strain components showed a relatively uniform distribution around the restoration and its magnitude equaled approximately to the volumetric shrinkage strain of the material.
Medicine, Issue 89, image processing, computer-assisted, polymer matrix composites, testing of materials (composite materials), dental composite restoration, polymerization shrinkage, digital image correlation, full-field strain measurement, interfacial debonding
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In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint
Authors: Andrew T. Jang, Jeremy D. Lin, Youngho Seo, Sergey Etchin, Arno Merkle, Kevin Fahey, Sunita P. Ho.
Institutions: University of California San Francisco, University of California San Francisco, Xradia Inc..
This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.
Bioengineering, Issue 85, biomechanics, bone-periodontal ligament-tooth complex, concentric loads, eccentric loads, contrast agent
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The Slice Culture Method for Following Development of Tooth Germs In Explant Culture
Authors: Sarah A. Alfaqeeh, Abigail S. Tucker.
Institutions: King's College London, King Saud University, Kingdom of Saudi Arabia.
Explant culture allows manipulation of developing organs at specific time points and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing. In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel's cartilage, nasal glands, tongue, and ear.
Anatomy, Issue 81, Tooth, Culture Techniques, Embryo Culture Techniques, Organ Culture Techniques, Developmental Biology, animal biology, animal models, Tooth germ, live slice, development, tissue chopper, lineage tracing, molar, incisor, gland
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Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface
Authors: Qichao Ruan, Janet Moradian-Oldak.
Institutions: University of Southern California.
Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.
Bioengineering, Issue 89, Enamel, Amelogenin, Chitosan hydrogel, Apatite, Biomimetic, Erosion, Superficial enamel reconstruction, Dense interface
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Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision
Authors: Andreas Ender, Albert Mehl.
Institutions: University of Zürich.
Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations. The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 ± 1.1 µm for trueness and 1.6 ± 0.6 µm for precision in case of full arch scans. Current dental impression methods showed much higher deviations (trueness: 20.4 ± 2.2 µm, precision: 12.5 ± 2.5 µm) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss. The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes.
Medicine, Issue 86, Laboratories, Dental, Calibration, Technology, Dental impression, Accuracy, Trueness, Precision, Full arch scan, Abrasion
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Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy
Authors: Víctor A. Lórenz-Fonfría, Joachim Heberle.
Institutions: Freie Universität Berlin.
Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards understanding its mechanism. Protonation and conformational changes affect the vibration pattern of amino acid side chains and of the peptide bond, respectively, both of which can be probed by infrared (IR) difference spectroscopy. For proteins whose function can be repetitively and reproducibly triggered by light, it is possible to obtain infrared difference spectra with (sub)microsecond resolution over a broad spectral range using the step-scan Fourier transform infrared technique. With ~102-103 repetitions of the photoreaction, the minimum number to complete a scan at reasonable spectral resolution and bandwidth, the noise level in the absorption difference spectra can be as low as ~10-4, sufficient to follow the kinetics of protonation changes from a single amino acid. Lower noise levels can be accomplished by more data averaging and/or mathematical processing. The amount of protein required for optimal results is between 5-100 µg, depending on the sampling technique used. Regarding additional requirements, the protein needs to be first concentrated in a low ionic strength buffer and then dried to form a film. The protein film is hydrated prior to the experiment, either with little droplets of water or under controlled atmospheric humidity. The attained hydration level (g of water / g of protein) is gauged from an IR absorption spectrum. To showcase the technique, we studied the photocycle of the light-driven proton-pump bacteriorhodopsin in its native purple membrane environment, and of the light-gated ion channel channelrhodopsin-2 solubilized in detergent.
Biophysics, Issue 88, bacteriorhodopsin, channelrhodopsin, attenuated total reflection, proton transfer, protein dynamics, infrared spectroscopy, time-resolved spectroscopy, step-scan, membrane proteins, singular value decomposition
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Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles
Authors: Eva Wagner, Sören Brandenburg, Tobias Kohl, Stephan E. Lehnart.
Institutions: Heart Research Center Goettingen, University Medical Center Goettingen, German Center for Cardiovascular Research (DZHK) partner site Goettingen, University of Maryland School of Medicine.
In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.
Bioengineering, Issue 92, cardiac myocyte, atria, ventricle, heart, primary cell isolation, fluorescence microscopy, membrane tubule, transverse-axial tubule system, image analysis, image processing, T-tubule, collagenase
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Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites
Authors: Ghania Nina Attik, Kerstin Gritsch, Pierre Colon, Brigitte Grosgogeat.
Institutions: UMR CNRS 5615, Université Lyon1, Hospices Civils de Lyon, APHP, Hôpital Rothschild.
It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.
