High-resolution Optical Technique Research Articles

Optical Imaging of Oral Squamous Cell Carcinoma Using Optical Coherence Tomography and Micro CT

Oral squamous cell carcinoma (OSCC) constitutes 90% of oral cancer. Early detection is a cornerstone to improve survival and to reduce diagnostic delay. We propose optical coherence tomography (OCT), as a non-invasive diagnosis method. OCT represents a new high-resolution optical technique that permits 3D, real-time imaging of near surface abnormalities in complex tissues. In the present study half of the excisional biopsy was directed to the pathologist and the other half was assigned for OCT and Micro-CT investigation. For the OCT imaging it was used an OCT prototype (1300 nm), Synchrotron Radiation Micro-CT and histopathology validated the results. Areas of OSCC of the buccal mucosa were identified in the OCT images by the disruption of the basement membrane, an epithelial layer that was highly variable in thickness, with areas of erosion, extensive epithelial down-growth and invasion into the sub-epithelial layers. In this respect, OCT seems to be a highly promising imaging modality.

Direct high-resolution label-free imaging of cellular nanostructure dynamics in living cells

We report the application of an optical microscope equipped with a high-resolution dark-field condenser for detecting dynamic responses of cellular nanostructures in real time. Our system provides an easy-to-use technique to visualize biological specimens without any staining. This system can visualize the dynamic behavior of nanospheres and nanofibers, such as F-actin, at the leading edges of adjacent neuronal cells. We confirmed that the nanofibers imaged with this high-resolution optical microscopic technique are F-actin by using fluorescence microscopy after immunostaining the F-actin of fixed cells. Furthermore, cellular dynamics are enhanced by applying noncontact electric field stimulation through a transparent graphene electric field stimulator. High-resolution label-free optical microscopy enables the visualization of nanofiber dynamics initiated by filopodial nanofiber contacts. In conclusion, our optical microscopy system allows the visualization of nanoscale cellular dynamics under various external stimuli in real time without specific staining.

High-Resolution Fluorescence Measurements Correlated to Cellular Traction Forces

Many cellular mechanosensory functions rely on molecular interactions related to cellular force exertion. Despite the rich biochemical knowledge in this field, the current mechanistic picture - that can be enhanced by high-resolution optical microscopy techniques - is still in its infancy. Here, we describe a simple yet universal approach to combine high-resolution optical microscopy techniques with traction force measurements. Etching 50 µm high spacers next to a micropillar array and inverting the array onto a thin coverslip before imaging enables the use of a short working distance objective. In our setup we use a spinning-disk confocal on an inverted microscope with a 100X, NA 1.4 objective. Our deflection detection resolution per micropillar is below 30 nm, corresponding to a force resolution of 500 pN. We validate our technique by imaging fixed and live mouse fibroblasts with dye-conjugated fibronectin micro-contact printed on the micropillars and immunostaining for paxillin and actin. We observe forces of up to 15 nN on individual pillars using a precise force-deflection relationship calibrated using scanning electron microscopy, tensile testing and finite element analysis. We quantified focal adhesion growth with increasing force of 4 ± 1 nN/µm2 and co-orientation of focal adhesion elongation and force direction within 2 ± 24 degrees. Furthermore, the cells remain viable during overnight live-cell imaging and stay adhered to the substrate. Our novel approach opens up the combined application of traction force measurements and super-resolution optical microscopy techniques.

Endogenous VIP May Contribute to Vagally Induced Electrophysiological Changes in Canine Atria

