Squamous Cell Carcinoma (SCC) is a common type of skin cancer usually detectable in body areas exposed to UV rays of sun such as head, neck and arms. Among the methods that are proposed in order to treat cancer is Photodynamic Therapy (PDT), an innovative therapy. Critical to the effectiveness of the treatment is a chemical molecule, the photosensitizer, which via excitation produces reactive oxygen species that destroy tumor lesions. Silicon phthalocyanines have drawn the attention of the scientific community since the presence of silicon ensures the increased production of reactive oxygen species. However due to their structure, they are insoluble in aqueous solutions. Silicon phthalocyanine dichloride, SiCl2Pc, is a hydrophobic second-generation photosensitizer showing aggregation in waterbased solutions. In order to overcome hydrophobicity, in this study, the encapsulation of SiCl2Pc in β-cyclodextrin and hydroxypropyl-β-cyclodextrin using the kneading method was proposed. Their action as photosensitizers was assessed by photophysical, photochemical and in vitro photobiological studies against squamous cell model skin cancer A431. The Dynamic Light Scattering (DLS) method was used to determine their size, polydispersity index and z-potential, while their structural characterization was performed by Infrared Spectroscopy (FT-IR). The results of photodynamic treatment showed that the encapsulation of SiCl2Pc into cyclodextrins improved its aqueous solubility enhancing its photodynamic action (50% cell viability for β-CD–SiCl2Pc, 57% for HP-β-CD–SiCl2Pc and 67% SiCl2Pc after irradiation for 3 min with 15 mW/cm2).
Collagen thin films consisting of randomly oriented and oriented collagen fibrils/fibers are fabricated by hydrodynamic flow and spin coating, and then they are characterized by atomic force microscopy (AFM). Fibroblasts are cultured on these films in order to correlate their morphology and alignment, which are assessed with fluorescence and AFM imaging with different film characteristics. The results showed that the formed films could be used as substrates for culturing cells. Furthermore, cells reacted to film nanocharacteristics and especially to the orientation of fibrils/fibers. The investigation of the influence that the substrate nanotopography has on cells will help to elucidate the mechanisms of cell–biomaterial interactions, and will enable the design of intelligent coatings for implants and tissue engineering purposes.
Collagen is the major fibrous protein in the extracellular matrix and consists a significant component of skin, bone,
cartilage and tendon. Due to its unique properties, it has been widely used as scaffold or culture substrate for tissue
regeneration or/and cell-substrate interaction studies. The ultraviolet light-collagen interaction investigations are crucial
for the improvement of many applications such as that of the UV irradiation in the field of biomaterials, as sterilizing and
photo-cross-linking method. The aim of this paper was to investigate the mechanisms of UV-collagen interactions by
developing a collagen-based, well characterized, surface with controlled topography of collagen thin films in the
nanoscale range. The methodology was to quantify the collagen surface modification induced on ultraviolet radiation and
correlate it with changes induced in cells. Surface nanoscale characterization was performed by Atomic Force
Microscopy (AFM) which is a powerful tool and offers quantitative and qualitative information with a non-destructive
manner. In order to investigate cells behavior, the irradiated films were used for in vitro cultivation of human skin
fibroblasts and the cells morphology, migration and alignment were assessed with fluorescence microscopy imaging and
image processing methods. The clarification of the effects of UV light on collagen thin films and the way of cells
behavior to the different modifications that UV induced to the collagen-based surfaces will contribute to the better
understanding of cell-matrix interactions in the nanoscale and will assist the appropriate use of UV light for developing
biomaterials.
In this paper the cytotoxic effect of m-THPC, Foscan®, as well as of the liposomal formulation of m-THPC, Fospeg®,(kind offer of Biolitec) were studied post PDT in the human prostate cancer cell line LNCaP. The cells were incubated for 24h with 0.15 μg/ml and 1.2 μg/ml Foscan® and Fospeg®. Irradiation was performed with a 652nm laser and energy doses 180, 360 and 540mJ/cm2. The effect was assessed by the MTT viability test 24h after irradiation. Also the intracellular localization of Foscan® and Fospeg® was monitored by using Laser Scanning Confocal Microscopy Imaging. The results showed no dark toxicity either with Foscan® or
Fospeg® at any concentration. Also irradiation at each energy dose in the absence of any photosensitizer, did not affect cellular
viability. The cellular death caused after Photodynamic Treatment was dependent on m-THPC concentration and formulation, as well as the delivered energy dose. Fospeg® was more effective as LD50 was achieved with 0.15μg/ml at 180mJ/cm2 while for the same cytotoxic result 1.2μg/ml Foscan® was needed. Images from confocal microscopy revealed higher fluorescence intensity in the cytoplasm after incubation with Fospeg®, than upon incubation with Foscan® under the same experimental conditions.