Medicine, Issue 93, In vitro biocompatibility, dental composites, Live/Deadstaining, 3D imaging, Confocal Microscopy, Time lapse imaging
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A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites
Authors: George Papadopoulos, Carolyn D. Kramer, Connie S. Slocum, Ellen O. Weinberg, Ning Hua, Cynthia V. Gudino, James A. Hamilton, Caroline A. Genco.
Institutions: Boston University School of Medicine, Boston University School of Medicine.
Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation. Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.
Immunology, Issue 90, Pathogen-Induced Chronic Inflammation; Porphyromonas gingivalis; Oral Bone Loss; Periodontal Disease; Atherosclerosis; Chronic Inflammation; Host-Pathogen Interaction; microCT; MRI
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In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions
Authors: Grant E. Johnson, K. Don Dasitha Gunaratne, Julia Laskin.
Institutions: Pacific Northwest National Laboratory.
Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3]2+ (bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.
Chemistry, Issue 88, soft landing, mass selected ions, electrospray, secondary ion mass spectrometry, infrared spectroscopy, organometallic, catalysis
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Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms
Authors: Derek S. Samarian, Nicholas S. Jakubovics, Ting L. Luo, Alexander H. Rickard.
Institutions: The University of Michigan, Newcastle University.
There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.
Bioengineering, Issue 94, Dental plaque, biofilm, confocal laser scanning microscopy, three-dimensional structure, pyrosequencing, image analysis, image reconstruction, saliva, modeling, COMSTAT, IMARIS, IMAGEJ, multi-species biofilm communities.
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Meal Duration as a Measure of Orofacial Nociceptive Responses in Rodents
Authors: Phillip R. Kramer, Larry L. Bellinger.
Institutions: Texas A&M University Baylor College of Dentistry.
A lengthening in meal duration can be used to measure an increase in orofacial mechanical hyperalgesia having similarities to the guarding behavior of humans with orofacial pain. To measure meal duration unrestrained rats are continuously kept in sound attenuated, computerized feeding modules for days to weeks to record feeding behavior. These sound-attenuated chambers are equipped with chow pellet dispensers. The dispenser has a pellet trough with a photobeam placed at the bottom of the trough and when a rodent removes a pellet from the feeder trough this beam is no longer blocked, signaling the computer to drop another pellet. The computer records the date and time when the pellets were taken from the trough and from this data the experimenter can calculate the meal parameters. When calculating meal parameters a meal was defined based on previous work and was set at 10 min (in other words when the animal does not eat for 10 min that would be the end of the animal's meal) also the minimum meal size was set at 3 pellets. The meal duration, meal number, food intake, meal size and inter-meal interval can then be calculated by the software for any time period that the operator desires. Of the feeding parameters that can be calculated meal duration has been shown to be a continuous noninvasive biological marker of orofacial nociception in male rats and mice and female rats. Meal duration measurements are quantitative, require no training or animal manipulation, require cortical participation, and do not compete with other experimentally induced behaviors. These factors distinguish this assay from other operant or reflex methods for recording orofacial nociception.
Behavior, Issue 83, Pain, rat, nociception, myofacial, orofacial, tooth, temporomandibular joint (TMJ)
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Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods
Authors: Razieh Karamzadeh, Mohamadreza Baghaban Eslaminejad, Reza Aflatoonian.
Institutions: Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers & also their differentiation capacity. Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 & CD44), hematopoietic/endothelial Markers (CD34, CD45 & CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) & Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE & dentin sialophosphoprotein; DSPP).14
Stem Cell Biology, Issue 69, Medicine, Developmental Biology, Cellular Biology, Bioengineering, Dental pulp tissue, Human third molar, Human dental pulp stem cells, hDPSC, Odontoblasts, Outgrown stem cells, MSC, differentiation
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Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging
Authors: Jill J. Weyers, Dara D. Carlson, Charles E. Murry, Stephen M. Schwartz, William M. Mahoney, Jr..
Institutions: University of Washington, University of Washington.
Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (μCT). μCT imaging can scan at resolutions <10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo μCT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by μCT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for μCT imaging. Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network – arteries, capillaries, and veins alike.
Medicine, Issue 60, Vascular biology, heart, coronary vessels, mouse, micro Computed Tomography (μCT) imaging, Microfil
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Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure
Authors: Bindesh Shrestha, Akos Vertes.
Institutions: George Washington University.
Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 μm wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.
Cellular Biology, Issue 43, single cell analysis, mass spectrometry, laser ablation electrospray ionization, LAESI, metabolomics, direct analysis
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Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography
Authors: Michael Heethoff, Lukas Helfen, Peter Cloetens.
Institutions: University of Tubingen, European Synchrotron Radiation Facility.
Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning. We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7µm. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.
Developmental Biology, Issue 15, Synchrotron X-ray tomography, Acari, Oribatida, micro-arthropods, non-invasive investigation
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.