Background: There has been increasing evidence that complex interactions among the various components of intracardiac neural network play an important role in atrial fibrillation (AF). Perfusion of vasoactive intestine polypeptide (VIP), a neural polypeptide, co-released with acetylcholine from intrinsic cardiac neurons during vagal stimulation, was shown to shorten the action potential duration (APD), decrease the intraatrial conduction velocity (CV), and promote induction of AF. However, the effect of endogenous VIP remains unclear. Methods: In 6 isolated arterially perfused canine left atria, high-resolution optical mapping techniques with di-4-ANEPPS and blebbistatin were used to measure APD and CV during fat-pad ganglion plexus stimulation (GPS, 30Hz, 10.2±2.3Volt validated with blockage of atrioventricular conduction), at during H9935, a VIP antagonist (1 μM) perfusion. The atria were paced at 200beats per minute. Metoprolol (1.8μM) was used to block the sympathetic effects. Results: Average APD was shorter (21%) during GPS compared to the baseline (100±8ms vs. 126±7ms, p<0.05), and average CV was slower than baseline (87±10cm/sec vs. 103±13cm/sec, p<0.05), which recovered within 2 min (APD: 128±8ms, p<0.05; CV: 105±13cm/sec, p<0.05). With H9335, the APD shortening effect (17%) of GPS persisted (GPS, 105±14ms, vs. 127±8ms at baseline and 125±10ms after recovery, p<0.05) with a trend towards being less pronounced as compared to GPS effect without H9335 (−18±6ms vs. −29±3ms, p=0.07). However, the GPS-induced CV slowing was abolished by H9335 (97±14cm/sec, vs. 103±15cm/sec at baseline and 101±16cm/sec after recovery, p=0.23). The effects of endogenous VIP were eliminated by atropine during GPS, indicating VIP release from intrinsic cardiac neurons may be mediated by muscarinic recpetors. Conclusion: Neuronally released VIP could contribute vagal effects and is primarily responsible for CV slowing during GPS. Neuronal release of VIP is likely mediated by muscarinic receptors in the intrinsic cardiac neurons.

Multicellular Tumor Spheroids as an in Vivo–Like Tumor Model for Three-Dimensional Imaging of Chemotherapeutic and Nano Material Cellular Penetration

We present a flexible and highly reproducible method using three-dimensional (3D) multicellular tumor spheroids to quantify chemotherapeutic and nanoparticle penetration properties in vitro. We generated HeLa cell-derived spheroids using the liquid overlay method. To properly characterize HeLa spheroids, scanning electron microscopy, transmission electron microscopy, and multiphoton microscopy were used to obtain high-resolution 3D images of HeLa spheroids. Next, pairing high-resolution optical characterization techniques with flow cytometry, we quantitatively compared the penetration of doxorubicin, quantum dots, and synthetic micelles into 3D HeLa spheroid versus HeLa cells grown in a traditional two-dimensional culturing system. Our data revealed that 3D cultured HeLa cells acquired several clinically relevant morphologic and cellular characteristics (such as resistance to chemotherapeutics) often found in human solid tumors. These characteristic, however, could not be captured using conventional two-dimensional cell culture techniques. This study demonstrated the remarkable versatility of HeLa spheroid 3D imaging. In addition, our results revealed the capability of HeLa spheroids to function as a screening tool for nanoparticles or synthetic micelles that, due to their inherent size, charge, and hydrophobicity, can penetrate into solid tumors and act as delivery vehicles for chemotherapeutics. The development of this image-based, reproducible, and quantifiable in vitro HeLa spheroid screening tool will greatly aid future exploration of chemotherapeutics and nanoparticle delivery into solid tumors.

Calculation of the Energy of a Bio-Polymer as a Function of End-To-End Extension

When considering the folding of a biopolymer (DNA, RNA or protein) it is natural to express the energy of the system as a function of conformation. However, in single molecule manipulation experiments the system is most naturally characterized in terms of the end-to-end distance of the polymer, and the energy landscape itself can be measured as a function of the end-to-end distance. The relationship between energy as a function of conformation and energy as a function of end-to-end distance is extremely complex. We introduce a physical model which allows the energy landscape as a function of end-to-end distance to be calculated from knowledge of the energy as a function of conformation of the polymer. Using optical trap based experiments we show that the measured energy landscape of a DNA stem-loop structure agrees with the landscape calculated from our model with extremely high precision. We believe the combination of our model with high resolution optical trap techniques will facilitate direct measurement of the hybridization energy of nucleic acid base pairs and sequencing of double stranded RNA and DNA.

3-D thermo-mechanical laboratory modeling of plate-tectonics: modeling scheme, technique and first experiments

Abstract. We present an experimental apparatus for 3-D thermo-mechanical analogue modeling of plate tectonic processes such as oceanic and continental subductions, arc-continent or continental collisions. The model lithosphere, made of temperature-sensitive elasto-plastic analogue materials with strain softening, is submitted to a constant temperature gradient causing a strength reduction with depth in each layer. The surface temperature is imposed using infrared emitters, which allows maintaining an unobstructed view of the model surface and the use of a high resolution optical strain monitoring technique (Particle Imaging Velocimetry). Subduction experiments illustrate how the stress conditions on the interplate zone can be estimated using a force sensor attached to the back of the upper plate and adjusted via the density and strength of the subducting lithosphere or the lubrication of the plate boundary. The first experimental results reveal the potential of the experimental set-up to investigate the three-dimensional solid-mechanics interactions of lithospheric plates in multiple natural situations.

