Lachezar A. Avramov

Tumor detection by exogenous fluorescent dyes using new generation photo-multiplier tubes

The easy and non-destructive fluorescence method for quantification of early changes in biological tissues improves the possibilities of the clinical research and diagnostics. Developments in this area are moving very rapidly in part because of advances in the technology and in part because of the numerous successful examples which are appearing. New family of photomultiplier tubes with a high detection sensitivity for near-infra red light (700-900 nm) were developed as a result of project IMPECABLE, which are valuable tools for early diagnosis of cutaneous pigmented melanoma using long-wave fluorescence dyes. Several phthalocyanines that are promising fluorophores for photodiagnosis of cutaneous malignant melanoma have been studied in different solvents for concentrations from 10-5 to 10-15 mol. Argon pumped dye laser as an excitation source was used. Three different wavelengths (613, 633 and 660 nm) in the red region, corresponding to first absorption peak, minimum of the absorption and near to the Q-band maximum of Pcs were applied. Fluorescence signals in the region of 700 to 800 nm were detected using spectrometric systems (Perkin-Elmer, UK-with conventional PMT as a detector, and PC2000, Ocean Optics, USA-with CCD-array as a detector) and a newly developed red-sensitive PMT. Detectable signal from other spectrometric systems was obtain up to 10-8 mol concentrations, which could be used for significant reduction of concentrations applied for in vivo applications. Fluorescence is a highly sensitive method of distinguishing between healthy and unhealthy tissue. The results demonstrate that extremely low concentrations of photosensitizers could be used to determine initial stages of melanoma. This application of PMT detectors will reduce extremely the negative side effects of higher concentrations of these drugs applied in the skin tissue. One can achieve high accuracy in the determination of pigmented malignant melanoma lesions with wide clinical applications.

Interaction of the IR laser radiation with human skin: Monte Carlo simulation

In this study, the Nd:YAG laser (1.06 μm and 1.32 μm) beam-skin tissue interaction is simulated by the MC method. Two-layer skin model-epidermis and dermis is used. The goal of the study is to explain the causes for obtaining of qualitatively different macro-effects in clinical applications of near situated IR wavelengths. The possibility to use the Nd:YAG laser in the cosmetics for skin rejuvenation by the thermal influence to collagen without any damaging effects to the surrounding tissues is shown.

Applications of laser-induced fluorescence for remote sensing

Recent experiments conducted using a ground-based lidar have shown that laser-induced fluorescence spectral emissions from green plants are detectable from a remote platform. A pulsed Nd-YAG laser emitting at 532 nm was used as an excitation source. The fluorescence maxima in Populus tree were found to be at 685 nm and 740 nm. An increase of the ratio 685/740 nm between the intensity maxima of the fluorescence spectra was observed for that tree in the case of water stress. The morphological differences or difference in the photosynthetic properties between the two sides of the leaves cause differences between the ratios of the intensity maxima at 685 nm and 740 nm of the fluorescence spectra observed in cases of different geometries of measurement during the laboratory study.

Laser-induced fluorescence spectroscopy of benign and malignant cutaneous lesions

The goals of this work were investigation of pigmented skin lesions by the method of laser-induced fluorescence spectroscopy. Fluorescence spectra were obtained from malignant and benign skin lesions after excitation with nitrogen laser at 337 nm, namely: benign nevi, dysplastic nevi, malignant melanoma (MM), keratopapilloma, base-cell papilloma and base-cell carcinoma, as well as from healthy skin areas near to the lesion that were used posteriori to reveal changes between healthy and lesion skin spectra. Initially lesions were classified by ABCD-dermatscopic method. All suspicious lesions were excised and were investigated histologically. Spectrum of healthy skin consists of one main maximum at 470-500 nm spectral region and secondary maxima at in the regions round 400 and 440 nm. In the cases of nevi and melanoma significant decrease of fluorescence intensity, which correlated with the type of pigment lesion was observed. This reduction of the signal is related to the accumulation of melanin in the lesions that re-absorb strongly the fluorescence from native skin fluorophores in whole visible spectral region. In cases of papilloma and base-cell carcinoma an intensity decrease was also observed, related to accumulation of pigments in these cutaneous lesions. An relative increase of the fluorescence peak at 440 nm were registered in the case of base-cell carcinoma, and appearance of green fluorescence, related to increase of keratin content in benign papilloma lesions were detected. The results, obtained in this investigation of the different pigment lesions could be used for better comprehension of the skin optical properties. The fluorescence spectroscopy of the human skin are very prominent for early diagnosis and differentiation of cutaneous diseases and gives a wide range of possibilities related to real-time determination of existing pathological condition.

