Atomic Force Microscopy Imaging of Elastin Nanofibers Self-Assembly.

Elastin is an extracellular matrix protein, providing elasticity to the organs, such as skin, blood vessels, lungs and elastic ligaments, presenting self-assembling ability to form elastic fibers. The elastin protein, as a component of elastin fibers, is one of the major proteins found in connective tissue and is responsible for the elasticity of tissues. It provides resilience to the human body, assembled as a continuous mesh of fibers that require to be deformed repetitively and reversibly. Thus, it is of great importance to investigate the development of the nanostructural surface of elastin-based biomaterials. The purpose of this research was to image the self-assembling process of elastin fiber structure under different experimental parameters such as suspension medium, elastin concentration, temperature of stock suspension and time interval after the preparation of the stock suspension. atomic force microscopy (AFM) was applied in order to investigate how different experimental parameters affected fiber development and morphology. The results demonstrated that through altering a number of experimental parameters, it was possible to affect the self-assembly procedure of elastin fibers from nanofibers and the formation of elastin nanostructured mesh consisting of naturally occurring fibers. Further clarification of the contribution of different parameters on fibril formation will enable the design and control of elastin-based nanobiomaterials with predetermined characteristics.

Assessment of natural antioxidants' effect on PDT cytotoxicity through fluorescence microscopy image analysis.

BACKGROUND AND OBJECTIVES: Photodynamic therapy (PDT) is a cancer treatment modality mediated by reactive oxygen species (ROS). However, the intracellular antioxidant defense system antagonizes PDT-generated ROS, impeding PDT efficacy. This study aimed to evaluate the enhancement of PDT cytotoxicity by its combination with natural antioxidants in pro-oxidant concentrations. METHODS: A rich natural antioxidant mixture originating from Pinus halepensis bark extract was studied for its potential to enhance the efficacy of m-tetrahydroxyphenylchlorin (m-THPC)-PDT on LNCaP prostate cancer cells, in vitro. Various P. halepensis concentrations, at two different incubation times, were used in combination with m-THPC-PDT. Assessment of cellular viability and intracellular ROS levels evaluated the treatments' outcome. A novel method was developed for the assessment of the intracellular ROS levels, based on image analysis and data extraction from fluorescence microscopy images. RESULTS: P. halepensis bark extract increased the intracellular ROS levels in a concentration-dependent but not in an incubation-dependent manner. The higher concentrations used (≥50 µg/ml) reduced cellular viability even by 50%. One hour pretreatment with 30 µg/ml P. halepensis before m-THPC-PDT exceeded the levels of cellular death by approximately 15%. CONCLUSIONS: The results provided evidence of the cytotoxic effect of P. halepensis bark extract on LNCaP cells, showing the potential of P. halepensis to be used as an anticancer agent in prostate cancer treatment. The results also provided evidence of enhancement of m-THPC-PDT by P. halepensis bark extract showed the potential to be used as a supplementary agent to improve prostate cancer PDT treatment.

A novel optical non-intrusive method for measuring acoustic startle reflex in humans.

OBJECTIVE: The development of a new non-intrusive optical system for remotely measuring acoustic startle reflex (ASR) in humans. APPROACH: The eye reflex movement during an acoustic stimulation session is recorded through a high-speed digital camera. The eyes region is isolated by the rest of the face by an advanced pyramid-like feature detection algorithm, which greatly reduces the number of false positives. A separate Lucas-Kanade optical flow routine is designed for the eyeblink movement detection and the startle eyeblink reflex (SEBR) curve extraction. Image masking is implemented for the elimination of unwanted artifacts caused mainly by voluntary eye movement. The proposed system was tested along with a valid EMG system on a sample of 32 healthy randomly selected adults, and the results were compared in order to measure the system's degree of reliability. MAIN RESULTS: To assess the proposed method's validity the EMG data was used as a benchmark. The results showed strong correlation between EMG and Camera acquired results, which proves the validity of the proposed method. Furthermore, by comparing the response probability and the signal to noise ratio (SNR) for the two techniques, we proved that the proposed method can surpass the traditional EMG system in terms of accuracy and reliability. SIGNIFICANCE: The proposed technique presents a simple, robust and reliable non-intrusive means of measuring ASR in humans, with the potential of future implementation on various ASR psychophysiology experiments, such as the study of PPI.

Atomic force microscopy investigation of the interaction of low-level laser irradiation of collagen thin films in correlation with fibroblast response.