Advancing systems medicine and therapeutics through biosimulation

This Interface Focus issue presents 15 different facets of the research conducted by the participants of the BioSim Network (www.biosim-network.net). BioSim was a network of excellence under the Life Sciences, Genomics and Biotechnology for Health Thematic Priority Area of the 6th European Framework Programme. The immediate goal of the network was to develop modelling and simulation approaches that can help the pharmaceutical industry achieve a more rational drug development process. In a broader perspective, the network aimed at contributing in an essential manner to the development of an integrated and quantitative understanding of biological processes. It included a total of 40 partners, including 26 academic research groups from various universities, nine SMEs and Novo Nordisk, with many of the partners having strong collaborations with the pharmaceutical industry. The network established a platform for very fruitful interdisciplinary collaboration with biological groups providing insights into genetics, biochemistry, cellular biology, pharmacology, endocrinology, neurology and physiology; clinicians and hospital groups bringing experience in treatments of depression, various forms of tremor, and cancer clinical interventions and theoreticians providing expertise in bioinformatics, systems biology (SB), network theory, statistical physics, nonlinear dynamics and complex systems theory, as well as pharmacokinetics and clinical trial planning.

Determination of crystalline perfection, optical indicatrix, birefringence and refractive-index homogeneity of ZTS crystals

Single crystals of the semi-organic non-linear optical material zinc tris (thiourea) sulphate (ZTS) were grown by slow evaporation solution growth technique. The crystals were characterized for crystalline perfection and optical homogeneity using high-resolution X-ray diffraction (HRXRD) and optical interferometric techniques. The FWHM of 6 arc sec in HRXRD rocking curve shows the good crystalline quality. Transmittance of ∼75% shows that the crystal is free from volume defects. Conoscopy was used to assess the optical quality and investigate the optical indicatrix of the grown crystal. Birefringence values of the crystal along the three principal crystallographic axes were measured using birefringence interferometry. Mach–Zehnder interferograms exhibit good refractive-index homogeneity of the grown crystal.

Interobserver variability for retreatment indications after Ranibizumab loading doses in neovascular age-related macular degeneration

To assess the interobserver variability (IOV) in indicating retreatment for neovascular Age-related macular degeneration 4 weeks after three Ranibizumab loading doses using spectral domain OCT (SD-OCT) as the primary objective diagnostic tool. Four observers decided for or against 4th Ranibizumab injection in 108 patients by six different rating rounds (RR) based on the SD-OCT findings after the loading doses. Postoperative OCT images were supplemented consecutively with information from a chart review as the 'patients subjective estimation of vision (SE)', the course of best-corrected visual acuity (BCVA) and the preoperative OCT as well as all information collectively. Agreement rates (AR) and Kappa statistics were calculated. Based on post-treatment OCT findings only (RR1), mean reinjection rate of all observers was 37.5%. Adding supplementary information, mean reinjection rate decreased to 20% when all information was available reflecting the 'real' situation (RR 6). Interobserver agreement rates varied from 66.7% to 90.7% depending on rating rounds and interobserver pairs. Mean AR and Kappa values (KV) were as following: AR 81.6%, KV 0.61 (RR1: 'only post-OP OCT'); AR 76.7%, KV 0.33 (RR2: post-OP OCT + SE); AR 80.3%, KV 0.45 (RR3: post-OP OCT + BCVA); AR 80.7%, KV 0.46 (RR4: pre- and post-OP OCT); AR 82.2%, KV 0.49 (RR5: post-OP OCT + SE + BCVA); and finally AR 83.6%, KV 0.47 (RR6: pre- and post-OP OCT + SE + BCVA). The overall mean agreement rate was 80.9% with a Kappa of 0.47. IOV for indicating retreatment after three Ranibizumab loading doses reveals only moderate agreement in Kappa statistics, which seems to be too low considering the high costs for retreatments. More concise guidelines based on the post-treatment OCT scans as the presumably most sensitive and noninvasive objective tool to follow choroidal neovascularization activity by judging the course of sub- and intraretinal fluid are necessary.

液晶涂层厚度对液晶热像校准及测量不确定度的影响

The liquid crystal thermography is a high-resolution and non-intrusive optical technique for full-field temperature measurement. We present detailed calibration data for a wide-bandwidth thermochromic liquid crystal (TLC) to investigate the effect of the coating thickness on the hue-temperature characteristics and the measurement uncertainty of the TLC. It is found that the coating thickness has appreciable effect on the TLC hue-temperature curve. For TLC coatings with the thickness over 20 μm, a thicker TLC coating shows a relatively smaller measurement uncertainty, but the effect of the coating thickness is non-distinctive on the measurement uncertainty.

