Kostas Politopoulos

Thermally Induced Irreversible Conformational Changes in Collagen Probed by Optical Second Harmonic Generation and Laser-induced Fluorescence

Irreversible thermal conformational changes induced to collagen have been studied by optical methods. More specifically, second harmonic generation (SHG) from incident nanosecond Ng:YAG 1064 nm radiation and laser-induced fluorescence by 337 nm, pulsed nanosecond nitrogen laser excitation, at 405, 410 and 415 nm emission wavelengths were registered at eight temperatures (40°, 50°, 55°, 60°, 65°, 70°, 75° and 80°C) and normalised with respect to the corresponding values at the ambient temperature of 30°C. The heating protocol used in this work, was selected to monitor only permanent changes reflecting in the optical properties of the samples under investigation. In this context, the SHG, directly related to the collagen fibril population in triple helix conformation, indicated on irreversible phase transition around 64°C. On the other hand fluorescence related to the destruction of cross-linked chromophores in collagen, some of which are related to the triple helix tertiary structure, also indicated a permanent phase transition around 63°C. These results are in agreement with previous results from studies with differential scanning calorimetry. However SHG and fluorescence, being non-invasive optical methods are expected to have a significant impact in the fields of laser ablative surgery and laser tissue welding.

Second and third optical harmonic generation in type I collagen, by nanosecond laser irradiation, over a broad spectral region

Abstract We report optical up-conversion of pulsed (ns) laser radiation in type I pure collagen, tuneable over a broad excitation spectrum covering the 760–1070 nm wavelength range. We investigated second harmonic generation (SHG) in collagen using λ p =1064, 901, 892, 828, 785 and 766 nm nanosecond pulsed laser excitation and recorded monochromatic signals at λ p /2, i.e. 532, 451, 446, 414, 393 and 383 nm, respectively, corresponding to the SHG optical process. The SHG signal intensity exhibited a quadratic dependence on the excitation radiation (log[ I 532 ]=1.92*log[ I 1064 ]). Furthermore, a weaker third harmonic generation (THG) signal from collagen was also observed at λ p /3 (355 nm) using 1064 nm nanosecond pulsed laser excitation. The THG signal was found to have near-cubic dependence upon the irradiation laser intensity (log[ I 356 ]=2.53*log[ I 1064 ]). The significance of collagen ability to exhibit broadly tuneable second harmonic generation is discussed.

A binocular machine vision system for three-dimensional surface measurement of small objects

Rendering three-dimensional information of a scene from optical measurements is very important for a wide variety of applications. However, computer vision advancements have not yet achieved the accurate three-dimensional reconstruction of objects smaller than 1 cm diameter. This paper describes the development of a novel volumetric method for small objects, using a binocular machine vision system. The achieved precision is high, providing a standard deviation of 0.04 mm. The robustness, of the system, issues from the lab prototype imaging system with the crucial z-axis movement without the need of further calibration and the fully automated volumetric algorithms.

Combined information from AFM imaging and SHG signal analysis of collagen thin films

Abstract Collagen being the most abundant protein in mammals is important for a variety of functions and its structure, concentration and orientation disturbance is associated with different pathological states. The use of the optical second harmonic generation (SHG) is emerging as a powerful non-invasive tool for assessing collagen modification in a variety of pathological conditions. The properties of second harmonic light from collagen structures have not yet been fully clarified due to a number of limitations, such as the difficulty to prepare collagen samples with well-known characteristics and optical properties, at a nanoscale resolution. The results of this paper suggest that some of these limitations can be overcome by using thin collagen films with pre-determined characteristics (PDC), which maintain or/and enhance their NLO properties capacities, as they have been checked by using atomic force microscopy (AFM). The collagen fiber structure and orientation was systematically altered by using thermal denaturation or different preparation methodologies (spin coating procedure, use of collagen solution hydrodynamic flow). These films can be used as prototypes and the combined information from AFM imaging and the one included in SHG signal, delivered from them, can significantly contribute to further understanding of the NLO properties of collagen and in the long term to take advantage as a non-invasive tool.

Atomic Force Microscopy surface nanocharacterization of UV-irradiated collagen thin films

