We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC) to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using “off-the-shelf” microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) μm in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.
25 Related JoVE Articles!
Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants
Institutions: Purdue University, Purdue University, Jiangsu Academy of Agricultural Sciences, Zhejiang University, Shanxi Academy of Agricultural Sciences, Chinese Academy of Sciences.
Developmental and environmental cues induce Ca2+
fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+
patterns are sensed by cellular Ca2+
binding proteins that initiate Ca2+
signaling cascades. However, we still know little about how stimulus specific Ca2+
signals are generated. The specificity of a Ca2+
signal may be attributed to the sophisticated regulation of the activities of Ca2+
channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+
signals at both the tissue and cellular levels. Genetically encoded Ca2+
indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+
signals. Here we describe instructions for the use of two Ca2+
detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+
imaging and case12 based live cell confocal fluorescence Ca2+
imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+
signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+
signals at a high resolution.
Plant Biology, Issue 91, Aequorin, Case12, abiotic stress, heavy metal stress, copper ion, calcium imaging, Arabidopsis
Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture
Institutions: Western Washington University, Washington State University Northwestern Research and Extension Center, Texas Tech University.
Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to degrade plastics. Typically, when formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones. However, this approach poorly mimics in situ
degradation of BDMs. First, BDMs are not dispersed as small particles throughout the soil matrix. Secondly, BDMs are not sold commercially as pure polymers, but rather as films containing additives (e.g.
fillers, plasticizers and dyes) that may affect microbial growth. The procedures described herein were used for isolates acquired from soil-buried mulch films. Fungal isolates acquired from excavated BDMs were tested individually for growth on pieces of new, disinfested BDMs laid atop defined medium containing no carbon source except agar. Isolates that grew on BDMs were further tested in liquid medium where BDMs were the sole added carbon source. After approximately ten weeks, fungal colonization and BDM degradation were assessed by scanning electron microscopy. Isolates were identified via analysis of ribosomal RNA gene sequences. This report describes methods for fungal isolation, but bacteria also were isolated using these methods by substituting media appropriate for bacteria. Our methodology should prove useful for studies investigating breakdown of intact plastic films or products for which plastic feedstocks are either unknown or not available. However our approach does not provide a quantitative method for comparing rates of BDM degradation.
Microbiology, Issue 75, Plant Biology, Environmental Sciences, Agricultural Sciences, Soil Science, Molecular Biology, Cellular Biology, Genetics, Mycology, Fungi, Bacteria, Microorganisms, Biodegradable plastic, biodegradable mulch, compostable plastic, compostable mulch, plastic degradation, composting, breakdown, soil, 18S ribosomal DNA, isolation, culture
Characterization of Electrode Materials for Lithium Ion and Sodium Ion Batteries Using Synchrotron Radiation Techniques
Institutions: Lawrence Berkeley National Laboratory, University of Illinois at Chicago, Stanford Synchrotron Radiation Lightsource, Haldor Topsøe A/S, PolyPlus Battery Company.
Intercalation compounds such as transition metal oxides or phosphates are the most commonly used electrode materials in Li-ion and Na-ion batteries. During insertion or removal of alkali metal ions, the redox states of transition metals in the compounds change and structural transformations such as phase transitions and/or lattice parameter increases or decreases occur. These behaviors in turn determine important characteristics of the batteries such as the potential profiles, rate capabilities, and cycle lives. The extremely bright and tunable x-rays produced by synchrotron radiation allow rapid acquisition of high-resolution data that provide information about these processes. Transformations in the bulk materials, such as phase transitions, can be directly observed using X-ray diffraction (XRD), while X-ray absorption spectroscopy (XAS) gives information about the local electronic and geometric structures (e.g.
changes in redox states and bond lengths). In situ
experiments carried out on operating cells are particularly useful because they allow direct correlation between the electrochemical and structural properties of the materials. These experiments are time-consuming and can be challenging to design due to the reactivity and air-sensitivity of the alkali metal anodes used in the half-cell configurations, and/or the possibility of signal interference from other cell components and hardware. For these reasons, it is appropriate to carry out ex situ
on electrodes harvested from partially charged or cycled cells) in some cases. Here, we present detailed protocols for the preparation of both ex situ
and in situ
samples for experiments involving synchrotron radiation and demonstrate how these experiments are done.