Laser system for optical biopsy and in-vivo study of the human skin

The aim of this study was to perform a preliminary evaluation of the diagnostic potential of noninvasive laser-induced autofluorescence spectroscopy (LIAFS) for human skin in vivo. The autofluorescence characterization of tissue relies on different spectral properties of tissue. It was demonstrated a differentiation between normal skin and skin with vitaligo. In our experimental investigation of the autofluorescence spectrum of human skin in vivo a nitrogen laser with excitation wavelength 337 nm was used. Two fluorescence bands were observed at 440 and 490 nm, these were attributed to reduced nicotinamide adenine dinucleotide (NADH) and collagen. The intensity of the NADH emission band was markedly reduced in the skin with vitaligo compared with the normal skin, which could indicate different redox conditions in skin with vitaligo. The autofluorescence spectrum of human skin depends on the main internal absorbers, which are blood and melanin. In this study was described the effect caused by melanin content on the shape of the autofluorescence spectrum of human skin. Human skin fluorescence spectrum might provide dermatologists with important information and such investigations are successfully used now in skin disease diagnostics, in investigation of the environmental factor impact or for evaluation of treatment efficiency. The goal of this work is optimization of detection and diagnosis of hollow organs and skin.

Investigation of Relations Between Skin Cancer Lesions‘ Images and Their Reflectance and Fluorescent Spectra

Biomedical optics is one of the fastest growing areas of research. The non-ionizing nature of light applied for investigation and detection of abnormalities in human tissues make this area very attractive for development of new diagnostic techniques and modalities (Wang, 2007). The optical spectra provide biochemical and morphological information about the tissue under investigation based on its absorption, reflectance, fluorescence and elastic scattering properties (Wang, 2007; Tuchin, 2002). Many techniques based on the recent progress in optics have been developed for biomedical applications. Fluorescence, absorption and diffuse scattering spectroscopy have been widely applied as probes acquiring information about physical, chemical, or physiological processes in the tissues. These methods have been proposed to be used by the medical community in view of extending the capabilities of the standard diagnostic modalities that have already been introduced in the clinical practice, as x-ray, magnetic resonance and ultrasound imaging. Laser-induced autofluorescence spectroscopy (LIAFS) could be utilized to quantify differences between normal and abnormal tissues in vivo, thus providing an appropriate method for detection of pathological lesions in real time. Diffuse reflectance spectroscopy (DRS) also allows distinguishing between pathological areas and normal tissue surroundings. In recent years, there has been growing interest in the combined use of laserinduced autofluorescence and reflectance spectroscopy to differentiate diseased from normal surrounding tissue – mainly for detection and differentiation of cancerous and precancerous changes in human body. Laser-induced autofluorescence spectroscopy is very promising from a technical point of view due to the easy coupling with optical fibres for delivering the excitation light to every part of the human body without significant losses of light power, as is the case of incoherent light sources, as well as because of the possibility to use information from naturally occurring endogenous fluorophores, without adding external fluorescent markers. LIAFS is also notable among the other non-invasive diagnostic techniques, as it offers real-time detection and differentiation of the lesion investigated with promising precision, selectivity

Early detection of the carious conditions by laser-induced fluorescence spectroscopy