Prostate cancer is a common disease among men with an increasing number of incidences during the last three decades.
Histopathological grading of prostate cancer is based on tissue structural abnormalities. Gleason grading system is the
gold standard and is based on the organization features of prostatic glands. However, till now there is an uncertainty
assign Gleason grade to intermediate stages of the disease, Gleason 3 and Gleason 4. The aim of this study was to
explore the possibility of introducing fluorescent probes in this prostate cancer Gleason grading problem. Propidium
Iodide with cellular nuclei binding pattern and Alexa 488-WGA with selectivity in polysaccharides with sialic acid
residues were finally chosen. Their localisation patterns were assessed using confocal microscopy. Their colocalisation
degree was quantified using special developed algorithms of image processing and analysis. The introduced metrics of
colocalisation were successfully used to correct classify samples in Gleason 3 and Gleason 4 grades. These metrics were
found appropriate to correctly classify 93.10 % of the images into the two classes using the logistic algorithm. The
integration of confocal microscopy along with fluorescent probes to pathologist routine, is an approach that cloud lead to
prognostic advances.
Prostate cancer is a common malignancy among maturing men and the second leading cause of cancer death in USA.
Histopathological grading of prostate cancer is based on tissue structural abnormalities. Gleason grading system is the
gold standard and is based on the organization features of prostatic glands. Although Gleason score has contributed on
cancer prognosis and on treatment planning, its accuracy is about 58%, with this percentage to be lower in GG2, GG3
and GG5 grading. On the other hand it is strongly affected by "inter- and intra observer variations", making the whole
process very subjective. Therefore, there is need for the development of grading tools based on imaging and computer
vision techniques for a more accurate prostate cancer prognosis.
The aim of this paper is the development of a novel method for objective grading of biopsy specimen in order to support
histopathological prognosis of the tumor. This new method is based on texture analysis techniques, and particularly on
Gray Level Co-occurrence Matrix (GLCM) that estimates image properties related to second order statistics.
Histopathological images of prostate cancer, from Gleason grade2 to Gleason grade 5, were acquired and subjected to
image texture analysis. Thirteen texture characteristics were calculated from this matrix as they were proposed by
Haralick. Using stepwise variable selection, a subset of four characteristics were selected and used for the description
and classification of each image field. The selected characteristics profile was used for grading the specimen with the
multiparameter statistical method of multiple logistic discrimination analysis. The subset of these characteristics
provided 87% correct grading of the specimens. The addition of any of the remaining characteristics did not improve
significantly the diagnostic ability of the method. This study demonstrated that texture analysis techniques could provide
valuable grading decision support to the pathologists, concerning prostate cancer prognosis.
Photodynamic therapy (PDT) has been used in the treatment of various skin diseases including non melanoma skin
carcinomas (NMSC). However, until now there are no publications concerning the efficacy of PDT after topical
application of mTHPC.
Although topical photosensitizer application presents many advantages over systemic drug administration, ALA-induced
protoporphyrin IX is the only sensitizer topically used so far.
In the present study photodynamic efficacy of the highly potent sensitizer meso-tetra(hydroxyphenyl)chlorin (mTHPC),
supplied in a novel liposome formulation is investigated after topical application in hairless SKH-HR1 mice, bearing non
melanoma skin carcinomas. The drug was applied topically for drug - light interval of 4 hours. The fluence rates were
100 and 50 mW/cm2 and two total energy doses, 10 J/cm2 and 100 J/cm2 were studied in groups of 5 animals. Three PDT
sessions were performed in each animal, once every 7 days. The final evaluation of PDT effects was performed 14 days
after the 3rd PDT treatment by measuring the geometrical characteristics of tumors.