Physics, Issue 81, X-Ray Absorption Spectroscopy, X-Ray Diffraction, inorganic chemistry, electric batteries (applications), energy storage, Electrode materials, Li-ion battery, Na-ion battery, X-ray Absorption Spectroscopy (XAS), in situ X-ray diffraction (XRD)
Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Institutions: University of Maine.
Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.
Basic Protocol, Issue 82, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
Designing Silk-silk Protein Alloy Materials for Biomedical Applications
Institutions: Rowan University, Rowan University, Cooper Medical School of Rowan University, Rowan University.
Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi
) and domestic mulberry silk (Bombyx mori
) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.
Bioengineering, Issue 90, protein alloys, biomaterials, biomedical, silk blends, computational simulation, implantable electronic devices
Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation
Institutions: Virginia Tech, Virginia Tech, Illinois Institute of Technology- Moffett Campus, University of Guadalajara, Virginia Tech, Virginia Tech.
Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall.
Plant Biology, Issue 88, nanocellulose, thin films, quartz crystal microbalance, layer-by-layer, LbL
A Technique to Functionalize and Self-assemble Macroscopic Nanoparticle-ligand Monolayer Films onto Template-free Substrates
Institutions: Naval Research Laboratory.
This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2
. The simple, robust and scalable technique efficiently functionalizes metallic nanoparticles with thiol-ligands in a miscible water/organic solvent mixture allowing for rapid grafting of thiol groups onto the gold nanoparticle surface. The hydrophobic ligands on the nanoparticles then quickly phase separate the nanoparticles from the aqueous based suspension and confine them to the air-fluid interface. This drives the ligand-capped nanoparticles to form monolayer domains at the air-fluid interface. The use of water-miscible organic solvents is important as it enables the transport of the nanoparticles from the interface onto template-free substrates. The flow is mediated by a surface tension gradient3,4
and creates macroscopic, high-density, monolayer nanoparticle-ligand films. This self-assembly technique may be generalized to include the use of particles of different compositions, size, and shape and may lead to an efficient assembly method to produce low-cost, macroscopic, high-density, monolayer nanoparticle films for wide-spread applications.
Chemistry, Issue 87, phase transfer, nanoparticle, self-assembly, bottom-up, fabrication, low-cost, monolayer, thin film, nanostructure, array, metamaterial
In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions
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
(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
Confocal Imaging of Confined Quiescent and Flowing Colloid-polymer Mixtures
Institutions: University of Houston.
The behavior of confined colloidal suspensions with attractive interparticle interactions is critical to the rational design of materials for directed assembly1-3
, drug delivery4
, improved hydrocarbon recovery5-7
, and flowable electrodes for energy storage8
. Suspensions containing fluorescent colloids and non-adsorbing polymers are appealing model systems, as the ratio of the polymer radius of gyration to the particle radius and concentration of polymer control the range and strength of the interparticle attraction, respectively. By tuning the polymer properties and the volume fraction of the colloids, colloid fluids, fluids of clusters, gels, crystals, and glasses can be obtained9
. Confocal microscopy, a variant of fluorescence microscopy, allows an optically transparent and fluorescent sample to be imaged with high spatial and temporal resolution in three dimensions. In this technique, a small pinhole or slit blocks the emitted fluorescent light from regions of the sample that are outside the focal volume of the microscope optical system. As a result, only a thin section of the sample in the focal plane is imaged. This technique is particularly well suited to probe the structure and dynamics in dense colloidal suspensions at the single-particle scale: the particles are large enough to be resolved using visible light and diffuse slowly enough to be captured at typical scan speeds of commercial confocal systems10
. Improvements in scan speeds and analysis algorithms have also enabled quantitative confocal imaging of flowing suspensions11-16,37
. In this paper, we demonstrate confocal microscopy experiments to probe the confined phase behavior and flow properties of colloid-polymer mixtures. We first prepare colloid-polymer mixtures that are density- and refractive-index matched. Next, we report a standard protocol for imaging quiescent dense colloid-polymer mixtures under varying confinement in thin wedge-shaped cells. Finally, we demonstrate a protocol for imaging colloid-polymer mixtures during microchannel flow.
Chemistry, Issue 87, confocal microscopy, particle tracking, colloids, suspensions, confinement, gelation, microfluidics, image correlation, dynamics, suspension flow
Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy
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
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
Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers
Institutions: The Children's Hospital of Philadelphia, University of Pennsylvania.
In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2
of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.