Collagen, the most abundant protein in mammals, is a basic component of the extracellular matrix and due to its unique properties it is widely used as biomaterial, scaffold and culture substrate for cell and tissue regeneration studies. Due to human skin chronic exposure to sun light and since UV rays are used as sterilizing and cross-linking methods the clarification of the UV light-collagen interactions are very crucial. Moreover, since the majority of the biological reactions occur on surfaces or interfaces the influence of UV light on the surface of collagen-based materials attracts the scientific interest, especially in the biomaterials science. Surface-nanoscale characterization could be performed with Atomic Force Microscopy (AFM), which is a powerful tool and offers quantitative and qualitative information. Its ability of high resolution imaging and non-destructive characterization makes it very attractive for biological samples investigation. The aim of this paper was to determine the surface properties and alterations of collagen thin films after UV-irradiations using AFM techniques. Furthermore, it was aimed to investigate the possible different influence on the surface when the collagen solution or the thin films were irradiated. In this paper topographic AFM images were acquired from thin films, formed from both irradiated and non-irradiated collagen solutions, with spin coating procedure. The results demonstrated that the UV irradiation have different results when it is applied in the collagen solution or in the film after the spin coating methodology. For short irradiation times (<;120 min) UV caused only rather small changes in the morphology of the studied films although fluorescence and absorption studies confirmed collagen photodegradation. The surface roughness and topography altered after 3 and 7 hours, respectively, while the fibrous structure was completely destroyed after 15 hours. Surface roughness of the films depends on whether the solution was irradiated or the film and on the time irradiation. The fully clarification of the role of the UV light on collagen thin films will enable the proper design and control of collagen based nanobiomaterials with appropriate and improved surface properties.

Atomic Force Microscopy Imaging of the Nanoscale Assembly of Type I Collagen on Controlled Polystyrene Particles Surfaces

The adsorption of collagen and the morphology of its assemblies at solid surfaces play an important role in a variety of research areas and applications, such as biomaterials, biocompatibility, tissue mechanics and cell studies. In this paper the nanoscale organization of type I collagen on polystyrene particle surfaces with controlled patterns was investigated by high-resolution atomic force microscopy. The results showed that patterned surfaces with a well-ordered morphology could be formed by spin coating polystyrene particles dispersions (20 sec at 1000 rpm) and appropriate annealing conditions (at 90°C for 2h). Collagen thin films consisting of fibers with natural characteristics, such as the typical 67nm periodicity (D-band), were formed on these nano-patterns. This kind of surfaces provides a useful substrate for studying the regulation of collagen assembly on substrates with different nano-topographical and environmental conditions and offers a potential way to create surfaces of functionalized and nano-patterend materials for biotechnological and biomedical applications.

Mechanical properties of collagen fibrils on thin films by Atomic Force Microscopy nanoindentation

Atomic Force Microscopy (AFM) is a powerful tool as far as surface characterization is concerned, due to its ability to relate high resolution imaging with mechanical properties. Furthermore biological samples, such as collagen, can be studied by AFM with a non-destructive manner. Collagen is the most abundant protein in mammals and because of its unique properties is widely used as biomaterial. Due to the human skin chronic exposure to sun light and since UV-rays are used in sterilizing and cross linking methods of collagen based biomaterials, the investigation of the influence of UV light on collagen, is crucial. The purpose of this paper was to investigate the topographic features and the mechanical properties of collagen fibrils prior and post ultraviolet (UV) by using the AFM indentation method. Hence, load-displacement curves were obtained on collagen fibrils in order to calculate the Young modulus for each case. Each curve presented, was the average of 10 curves. The results showed that Young modulus value increased after 4 hours of UV irradiation from 0.5 GPa to 1.53 GPa. After 8 hours of UV irradiation the Young modulus value was calculated equal to 3.2 GPa. These experiments yielded a clear stiffening of collagen fibrils as a result of UV exposure. Moreover, after 8 hours of UV exposure, collagen fibrillar structure started to deform and the characteristic D-band of collagen fibrils deteriorated. The investigation of the alterations of the modulus under UV irradiation, will contribute to the clarification of the impact that different physical and chemical parameters have on the mechanical properties of collagen-based materials. In addition, this will enable the design and development of biomaterials with improved properties.

Combined SHG signal information with AFM imaging to assess conformational changes in collagen

Collagen is the most abundant protein in mammals and is important for a variety of functions and its concentration, structure and function is associated with different pathological states. In this research we correlate structural changes with those changes in the Second Harmonic Generation (SHG) signal. The combination of Atomic Force Microscopy (AFM) imaging with information included in SHG signal can significantly contribute to further understanding of the nonlinear optical properties of collagen.

Max-Density Revisited: a Generalization and a More Efficient Algorithm

We present an algorithm that given a graph computes a subgraph of maximum ‘density’. (For unweighed graphs, density is the edges-to-vertices ratio). The proposed algorithm is asymptotically more efficient than the currently available ones. Our approach remains efficient for weighed graphs and more generally for weighed set-systems. Two faster approximation algorithms are offered, and a number of applications are discussed.

Precedence Constrained Scheduling: A Case in P

‘Unit execution time’ precedence constrained scheduling (UET) is an NP-complete problem with very few special cases known to be solvable in P-time. In this article we present a practically useful case of UET solvable in P-time: we show that if the task graph is given in levels that are ‘locally’ of in-degree two and of ‘width’ more than 1.55 times the number of processors (plus 1), then an optimal schedule can be found in P-time. Task graphs which represent algebraic computations fall ordinarily in this category. Our algorithm is based on a limited look-ahead technique which allows us to use it in an on-line fashion. In the appendix we give two short NP-completeness proofs which suggest that both ‘width’ and ‘degree’ restrictions are needed to get a polynomially solvable subcase.