The aim of this study was to investigate the natural intrinsic fluorescence in sound and diseased human teeth and the correspondence of such autofluorescence to the stages of the dental lesions. Direct visual examination was used for comparison. Different stages of caries lesions are detected, by using a nitrogen laser (337 nm), light-emitting diode (LED) (440 nm) and argon laser (488 nm). Besides caries, there were investigated samples of the fluorosa dentis and odontolithiasis, for better determination of the influence of other teeth pathologies over the teeth autofluorescence spectra. There was observed a significant decrease in the intensity of the autofluorescence signal in the case of caries. The carious lesions revealed characteristic emission of endogenous fluorophores with fluorescence band in the red spectral region. Healthy hard dental tissue exhibited no emission bands in the red. An algorithm for early diagnosis is created on the basis of collected significant statistical material.

Light enhancement of in vitro antitumor activity of galactosylated phthalocyanines

Abstract Background and objectives: Intensive research in the area of photodynamic therapy (PDT) has been made in recent years revealing it as a promising method for the treatment of tumors and inactivation of pathogenic microorganisms. However, for a broader application of this therapy one major challenge, namely a significant improvement of the targeted drug delivery and uptake, still remains. A possible solution of the selectivity problem could be the application of specifically functionalized photosensitizers, in particular phthalocyanine dyes. Materials and methods: Water-soluble Zn(II) phthalocyanines (ZnPcs) with four galactose moieties on non-peripheral and peripheral positions and a non-substituted Zn(II) phthalocyanine were studied for in vitro antitumor activity on three breast cancer cell lines (MCF-7, MDA-MB-231 and HBL-100). The influence of the exposure to ultraviolet (UV) (365 nm) and red (635 nm) light in non-therapeutic doses on the cellular uptake, binding and subcellular localization of three photosensitizers was investigated by confocal laser scanning microscopy. In addition, phototoxicity studies with the tested phthalocyanines on the non-tumorigenic mouse embryo cell line Balb c/3T3 (clone 31) were carried out. Results: The results indicate that the pre-treatment, namely exposure to UV or red light, influences the localization properties of the used dyes. The positions of galactose units to the ZnPc ring also influenced the uptake, localization and the photodynamic response of breast cancer cells. The results show that the galactose substitution, together with exposure to UV or red light in non-therapeutic doses, are important factors for the photodynamic effect. Conclusion: Experimental PDT with galactose-substituted ZnPcs accompanied by UV and red light pre-irradiation leads to a higher photodynamic effect towards breast tumor cells. Thus, the investigated galactopyranosyl-substituted phthalocyanines could be used as a part of the design of intelligent, stimuli-responsive nanosystems for medical applications.

Classification of dental lesions fluorescence spectra using support vector machine method

We use the Support Vector Machine method to distinguish fluorescence spectra obtained from demineralized teeth from those obtained from healthy teeth. We find intervals of values for the various parameters of the algorithm, such as dimension, degree of smoothness, etc, as to achieve at least 90% diagnostic accuracy. We argue that the success of the methodology is determined by its multidimensional nature.

Laser-induced fluorescence studies of caries model

The goal of this study is fluorescence investigations in the process of caries growth in presence of bacteria metabolism products. Spectra of teeth, illuminated with 337 nm nitrogen laser were measured during in vitro caries formation. Spectra, obtained from sound tooth consist of one intensive peak at 480-500 nm and one secondary peak at 430-450 nm. In the process of the caries formation of the teeth we observed increase in intensity at 430-450 nm and appearance of two peaks in the red spectral region at 590-650 nm. A general decrease in the intensity of the fluorescence signal is observed, but changes in spectral shape are more significant. We observe that the peak increase at 430-450 nm is related to the tooth demineralization. Bacteria presence and their metabolism products induce fluorescence signal in the red region. These results allow consideration of caries in terms of two different processes, which lead to its formation: demineralization and metabolism products increase, which are caused by bacteria activities, and could be used to obtain a more complete picture of caries formation.