Medicine, Issue 90, gene therapy, bioconjugation, adenoviral vectors, stents, local gene delivery, smooth muscle cells, endothelial cells, bioluminescence imaging
Concurrent Quantitative Conductivity and Mechanical Properties Measurements of Organic Photovoltaic Materials using AFM
Institutions: Argonne National Laboratory, University of Chicago.
Organic photovoltaic (OPV) materials are inherently inhomogeneous at the nanometer scale. Nanoscale inhomogeneity of OPV materials affects performance of photovoltaic devices. Thus, understanding of spatial variations in composition as well as electrical properties of OPV materials is of paramount importance for moving PV technology forward.1,2
In this paper, we describe a protocol for quantitative measurements of electrical and mechanical properties of OPV materials with sub-100 nm resolution. Currently, materials properties measurements performed using commercially available AFM-based techniques (PeakForce, conductive AFM) generally provide only qualitative information. The values for resistance as well as Young's modulus measured using our method on the prototypical ITO/PEDOT:PSS/P3HT:PC61
BM system correspond well with literature data. The P3HT:PC61
BM blend separates onto PC61
BM-rich and P3HT-rich domains. Mechanical properties of PC61
BM-rich and P3HT-rich domains are different, which allows for domain attribution on the surface of the film. Importantly, combining mechanical and electrical data allows for correlation of the domain structure on the surface of the film with electrical properties variation measured through the thickness of the film.
Materials Science, Issue 71, Nanotechnology, Mechanical Engineering, Electrical Engineering, Computer Science, Physics, electrical transport properties in solids, condensed matter physics, thin films (theory, deposition and growth), conductivity (solid state), AFM, atomic force microscopy, electrical properties, mechanical properties, organic photovoltaics, microengineering, photovoltaics
Characterization of Surface Modifications by White Light Interferometry: Applications in Ion Sputtering, Laser Ablation, and Tribology Experiments
Institutions: Argonne National Laboratory, Argonne National Laboratory, MassThink LLC.
In materials science and engineering it is often necessary to obtain quantitative measurements of surface topography with micrometer lateral resolution. From the measured surface, 3D topographic maps can be subsequently analyzed using a variety of software packages to extract the information that is needed.
In this article we describe how white light interferometry, and optical profilometry (OP) in general, combined with generic surface analysis software, can be used for materials science and engineering tasks. In this article, a number of applications of white light interferometry for investigation of surface modifications in mass spectrometry, and wear phenomena in tribology and lubrication are demonstrated. We characterize the products of the interaction of semiconductors and metals with energetic ions (sputtering), and laser irradiation (ablation), as well as ex situ
measurements of wear of tribological test specimens.
Specifically, we will discuss:
Aspects of traditional ion sputtering-based mass spectrometry such as sputtering rates/yields measurements on Si and Cu and subsequent time-to-depth conversion.
Results of quantitative characterization of the interaction of femtosecond laser irradiation with a semiconductor surface. These results are important for applications such as ablation mass spectrometry, where the quantities of evaporated material can be studied and controlled via pulse duration and energy per pulse. Thus, by determining the crater geometry one can define depth and lateral resolution versus experimental setup conditions.
Measurements of surface roughness parameters in two dimensions, and quantitative measurements of the surface wear that occur as a result of friction and wear tests.
Some inherent drawbacks, possible artifacts, and uncertainty assessments of the white light interferometry approach will be discussed and explained.
Materials Science, Issue 72, Physics, Ion Beams (nuclear interactions), Light Reflection, Optical Properties, Semiconductor Materials, White Light Interferometry, Ion Sputtering, Laser Ablation, Femtosecond Lasers, Depth Profiling, Time-of-flight Mass Spectrometry, Tribology, Wear Analysis, Optical Profilometry, wear, friction, atomic force microscopy, AFM, scanning electron microscopy, SEM, imaging, visualization
Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres
Institutions: Agriculture and Agri-Food Canada, University of Saskatchewan , National Research Council of Canada.
Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus
-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis
, Atopobium vaginae
, and others1-3
. This condition is associated with a range of negative health outcomes, including HIV acquisition4
, and it can be difficult to manage clinically5
. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7
. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8
. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10
, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12
. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13
. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14
. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.
Immunology, Issue 56, Medicine, chaperonin-60, hsp60, luminex, multiplex, diagnostics, bacterial vaginosis, PCR
Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis
Institutions: Louisiana State University Health Sciences Center.
