Optical tweezers reveal force plateau and internal friction in PEG-induced DNA condensation.

The simplified artificial environments in which highly complex biological systems are studied do not represent the crowded, dense, salty, and dynamic environment inside the living cell. Consequently, it is important to investigate the effect of crowding agents on DNA. We used a dual-trap optical tweezers instrument to perform force spectroscopy experiments at pull speeds ranging from 0.3 to 270 µm/s on single dsDNA molecules in the presence of poly(ethylene glycol) (PEG) and monovalent salt. PEG of sizes 1,500 and 4,000 Da condensed DNA, and force-extension data contained a force plateau at approximately 1 pN. The level of the force plateau increased with increasing pull speed. During slow pulling the dissipated work increased linearly with pull speed. The calculated friction coefficient did not depend on amount of DNA incorporated in the condensate, indicating internal friction is independent of the condensate size. PEG300 had no effect on the dsDNA force-extension curve. The force plateau implies that condensation induced by crowding agents resembles condensation induced by multivalent cations.

Evaluation of bone growth around bioactive glass S53P4 by scanning acoustic microscopy co-registered with optical interferometry and elemental analysis.

Bioactive glass (BAG) is a bone substitute that can be used in orthopaedic surgery. Following implantation, the BAG is expected to be replaced by bone via bone growth and gradual degradation of the BAG. However, the hydroxyapatite mineral forming on BAG resembles bone mineral, not providing sufficient contrast to distinguish the two in X-ray images. In this study, we co-registered coded-excitation scanning acoustic microscopy (CESAM), scanning white light interferometry (SWLI), and scanning electron microscopy with elemental analysis (Energy Dispersive X-ray Spectroscopy) (SEM-EDX) to investigate the bone growth and BAG reactions on a micron scale in a rabbit bone ex vivo. The acoustic impedance map recorded by the CESAM provides high elasticity-associated contrast to study materials and their combinations, while simultaneously producing a topography map of the sample. The acoustic impedance map correlated with the elemental analysis from SEM-EDX. SWLI also produces a topography map, but with higher resolution than CESAM. The two topography maps (CESAM and SWLI) were in good agreement. Furthermore, using information from both maps simultaneously produced by the CESAM (acoustic impedance and topography) allowed determining regions-of-interest related to bone formation around the BAG with greater ease than from either map alone. CESAM is therefore a promising tool for evaluating the degradation of bone substitutes and the bone healing process ex vivo.

Rapid interferometric imaging of printed drug laden multilayer structures.

The developments in printing technologies allow fabrication of micron-size nano-layered delivery systems to personal specifications. In this study we fabricated layered polymer structures for drug-delivery into a microfluidic channel and aimed to interferometrically assure their topography and adherence to each other. We present a scanning white light interferometer (SWLI) method for quantitative assurance of the topography of the embedded structure. We determined rapidly in non-destructive manner the thickness and roughness of the structures and whether the printed layers containing polymers or/and active pharmaceutical ingredients (API) adhere to each other. This is crucial in order to have predetermined drug release profiles. We also demonstrate non-invasive measurement of a polymer structure in a microfluidic channel. It shown that traceable interferometric 3D microscopy is a viable technique for detailed structural quality assurance of layered drug-delivery systems. The approach can have impact and find use in a much broader setting within and outside life sciences.

Photo-acoustic excitation and optical detection of fundamental flexural guided wave in coated bone phantoms.

Photo-acoustic (PA) imaging was combined with skeletal quantitative ultrasound (QUS) for assessment of human long bones. This approach permitted low-frequency excitation and detection of ultrasound so as to efficiently receive the thickness-sensitive fundamental flexural guided wave (FFGW) through a coating of soft tissue. The method was tested on seven axisymmetric bone phantoms, whose 1- to 5-mm wall thickness and 16-mm diameter mimicked those of the human radius. Phantoms were made of a composite material and coated with a 2.5- to 7.5-mm layer of soft material that mimicked soft tissue. Ultrasound was excited with a pulsed Nd:YAG laser at 1064-nm wavelength and received on the same side of the coated phantom with a heterodyne interferometer. The FFGW was detected at 30-kHz frequency. Fitting the FFGW phase velocity by the FLC(1,1) tube mode provided an accurate (9.5 ± 4.0%) wall thickness estimate. Ultrasonic in vivo characterization of cortical bone thickness may thus become possible.

Nanometer depth resolution in 3D topographic analysis of drug-loaded nanofibrous mats without sample preparation.