The groups treated with 100 mW/cm2 presented a higher complete tumor remission than the group of 50 mW/cm2 but an
unusual high mortality. In the group of 50 mW/cm2 and 100 J/cm2, although the complete tumor remission percentage is
poor, the tumor growth rate was decreased. No lesion, papilloma, or tumor was observed in the treated area even six
months after tumor remission. Furthermore tumours up to 7 mm were achieved to be treated, indicating that this novel
mTHPC formulation could be used for deeper and not only superficial carcinomas or lesions.
The aim of this work is to study pharmacokinetics and photodynamic efficiency of aluminium phthalocyanine chloride
(AlClPc) in dimethylsulfoxide/Tween 80/water solution, after topical application on hairless mice bearing non-melanoma
skin carcinomas. The concentration of photosensitizer in normal skin and tumor biopsies 1-6 hours after application was
assessed by fluorescence spectroscopy of chemical extractions. The concentration of photosensitizer was 40 times higher
in tumor than in normal skin even 1 h after application.
For photodynamic therapy (PDT) AlClPc was excited by a diode laser emitting at 670 nm, 1 h after application. Seven
different combinations of therapeutic parameters were chosen. The efficiency was assessed as the percentage of complete
tumor remission, the tumor growth retardation and the cosmetic outcomes. The highest complete remission 60% was
achieved with the combination of 75 mW/cm2 with 150 J/cm2. No recurrence rate was observed in any treatment
parameters group and the cosmetic outcome in all completely treated tumors was excellent. The results show that the
effectiveness of PDT is highly dependent on fluence rate. In addition, they are promising for further investigation of this
PDT scheme in preclinical studies mainly in non-melanoma skin carcinomas up to 7mm.
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) ng/mg 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.
m-THPC photodynamic therapy has been successfully studied in skin cancer, but no research effort concerning its topical application has been performed until now. Determination of the biodistribution of a special m-THPC thermogel formulation and its tumour selectivity was studied after topical application on hairless SKH-1 mice bearing non-melanoma skin carcinomas. 20 μl/cm2 of m-THPC thermogel (0.5 mg m-THPC/ml) were applied on normal and tumour area and the concentration or demarcation of tumor by mTHPC fluorescence was measured at 2, 4 and 6 hours after drug application by three methods: 1. A fluorescence imaging system capturing images at two emission wavelengths (500 and 654 nm) following 405 nm excitation. Signals from different regions of interest were averaged and the intensity ratio at 654 to 500 was calculated. 2. A fluorescence spectrometer with a fiber bundle for in vivo spectra recording after 420 nm excitation. 3 Each animal was euthanized and the photosensitizer was chemically extracted from liver, spleen, muscle, normal skin and tumour. The photosensitizer concentrations in the extracts and in plasma were determined by fluorescence spectroscopy. The in vivo methods showed a remarkable difference in the concentration of photosensitizer in normal skin and tumour. The highest concentration in tumour was observed 6h after drug application and the highest fluorescence intensity ratio of m-THPC in tumour to normal tissue was observed at 4 hours. Furthermore, no m-THPC was detected in normal tissues or plasma after drug topical application. In vivo and ex vivo results were consistent.
Measurements of concentration of sensitizers for photodynamic therapy can provide important information in the dosimetry planning and can also give input to the optimal time for treatment. There has been skepticism towards fluorescence techniques for this purpose, as the signal depends on the fluorescence yield and optical properties of the
tissue. Absorption based techniques, lack on the other hand, often the sensitivity required for many sensitizers with relative weak absorption in a wavelength region where hemoglobin absorption is dominant. A direct comparison between absorption and fluorescence techniques for measuring mTHPC concentration after topical application on hairless SKH-1 mice bearing skin carcinomas has been performed. 20 μl/cm2 of m-THPC thermogel (0.5 mg m-THPC/ml) were applied on normal and tumor area and the concentration of mTHPC was measured at 4 and 6 hours after drug application by two methods: 1. A fluorescence imaging system capturing images at two wavelengths (500 and 650 nm) following 405 nm excitation. Signals from different regions of interest were averaged and the intensity ratio at 650 to 500 was calculated. 2. A diffuse reflectance spectroscopy system with a fiber separation of 2 mm, providing the absorbance at 652 nm. Both
systems provided consistent results related to the photosensitizer concentration. The methods show a remarkable difference in the concentration of photosensitizer in normal skin and tumor. No significant difference in mTHPC concentration in tumor could be observed between the 4 and 6h groups after drug application.