Vulvovaginal candidiasis (VVC), caused by Candida
species, is a fungal infection of the lower female genital tract that affects approximately 75% of otherwise healthy women during their reproductive years18,32-34
. Predisposing factors include antibiotic usage, uncontrolled diabetes and disturbance in reproductive hormone levels due to pregnancy, oral contraceptives or hormone replacement therapies33,34
. Recurrent VVC (RVVC), defined as three or more episodes per year, affects a separate 5 to 8% of women with no predisposing factors33
An experimental mouse model of VVC has been established and used to study the pathogenesis and mucosal host response to Candida3,4,11,16,17,19,21,25,37
. This model has also been employed to test potential antifungal therapies in vivo13,24
. The model requires that the animals be maintained in a state of pseudoestrus for optimal Candida
. Under such conditions, inoculated animals will have detectable vaginal fungal burden for weeks to months. Past studies show an extremely high parallel between the animal model and human infection relative to immunological and physiological properties3,16,21
. Differences, however, include a lack of Candida
as normal vaginal flora and a neutral vaginal pH in the mice.
Here, we demonstrate a series of key methods in the mouse vaginitis model that include vaginal inoculation, rapid collection of vaginal specimens, assessment of vaginal fungal burden, and tissue preparations for cellular extraction/isolation. This is followed by representative results for constituents of vaginal lavage fluid, fungal burden, and draining lymph node leukocyte yields. With the use of anesthetics, lavage samples can be collected at multiple time points on the same mice for longitudinal evaluation of infection/colonization. Furthermore, this model requires no immunosuppressive agents to initiate infection, allowing immunological studies under defined host conditions. Finally, the model and each technique introduced here could potentially give rise to use of the methodologies to examine other infectious diseases of the lower female genital tract (bacterial, parasitic, viral) and respective local or systemic host defenses.
Immunology, Issue 58, Candida albicans, vaginitis, mouse, lumbar lymph nodes, vaginal tissues, vaginal lavage
A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium
Institutions: The University of Exeter.
The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1
. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2
. We describe the protocol for use of PFD with Arabidopsis thaliana
leaf tissue, which is optically complex as a result of its structure (Figure 1
). PFD has a number of attributes that make it suitable for this use3
. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1
) is lower than that of water (72 dynes cm-1
) and also below the limit (25 - 30 dyne cm-1
) for stomatal penetration4
, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2
, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6
, artificial blood8
, oxygenation of growth media9
, and studies of ice crystal formation in plants10
. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.
Plant Biology, Issue 59, Arabidopsis, leaf, confocal, microscopy, perfluorocarbon, perfluorodecalin, PFD, mesophyll, imaging depth
Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue
Institutions: University of Missouri, University of Missouri.
Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of lesions, and/or chronic pharmaceutical management of pain and infertility symptoms. The cost associated with endometriosis has been estimated at 22 billion dollars per year in the United States1
. To further our understanding of mechanisms underlying this enigmatic disease, animal models have been employed. Primates spontaneously develop endometriosis and therefore primate models most closely resemble the disease in women. Rodent models, however, are more cost effective and readily available2
. The model that we describe here involves an autologous transfer of uterine tissue to the intestinal mesentery (Figure 1) and was first developed in the rat3
and later transferred to the mouse4
. The goal of the autologous rodent model of surgically-induced endometriosis is to mimic the disease in women. We and others have previously shown that the altered gene expression pattern observed in endometriotic lesions from mice or rats mirrors that observed in women with the disease5,6
. One advantage of performing the surgery in the mouse is that the abundance of transgenic mouse strains available can aid researchers in determining the role of specific components important in the establishment and growth of endometriosis. An alternative model in which excised human endometrial fragments are introduced to the peritoneum of immunocompromised mice is also widely used but is limited by the lack of a normal immune system which is thought to be important in endometriosis2,7
. Importantly, the mouse model of surgically induced endometriosis is a versatile model that has been used to study how the immune system8
and environmental factors11,12
affect endometriosis as well as the effects of endometriosis on fertility13
Medicine, Issue 59, mouse, rat, endometriosis, surgery, uterus, ectopic, endometriotic lesion
Silk Film Culture System for in vitro Analysis and Biomaterial Design
Institutions: Weill Cornell Medical College , Tufts University.
Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2
. These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8
. In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro
or in vivo
, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13
. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14
. The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro
culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo
models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.
Bioengineering, Issue 62, silk, fibroin, film, biomaterial, surface patterning, in vitro, epithelium, cell culture
Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models
Institutions: University of Arizona College of Medicine - Phoenix.
Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro
cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment.
Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues 1-6
. The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A)
. The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B)
. The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-
like characteristics often not observed under standard 2-D culture conditions (Figure 1D)
. These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo
protein localization, and additional epithelial cell-type specific properties.
The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type1, 7-13
. Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability (Figure 1D)
. Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G)
. In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/drug analysis and host-pathogen interactions. The utilization of these assays set the foundation for more in-depth and expansive studies such as metabolomics, transcriptomics, proteomics and other array-based applications. Our goal is to present a non-conventional means of culturing human epithelial cells to produce organotypic 3-D models that recapitulate the human in vivo
tissue, in a facile and robust system to be used by researchers with diverse scientific interests.
Cellular Biology, Issue 62, Rotating wall vessel bioreactor, female reproductive tract, human epithelial cells, three-dimensional in vitro cell culture, organotypic mucosal models, vaginal epithelial cells, microbicide, herpes simplex virus, toxicology, host-pathogen interactions, hormone receptors
Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries
Institutions: Iowa State University.
Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1
. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1
), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2
) as described previously1
. This combinatorial platform has been validated with conventional methods2
and the polyanhydride film and nanoparticle libraries have been characterized with 1
H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro
cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo
biodistribution and mucoadhesion1-11
. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro
and in vivo
for optimization of biomaterial performance.
Bioengineering, Issue 67, combinatorial, high-throughput, polymer synthesis, polyanhydrides, nanoparticle fabrication, release kinetics, protein delivery
Polycrystalline Silicon Thin-film Solar cells with Plasmonic-enhanced Light-trapping
Institutions: University of New South Wales .
One of major approaches to cheaper solar cells is reducing the amount of semiconductor material used for their fabrication and making cells thinner. To compensate for lower light absorption such physically thin devices have to incorporate light-trapping which increases their optical thickness. Light scattering by textured surfaces is a common technique but it cannot be universally applied to all solar cell technologies. Some cells, for example those made of evaporated silicon, are planar as produced and they require an alternative light-trapping means suitable for planar devices. Metal nanoparticles formed on planar silicon cell surface and capable of light scattering due to surface plasmon resonance is an effective approach.
The paper presents a fabrication procedure of evaporated polycrystalline silicon solar cells with plasmonic light-trapping and demonstrates how the cell quantum efficiency improves due to presence of metal nanoparticles.
To fabricate the cells a film consisting of alternative boron and phosphorous doped silicon layers is deposited on glass substrate by electron beam evaporation. An Initially amorphous film is crystallised and electronic defects are mitigated by annealing and hydrogen passivation. Metal grid contacts are applied to the layers of opposite polarity to extract electricity generated by the cell. Typically, such a ~2 μm thick cell has a short-circuit current density (Jsc
) of 14-16 mA/cm2
, which can be increased up to 17-18 mA/cm2
(~25% higher) after application of a simple diffuse back reflector made of a white paint.
To implement plasmonic light-trapping a silver nanoparticle array is formed on the metallised cell silicon surface. A precursor silver film is deposited on the cell by thermal evaporation and annealed at 23°C to form silver nanoparticles. Nanoparticle size and coverage, which affect plasmonic light-scattering, can be tuned for enhanced cell performance by varying the precursor film thickness and its annealing conditions. An optimised nanoparticle array alone results in cell Jsc
enhancement of about 28%, similar to the effect of the diffuse reflector. The photocurrent can be further increased by coating the nanoparticles by a low refractive index dielectric, like MgF2
, and applying the diffused reflector. The complete plasmonic cell structure comprises the polycrystalline silicon film, a silver nanoparticle array, a layer of MgF2
, and a diffuse reflector. The Jsc
for such cell is 21-23 mA/cm2
, up to 45% higher than Jsc
of the original cell without light-trapping or ~25% higher than Jsc
for the cell with the diffuse reflector only.
Light-trapping in silicon solar cells is commonly achieved via light scattering at textured interfaces. Scattered light travels through a cell at oblique angles for a longer distance and when such angles exceed the critical angle at the cell interfaces the light is permanently trapped in the cell by total internal reflection (Animation 1: Light-trapping)
. Although this scheme works well for most solar cells, there are developing technologies where ultra-thin Si layers are produced planar (e.g. layer-transfer technologies and epitaxial c-Si layers) 1
and or when such layers are not compatible with textures substrates (e.g. evaporated silicon) 2
. For such originally planar Si layer alternative light trapping approaches, such as diffuse white paint reflector 3
, silicon plasma texturing 4
or high refractive index nanoparticle reflector 5
have been suggested.