We showed that scanning white light interferometry (SWLI) can provide nanometer depth resolution in 3D topographic analysis of electrospun drug-loaded nanofibrous mats without sample preparation. The method permits rapidly investigating geometric properties (e.g. fiber diameter, orientation and morphology) and surface topography of drug-loaded nanofibers and nanomats. Electrospun nanofibers of a model drug, piroxicam (PRX), and hydroxypropyl methylcellulose (HPMC) were imaged. Scanning electron microscopy (SEM) served as a reference method. SWLI 3D images featuring 29 nm by 29 nm active pixel size were obtained of a 55 µm × 40 µm area. The thickness of the drug-loaded non-woven nanomats was uniform, ranging from 2.0 µm to 3.0 µm (SWLI), and independent of the ratio between HPMC and PRX. The average diameters (n=100, SEM) for drug-loaded nanofibers were 387 ± 125 nm (HPMC and PRX 1:1), 407 ± 144 nm (HPMC and PRX 1:2), and 290 ± 100 nm (HPMC and PRX 1:4). We found advantages and limitations in both techniques. SWLI permits rapid non-contacting and non-destructive characterization of layer orientation, layer thickness, porosity, and surface morphology of electrospun drug-loaded nanofibers and nanomats. Such analysis is important because the surface topography affects the performance of nanomats in pharmaceutical and biomedical applications.

Scratch resistance of plasticized hydroxypropyl methylcellulose (HPMC) films intended for tablet coatings.

Scratch resistance (SR) of externally plasticized hydroxypropyl methylcellulose (HPMC) films intended for tablet film coatings was studied. Special attention was paid to effects of short-term aging and ultraviolet (UV) treatment on the SR properties of these films. Controlled scratching of the films was performed with a Lloyd LRX materials tester featuring a spherical steel tip. Scratch surface profiles were measured by scanning white light interferometry (SWLI). The influence of using an external plasticizer on the SR was studied by comparing scratch dimensions in non-plasticized films to samples plasticized either with glycerol or polyethylene glycol (PEG) 400. The study demonstrates that both the amount and type of plasticizer influences the SR of aqueous HPMC films. It also shows that SWLI can quantitatively evaluate the effect of plasticizer content and aging on the SR of pharmaceutical films. This knowledge could be used to optimize pharmaceutical film coating formulations.

Layer by layer three-dimensional tissue epitaxy by cell-laden hydrogel droplets.

The ability to bioengineer three-dimensional (3D) tissues is a potentially powerful approach to treat diverse diseases such as cancer, loss of tissue function, or organ failure. Traditional tissue engineering methods, however, face challenges in fabricating 3D tissue constructs that resemble the native tissue microvasculature and microarchitectures. We have developed a bioprinter that can be used to print 3D patches of smooth muscle cells (5 mm x 5 mm x 81 microm) encapsulated within collagen. Current inkjet printing systems suffer from loss of cell viability and clogging. To overcome these limitations, we developed a system that uses mechanical valves to print high viscosity hydrogel precursors containing cells. The bioprinting platform that we developed enables (i) printing of multilayered 3D cell-laden hydrogel structures (16.2 microm thick per layer) with controlled spatial resolution (proximal axis: 18.0 +/- 7.0 microm and distal axis: 0.5 +/- 4.9 microm), (ii) high-throughput droplet generation (1 s per layer, 160 droplets/s), (iii) cell seeding uniformity (26 +/- 2 cells/mm(2) at 1 million cells/mL, 122 +/- 20 cells/mm(2) at 5 million cells/mL, and 216 +/- 38 cells/mm(2) at 10 million cells/mL), and (iv) long-term viability in culture (>90%, 14 days). This platform to print 3D tissue constructs may be beneficial for regenerative medicine applications by enabling the fabrication of printed replacement tissues.

Capacitive micromachined ultrasonic transducers with piston-shaped membranes: fabrication and experimental characterization.

Capacitive micromachined ultrasonic transducers (CMUTs) featuring piston-shaped membranes (piston CMUTs) were developed to improve device performance in terms of transmission efficiency, reception sensitivity, and fractional bandwidth (FBW). A piston CMUT has a relatively flat active moving surface whose membrane motion is closer to ideal piston-type motion compared with a CMUT with uniformly thick membranes (classical CMUT). Piston CMUTs with a more uniform surface displacement profile can achieve high output pressure with a relatively small electrode separation. The improved device capacitance and gap uniformity also enhance detection sensitivity. By adding a center mass to the membrane, a large ratio of second-order resonant frequency to first-order resonant frequency was achieved. This improved the FBW. Piston CMUTs featuring membranes of different geometric shapes were designed and fabricated using wafer bonding. Fabricating piston CMUTs is a more complex process than fabricating CMUTs with uniformly thick membranes. However, no yield loss was observed. These devices achieved ~100% improvement in transduction performance (transmission and reception) over classical CMUTs. For CMUTs with square and rectangular membranes, the FBW increased from ~110% to ~150% and from ~140% to ~175%, respectively, compared with classical CMUTs. The new devices produced a maximum output pressure exceeding 1 MPa at the transducer surface. Performance optimization using geometric membrane shape configurations was the same in both piston CMUTs and classical CMUTs.