Optical birefringence study of AgNa(NO2)2 crystals near the paraelectric-to-ferroelectric phase transition

The paper reports nearly tricritical behaviour of AgNa(NO2)2 crystal in the region of its paraelectric-to-ferroelectric phase transition studied by a highresolution optical birefringence technique. The results obtained are analysed within the phenomenological approach based on the Landau theory for secondorder phase transitions. Anomalous changes in the optical birefringence below the Curie point are found to be driven by the spontaneous polarisation via a spontaneous quadratic electrooptic effect. In the region of the Curie point AgNa(NO2)2 exhibits slightly discontinuous behaviour at cooling and practically continuous behaviour at heating, with the critical exponent for the order parameter equal to 0.25, as expected for the tricritical point.

Microstructures produced during the epitaxial growth of InGaN alloys

Effects due to phase separation in InGaN have been identified as having major effects on the performance of devices, in particular light-emitting diodes (LEDs) and injection lasers. However, the complexity of the various materials phenomena that can occur has led to a great deal of recent confusion. Much of this confusion can be eliminated by considering the experimentally measured materials properties in the context of the set of physical phenomena occurring during epitaxial growth, including coupling that exists between the various effects. Spinodal decomposition is expected to produce phase separation due to the miscibility gap in InGaN alloys. However, the actual occurrence of this phenomenon has been disputed due to the complexity of real systems. For example, the region of solid immiscibility for InGaN is strongly dependent on elastic strain. In addition, the strain, itself, affects properties such as the bandgap energy. Complicating the analysis of these phenomena is that the solid composition can be affected by elastic strain due to the well-known thermodynamic phenomenon of “compositional pulling”. An additional factor must be considered if the experimentally observed phenomena are to be understood. Thin, lattice mismatched epitaxial layers are coherent with the substrate (or underlying layer). Thus, the actual growth process for the formation of lattice mismatched layers, namely the Stranski–Krastanov (S–K) formation of islands, must be included in any realistic growth model. By considering all the phenomena together, including the coupling between them, it becomes clear that several separate mechanisms exist for phase separation. The focus of this paper is the analysis of the thin (2–3 nm), coherent InGaN layers used in the quantum well structures used for virtually all LEDs and lasers produced by the S–K mechanism. By considering these coupled phenomena together it is possible to arrive at a coherent interpretation of the various materials properties measured using techniques such as high resolution transmission electron microscopy, X-ray diffraction, and optical techniques as well as the device characteristics.

High-Resolution Dual-Trap Optical Tweezers with Differential Detection: An Introduction: Figure 1.

Optical traps or "optical tweezers" have become an indispensable tool in understanding fundamental biological processes. The ability to manipulate and probe individual molecules or molecular complexes has led to a new, more refined understanding of the mechanical properties of the fundamental building blocks of the cell, and of the mechanism by which molecular machines function. The field has seen a steady stream of technological advances that have greatly refined the technique. One major effort has been in developing methods to resolve motions at the angstrom level--the fundamental length scale for many biological processes. This drive has only recently come to fruition with the advent of high-resolution optical trapping techniques that can now detect movements on the scale of a single base pair of DNA, 3.4 A. Here we briefly review the basic concepts and components of optical traps and the single-molecule experiments in which they are used.

Nonlinear Cone Penetration Test-Based Method for Predicting Footing Settlements on Sand

This paper presents centrifuge data from model footing tests on dry sand, where a high resolution optical displacement measurement technique was employed to record subsurface soil displacements beneath the centerline of loaded strip footings. These measurements allow derivation of vertical strain profiles, which are then used to estimate operational soil stiffness values. The stiffness values, which were assessed assuming a dependence on cone penetration test tip resistance and initial vertical effective stress level, are shown to degrade rapidly with increasing strain level. Despite such nonlinearity, the experimental strain data can be represented using an updated form of the well known Schmertmann strain influence profile. Settlements calculated using this profile are shown to be in agreement with subsurface settlements when appropriate soil stiffness values are employed.

