Dido Yova

Human fibroblast alterations induced by low power laser irradiation at the single cell level using confocal microscopy

Low power laser irradiation is regarded to have a significant role in triggering cellular proliferation and in treating diseases of diverse etiologies. The present work contributes to the understanding of the mechanisms of action by studying low power laser effects in human fibroblasts. Confocal laser scanning microscopy is used for irradiation and observation of the same area of interest allowing the imaging of laser effects at the single cell level and in real time. Coverslip cultures were placed in a small incubation chamber for in vivo microscopic observation. Laser stimulation of the cells was performed using the 647 nm line of the confocal laser through the objective lens of the microscope. Mitochondrial membrane potential (delta psi(m)), intracellular pH, calcium alterations and generation of reactive oxygen species (ROS) were monitored using specific fluorescent vital probes. The induced effects were quantified using digital image processing techniques. After laser irradiation, a gradual alkalinization of the cytosolic pH and an increase in mitochondrial membrane potential were observed. Recurrent spikes of intracellular calcium concentration were also triggered by laser. Reactive oxygen species were generated as a result of biostimulation. No such effects were monitored in microscopic fields other than the irradiated ones.

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.

Surface nanoscale imaging of collagen thin films by Atomic Force Microscopy

Collagen, the most abundant protein in mammals, due to its unique properties is widely used as biomaterial, scaffold and culture substrate for cell and tissue regeneration studies. Since the majority of biological reactions occur on surfaces and structures at the nanoscale level it is of great importance to image the nanostructural surface of collagen based materials. The aim of this paper was to characterize, with Atomic Force Microscopy (AFM), collagen thin films formed on different substrates (glass, mica, polystyrene latex particle surfaces) and correlate their morphology with the used substrates, formation methodologies (spin coating, hydrodynamic flow) and original collagen solution. The results demonstrated that, by altering a number of parameters, it was possible to control the formation of collagen nanostructured films consisting of naturally occurring fibrils. The spin coating procedure enabled the formation of films with random oriented fibrils, while substrates influenced the fibril packing and surface roughness. The hydrodynamic flow was used for guiding fibril major orientation, while adsorption time, rinsing with buffer and solution concentration influenced the fibril orientation. The clarification of the contribution that different parameters had on thin film formation will enable the design and control of collagen nanobiomaterials with pre-determined characteristics.

Topical photodynamic therapy of murine non-melanoma skin carcinomas with aluminum phthalocyanine chloride and a diode laser: pharmacokinetics, tumor response and cosmetic outcomes

Background/purpose: Topical photodynamic therapy (PDT) is potentially useful for the treatment of non‐melanoma skin cancer and other skin diseases. We investigated the therapeutic effects of PDT using topical application of aluminum phthalocyanine chloride (AlClPc) and a diode laser emitting at 670 nm in murine non‐melanoma skin carcinomas.

Texture analysis of fluorescence microscopic images of colonic tissue sections

The aim of this study was to assess the potential of texture analysis for the characterization of fluorescence images from colonic tissue sections stained with a novel and selective fluoroprobe, Rhodamine B-phenylboronic acid. Fluorescence microscopy images of colonic healthy mucosa (n=35) and adenocarcinomas (n=35) were digitally captured and subjected to image texture analysis. Textural features derived from the grey level co-occurrence matrix were calculated. A modified version of the multiple discriminant analysis criterion was used to choose an appropriate subset of features. A minimum Mahalanobis distance, linear discriminant classifier and a simple evaluation ‘score’ method were used to classify image feature data into the two categories. A subset of four textural features was selected and used for the description and classification of each image field. They were found appropriate to correctly classify 95% of the images into the two classes, using two different classifiers. These features contained information about local homogeneity and grey level linear dependencies of the image. This study demonstrated that texture analysis techniques could provide valuable diagnostic decision support in a complex domain such as colorectal tissue.

Comparative characterization of the cellular uptake and photodynamic efficiency of Foscan® and Fospeg in a human prostate cancer cell line

BACKGROUND m-THPC (Foscan(®)) is one of the most potent second generation photosensitizers used in photodynamic therapy, photoactivated at higher wavelengths (652 nm). However, its strongly hydrophobic nature causes aggregation of the molecules and prevents its unbiased bioavailability in the biological media, resulting in lower accumulation in the tumor cells. Several strategies have been adopted to improve the photodynamic characteristics of the photosensitizer. Among them, very promising seems to be the encapsulation of the molecule into liposomes, due to the superior properties of liposomes as drug carriers. METHODS In this paper the photodynamic characteristics of the PEGylated liposomal formulation of m-THPC, Fospeg, using the human prostate cancer cell line LNCaP, as an in vitro model, were investigated. In addition the spectral characteristics, cellular uptake and localization, dark and light induced cytotoxicity and photodynamic efficacy of Foscan(®) and Fospeg were compared. RESULTS Fospeg, compared with Foscan, showed higher intracellular uptake at any concentration and incubation time. Regarding PDT efficacy, Fospeg produced more severe cytotoxicity than Foscan(®) at any concentration and energy dose. Using Fospeg, the lowest concentration (0.22 μM) and energy dose (180 mJ/cm(2)) was adequate to result in the death of 50% of the cells 24h post PDT while an approximately 10 times higher Foscan(®) concentration (1.8 μM) was needed to result in the same cytotoxicity. CONCLUSIONS The use of the PEGylated liposomal formulation of m-THPC resulted in the improvement of its intracellular uptake and the enhancement of its photodynamic activity. Fospeg, compared to Foscan(®), proved to be a more advantageous photosensitizer for photodynamic therapy.

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.

Fluorescence and absorption assessment of a lipid mTHPC formulation following topical application in a non-melanotic skin tumor model.

Although the benefits of topical sensitizer administration have been confirmed for photodynamic therapy (PDT), ALA-induced protoporphyrin IX is the only sensitizer clinically used with this administration route. Unfortunately, ALA-PDT results in poor treatment response for thicker lesions. Here, selectivity and depth distribution of the highly potent sensitizer meso-tetra(hydroxyphenyl)chlorin (mTHPC), supplied in a novel liposome formulation was investigated following topical administration for 4 and 6 h in a murine skin tumor model. Extraction data indicated an average [+/- standard deviation (SD)] mTHPC concentration within lesions of 6.0(+/-3.1) ngmg tissue with no significant difference (p<0.05) between 4- and 6-h application times and undetectable levels of generalized photosensitivity. Absorption spectroscopy and chemical extraction both indicated a significant selectivity between lesion and normal surrounding skin at 4 and 6 h, whereas the more sensitive fluorescence imaging setup revealed significant selectivity only for the 4-h application time. Absorption data showed a significant correlation with extraction, whereas the results from the fluorescence imaging setup did not correlate with the other methods. Our results indicate that this sensitizer formulation and administration path could be interesting for topical mTHPC-PDT, decreasing the effects of extended skin photosensitivity associated with systemic mTHPC administration.