Low-level red laser (LLRL)-tissue interactions have a wide range of medical applications and are garnering increased attention. Although the positive effects of low-level laser therapy (LLLT) have frequently been reported and enhanced collagen accumulation has been identified as one of the most important mechanisms involved, little is known about LLRL-collagen interactions. In this study, we aimed to investigate the influence of LLRL irradiation on collagen, in correlation with fibroblast response. Atomic force microscopy (AFM) and fluorescence spectroscopy were used to characterize surfaces and identify conformational changes in collagen before and after LLRL irradiation. Irradiated and non-irradiated collagen thin films were used as culturing substrates to investigate fibroblast response with fluorescence microscopy. The results demonstrated that LLRL induced small alterations in fluorescence emission and had a negligible effect on the topography of collagen thin films. However, fibroblasts cultured on LLRL-irradiated collagen thin films responded to LRLL. The results of this study show for the first time the effect of LLRL irradiation on pure collagen. Although irradiation did not affect the nanotopography of collagen, it influenced cell behavior. The role of collagen appears to be crucial in the LLLT mechanism, and our results demonstrated that LLRL directly affects collagen and indirectly affects cell behavior.

The effects of UV irradiation on collagen D-band revealed by atomic force microscopy.

The objective of this paper was to investigate the influence of UV irradiation on collagen D-band periodicity by using the AFM imaging and nanoindentation methods. It is well known than UV irradiation is one of the main factors inducing destabilization of collagen molecules. Due to the human's skin chronic exposure to sun light, the research concerning the influence of UV radiation on collagen is of great interest. The impact of UV irradiation on collagen can be studied in nanoscale using Atomic Force Microscopy (AFM). AFM is a powerful tool as far as surface characterization is concerned, due to its ability to relate high resolution imaging with mechanical properties. Hence, high resolution images of individual collagen fibrils and load-displacement curves on the overlapping and gap regions, under various time intervals of UV exposure, were obtained. The results demonstrated that the UV rays affect the height level differences between the overlapping and gap regions. Under various time intervals of UV exposure, the height difference between overlaps and gaps reduced from ~3.7 nm to ~0.8 nm and the fibril diameters showed an average of 8-10% reduction. In addition, the irradiation influenced the mechanical properties of collagen fibrils. The Young's modulus values were reduced per 66% (overlaps) and 61% (gaps) compared to their initial values. The observed alterations on the structural and the mechanical properties of collagen fibrils are probably a consequence of the polypeptide chain scission due to the impact of the UV irradiation.

Investigation of the influence of UV irradiation on collagen thin films by AFM imaging.

Collagen is the major fibrous extracellular matrix protein and due to its unique properties, it has been widely used as biomaterial, scaffold and cell-substrate. The aim of the paper was to use Atomic Force Microscopy (AFM) in order to investigate well-characterized collagen thin films after ultraviolet light (UV) irradiation. The films were also used as in vitro culturing substrates in order to investigate the UV-induced alterations to fibroblasts. A special attention was given in the alteration on collagen D-periodicity. For short irradiation times, spectroscopy (fluorescence/absorption) studies demonstrated that photodegradation took place and AFM imaging showed alterations in surface roughness. Also, it was highlighted that UV-irradiation had different effects when it was applied on collagen solution than on films. Concerning fibroblast culturing, it was shown that fibroblast behavior was affected after UV irradiation of both collagen solution and films. Furthermore, after a long irradiation time, collagen fibrils were deformed revealing that collagen fibrils are consisting of multiple shells and D-periodicity occurred on both outer and inner shells. The clarification of the effects of UV light on collagen and the induced modifications of cell behavior on UV-irradiated collagen-based surfaces will contribute to the better understanding of cell-matrix interactions in the nanoscale and will assist in the appropriate use of UV light for sterilizing and photo-cross-linking applications.

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.

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.

Combination of Fospeg-IPDT and a natural antioxidant compound prevents photosensitivity in a murine prostate cancer tumour model.