Metal nanoparticles can effectively scatter incident light into a higher refractive index material, like silicon, due to the surface plasmon resonance effect 6
. They also can be easily formed on the planar silicon cell surface thus offering a light-trapping approach alternative to texturing. For a nanoparticle located at the air-silicon interface the scattered light fraction coupled into silicon exceeds 95% and a large faction of that light is scattered at angles above critical providing nearly ideal light-trapping condition (Animation 2: Plasmons on NP)
. The resonance can be tuned to the wavelength region, which is most important for a particular cell material and design, by varying the nanoparticle average size, surface coverage and local dielectric environment 6,7
. Theoretical design principles of plasmonic nanoparticle solar cells have been suggested 8
. In practice, Ag nanoparticle array is an ideal light-trapping partner for poly-Si thin-film solar cells because most of these design principle are naturally met. The simplest way of forming nanoparticles by thermal annealing of a thin precursor Ag film results in a random array with a relatively wide size and shape distribution, which is particularly suitable for light-trapping because such an array has a wide resonance peak, covering the wavelength range of 700-900 nm, important for poly-Si solar cell performance. The nanoparticle array can only be located on the rear poly-Si cell surface thus avoiding destructive interference between incident and scattered light which occurs for front-located nanoparticles 9
. Moreover, poly-Si thin-film cells do not requires a passivating layer and the flat base-shaped nanoparticles (that naturally result from thermal annealing of a metal film) can be directly placed on silicon further increases plasmonic scattering efficiency due to surface plasmon-polariton resonance 10
The cell with the plasmonic nanoparticle array as described above can have a photocurrent about 28% higher than the original cell. However, the array still transmits a significant amount of light which escapes through the rear of the cell and does not contribute into the current. This loss can be mitigated by adding a rear reflector to allow catching transmitted light and re-directing it back to the cell. Providing sufficient distance between the reflector and the nanoparticles (a few hundred nanometers) the reflected light will then experience one more plasmonic scattering event while passing through the nanoparticle array on re-entering the cell and the reflector itself can be made diffuse - both effects further facilitating light scattering and hence light-trapping. Importantly, the Ag nanoparticles have to be encapsulated with an inert and low refractive index dielectric, like MgF2
, from the rear reflector to avoid mechanical and chemical damage 7
. Low refractive index for this cladding layer is required to maintain a high coupling fraction into silicon and larger scattering angles, which are ensured by the high optical contrast between the media on both sides of the nanoparticle, silicon and dielectric 6
. The photocurrent of the plasmonic cell with the diffuse rear reflector can be up to 45% higher than the current of the original cell or up to 25% higher than the current of an equivalent cell with the diffuse reflector only.
Physics, Issue 65, Materials Science, Photovoltaics, Silicon thin-film solar cells, light-trapping, metal nanoparticles, surface plasmons
Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution
Institutions: University of California, Los Angeles , University of California, Los Angeles , University of California, Los Angeles .
Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. 1-6
These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans
, zebrafish and mouse embryos.
Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7
LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of <1 μm x <1 μm x <3 μm in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3
, and can be particularly useful for lab-on-a-chip platforms.
Bioengineering, Issue 66, Electrical Engineering, Mechanical Engineering, lensfree imaging, lensless imaging, on-chip microscopy, lensfree tomography, 3D microscopy, pixel super-resolution, C. elegans, optical sectioning, lab-on-a-chip
Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology
Institutions: University of Washington.
The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes >10 μm in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.
Physiology, Issue 76, Biophysics, Molecular Biology, Biochemistry, Genetics, Cellular Biology, Proteins, Membranes, Artificial, Lipid Bilayers, Liposomes, Phospholipids, biochemistry, Lipids, Giant Unilamellar Vesicles, liposome, electrophysiology, electroformation, reconstitution, patch clamp
Phase Contrast and Differential Interference Contrast (DIC) Microscopy
Institutions: University of Texas Health Science Center at San Antonio (UTHSCSA).
Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the phase halos typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.
Basic protocols, Issue 18, Current Protocols Wiley, Microscopy, Phase Contrast, Difference Interference Contrast
Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation
Institutions: Karlsruhe Research Centre, University of Twente, Institute for Heavy Ion Research, Karlsruhe Research Centre, Karlsruhe Research Centre.
Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.
Cellular Biology, Issue 15, SMART, microthermoforming, microfabrication, scaffolds, polymer