Modulation of rabbit sinoatrial node activation sequence by acetylcholine and isoproterenol investigated with optical mapping technique

Changes in the rabbit sinoatrial node (SAN) activation sequence with the cholinergic and adrenergic factors were studied. The correlation between the sinus rhythm rate and the leading pacemaker site shift was determined. The hypothesis concerning the cholinergic suppression of nodal cell excitability as one of the mechanisms associated with pacemaker shift was tested. A high-resolution optical mapping technique was used to register beat-to-beat changes in the SAN activation pattern under the influence of the cholinergic and adrenergic factors. Acetylcholine (10 microm) and strong intramural parasympathetic nerve stimulation caused a pacemaker shift as well as rhythmic slowing and the formation of an inexcitable region in the central part of SAN. In this region the generation of action potentials was suppressed. The slowing of the sinus rhythm (which exceeded 12.8 +/- 3.1% of the rhythm control rate) always accompanied the pacemaker shift. Isoproterenol (10, 100 nm, 1 microm) and sympathetic postganglionic nerve stimulation also evoked a pacemaker shift but without formation of an inexcitable zone. The acceleration of the sinus rhythm, which exceeded 10.5 +/- 1.3% of the control rate of the rhythm, always accompanied the shift. Both cholinergic and adrenergic factors cause pacemaker shifts in the rabbit SAN. While modest changes in the sinus rhythm do not coincide with the pacemaker shift, greater changes always accompany the shift and may be caused by it, according to one hypothesis. The formation of an inexcitable zone at the place where the leading pacemaker is situated is one of the mechanisms associated with pacemaker shift.

Knocking diagnostics in the combustion chamber of boosted port fuel injection spark ignition optical engine

High spatial resolution optical techniques have been used to get information about the timing and the location of knocking and about the chemical species involved in this phenomenon. The experiments were realised in the combustion chamber of a boosted single-cylinder spark ignition port-fuel injection optical engine fuelled with commercial gasoline. Engine conditions with different levels of knock were considered from the borderline case onto standard knocking and heavy knocking. Cycle-resolved digital imaging was used to follow the combustion and the flame propagation in normal combustion and knocking conditions. Moreover, the effects of an abnormal combustion due to the firing of fuel deposition near the intake valves and on the piston surface were investigated. The knocking influence on the flame front propagation and combustion speed was investigated following the time evolution of the mean flame radius in the different engine conditions. The appearance of the auto-ignition centres in the end gas during the knock was evaluated in terms of timing, location and frequency of occurrence. Finally, UV-visible natural emission spectroscopy was applied to detect radical species that marked the knock. HCO and OH were identified as markers of the knocking onset and OH of its intensity.

Second‐harmonic microscopy of ex vivo porcine corneas

The ex vivo cornea of porcine eyes has been studied with second-harmonic microscopy with a laboratory-built system to examine the structure of collagen fibrils at different length scales, as well as the image dependence on polarization and wavelength of the illumination source. We found that collagen fibrils can effectively be visualized with second-harmonic microscopy, in agreement with previous findings, at different wavelengths of the illumination. The same laser source used for imaging may also be used to induce changes to the corneal tissues that are observable both in the linear and second-harmonic imaging channels. Such studies are essential first steps towards a future high-resolution optical characterization technique for simultaneous corneal surgery and wound healing of the human eye.

Nondestructive high-resolution soft-boundary profiling based on forward shadow diffraction

Nondestructive noncontact high-resolution optical technique for profiling soft or fluidic boundary of an opaque object is presented. Our technique utilizes the fact that the angle width, the angular separation between two adjacent intensity minima in the forward shadow diffraction, is inversely proportional to the projected width of the object in the same direction. An analytic formula for reconstructing the boundary shape is obtained for an object with two-fold symmetry in terms of the angle widths measured for various rotational angles of the object. The typical error in determining the object shape parameter is less than 0.2%, which corresponds to 20 nm of radial accuracy when applied to an object with a mean radius of 10 microns.

Application of three-dimensional spline variational analysis for composite repair

Abstract The purpose of this research was to apply a non-commercialized spline-based (B-spline analysis method or BSAM) computer program to model and predict strain fields in two composite repairs, the scarf joint and the stepped-lap joint, subjected to static tensile loading. Test specimens with scarf and stepped-lap joints were fabricated using quasi-isotropic 16 ply panels made from IM6/3501-6 pregreg with a [+45 2 /0 2 /−45 2 /90 2 ] S lay-up. Moire interferometry, a high-resolution full-field optical technique used to measure displacements on a surface, was used to determine experimental strain data. Full-field strains along the free-edge were predicted and compared to experimental strain data. Line plots are generated from predicted and experimental data extracted at several points across the width of the repair to provide a quantitative comparison. Overall, both the full-field strain images and line plots show good agreement between the BSAM analysis and the experimental data.