BACKGROUND: The aim of the present research was to investigate the potential use of a natural compound rich in antioxidant agents, derived from Pinus halepensis (P. halepensis), to prevent PDT induced photosensitivity. The present research progressed in two levels. The first one evolved the optimization of Fospeg-interstitial photodynamic therapy (IPDT) in a prostate cancer animal model. In the second one, P. halepensis bark extract, was evaluated for its potential use to prevent photosensitivity. METHODS: Two sets of experiments were performed, IPDT only and IPDT in the presence of antioxidant. For both of them, Fospeg was administrated intravenously to SCID mice bearing prostate cancer, followed by IPDT after 6 h. For the IPDT+antioxidant experiments, P. halepensis was injected intratumourously 1 h prior the tumour illumination. Treatment outcome was monitored twice a week by an imaging system and by measuring tumour dimensions using a caliper. Photosensitivity was assessed by monitoring erythema of the tail using the imaging system. RESULTS: IPDT with Fospeg and 15 J total light energy is a therapeutic scheme that can eliminate tumours in the murine model of prostate cancer. Two months after complete tumour remission no tumour recurrence was observed. Also, the cosmetic outcome of the research was excellent. The major drawback of this treatment scheme was that 90% of the animals developed photosensitivity. The addition of P. halepensis bark extract resulted in prevention of the photosensitivity, leaving PDT outcome unaffected. CONCLUSIONS: The combined use of PDT and the used antioxidant agent could broaden the implementation of photodynamic therapy, by eliminating photosensitivity.

A priori fluorophore distribution estimation in fluorescence imaging through application of a segmentation process and a data fitting technique.

During the last few years a quite large number of fluorescence imaging applications have been reported in the literature, as one of the most challenging problems in medical imaging is to "see" a tumor embedded in tissue, which is a turbid medium. This problem has not been fully encountered yet, due to the non-linear nature of the inverse problem. In this paper, a novel method for processing the forward solver outcomes is presented. Through this technique the comparison between the simulated and the acquired data can be performed only at the region-of-interest, minimizing time-consuming pixel-to-pixel comparison. With this modus operandi a-priori information about the initial fluorophore distribution becomes available, leading to a more feasible inverse problem solution.

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. METHODS: AlClPc solution (0.7% w/v) was applied to tumors in mice for 1-6 h. The penetration depth and the optimum drug-light interval were assessed using pharmacokinetic studies. Then, PDT was performed on a murine model of non-melanoma skin cancer using seven different combinations of therapeutic parameters (fluence rate and energy dose). RESULTS: Pharmacokinetic studies revealed that AlClPc was absorbed 40 times more and penetrated 19 times deeper in tumors than normal skin. PDT using AlClPc (0.7% w/v) and a diode laser (75 mW/cm(2), 150 J/cm(2)) resulted in complete tumor remission in 60% of the mice, excellent cosmetic outcomes and growth retardation of tumors with partial remission. CONCLUSIONS: The results indicate that AlClPc-PDT is an effective treatment for non-melanoma skin carcinomas in experimental mouse models.

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.

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.

Cancer chemopreventive effects of Pinus Maritima bark extract on ultraviolet radiation and ultraviolet radiation-7,12,dimethylbenz(a)anthracene induced skin carcinogenesis of hairless mice.

The bark extract of Pinus Maritima (PBE), a rich in phenolic acids, polyphenols and in particular flavonoids mixture, was examined for skin cancer preventive action that was evaluated in two different experimental animal tumor models induced by ultraviolet radiation (UVR) and combination of UVR with 7,12-dimethylbenz[a]anthracene. Significant decrease in the number of animals bearing tumors and the number of tumors per animal was observed in the PBE treated animals. In the same time significant increase in the viability of these animals was also observed. Furthermore, PBE delayed the appearance of tumors. These results provide strong evidence about the preventive anticancer activity of this extract on non-melanoma skin cancer and its protective effect not only from UVR, but also from more potent carcinogenic agents.

A confocal microscopy study of the very early cellular response to oxidative stress induced by zinc phthalocyanine sensitization.

Oxidative stress has been involved in several biological and pathological processes. Reactive oxygen species have been shown to play both beneficial and deleterious roles. The present work contributes to the understanding of the very early events of cellular response to oxidative stress. Oxidative stress was produced intracellularly by light activation of zinc phthalocyanine (ZnPc) at a light dose that did not lead to apoptosis or necrosis. Phthalocyanine was photoactivated using the 647-nm laser line of a confocal microscope through the objective lens causing oxidative stress and allowing observation of the evoked phenomena at the single cell level and in real time. Mitochondria membrane potential (DeltaPsi(m)), intracellular pH, calcium concentration, and generation of reactive oxygen species (ROS) were recorded using specific vital fluorescent probes and quantified by image processing and analysis. Subcellular localization of ZnPc was also studied in order to determine the primary and intermediate ROS target.

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.