Collagen is known to be a very efficient producer of both second harmonic generation (SHG) and two-photon excited
fluorescence and the combined use of those nonlinear signals is emerging as a new imaging probe to be used as a
diagnostic tool. By recording structural information of collagen between different samples, the technique shows
promising for the study of the distribution of collagen in tissue and for identifying pathologic conditions. Unique
information about the molecular organization of collagen can be extracted from SHG and TPEF imaging data in several
ways and we have initiated a systematic study of these issues. The main objectives of this work are to combine TPEF
and SHG methodologies, in order to elucidate and quantify cross-linking and to describe a model of fibrils orientation
within different samples. In this early approach we discuss fundamental principles governing SHG and TPEF and
present the first results of applying these rules to collagen type I images analysis. By comparing signals between
lyophilized and soluble collagen we validate that the SHG signal arises from dipolar interactions that are enhanced by
the quaternary structure of collagen fibrils, while TPEF arises from fluorophores which are suggested to be products of
cross-linking. Using a homogenization protocol of acid treated collagen gels we manage to produce SHG and TPEF
active thin films, which characterized by means of their contrast capability. A home-built scanning microscope
employing SHG and TPEF was used for the high-resolution imaging of endogenous SHG and TPEF signals, without
exogenous dyes.
The high incidence and mortality rates of prostate cancer have stimulated research for prevention, early diagnosis and appropriate treatment. DNA ploidy status of tumour cells is an important parameter with diagnostic and prognostic significance. In the current study, DNA ploidy analysis was performed using image cytometry technique and digital image processing and analysis. Tissue samples from prostate patients were stained using the Feulgen method. Images were acquired using a digital imaging microscopy system consisting of an Olympus BX-50 microscope equipped with a color CCD camera. Segmentation of such images is not a trivial problem because of the uneven background, intensity variations within the nuclei and cell clustering. In this study specific algorithms were developed in Matlab based on the most prominent image segmentation approaches that emanate from the field of Mathematical Morphology, focusing on region-based watershed segmentation. First biomedical images were simplified under non-linear filtering (alternate sequential filters, levelings), and next image features such as gradient information and markers were extracted so as to lead the segmentation process. The extracted markers are used as seeds; watershed transformation was performed to the gradient of the filtered image. Image flooding was performed isotropically from the markers using hierarchical queues based on Beucher and Meyer methodology. The developed algorithms have successfully segmented the cell from its background and from cells clusters as well. To characterize the nuclei, we attempt to derive a set of effective color features. By analyzing more than 50 color features, we have found that a set of color features, hue, saturation-weighted hue, I1=(R+G+B)/3, I2=(R-B),I3=(2G-R-B)/2, Karhunen-Loeve transformation and energy operator, are effective.
Computer vision advancements have not till now achieved the accurate 3D reconstruction of objects smaller than 1cm diameter. Although this problem is of great importance in dermatology for Non Melanoma Skin Cancer diagnosis and therapy, has not yet been solved. This paper describes the development of a novel volumetric method for NMSC animal model tumors, using a binocular vision system. Monitoring NMSC tumors volume changes during PDT will grant important information for the assessment of the therapeutic progress and the efficiency of the applied drug. The vision system was designed taking into account the targets size and the flexibility. By using high resolution cameras with telecentric lenses most distortion factors were reduced significantly. Furthermore, z-axis movement was possible without requiring calibration, in contrary to wide angle lenses. The calibration was achieved by means of adapted photogrammetric technique. The required time for calibrating both cameras was less than a minute. For accuracy expansion, a structured light projector was used. The captured stereo-pair images were processed with modified morphological filters to improve background contrast and minimize noise. The determination of conjugate points was achieved via maximum correlation values and region properties, thus decreasing significantly the computational cost. The 3D reconstruction algorithm has been assessed with objects of known volumes and applied to animal model tumors with less than 0.6cm diameter. The achieved precision was at very high levels providing a standard deviation of 0.0313mm. The robustness of our system is based on the overall approach and on the size of the targets.
The scope of this work was to determine the Kubelka-Munk scattering and absorption coefficients of healthy and atherosclerotic animal model aorta, from 200 to 1100 nm. Furthermore, using the measured and calculated optical properties, special algorithms were developed in order to discriminate healthy from diseased aorta. Diffuse reflectance and total transmittance were measured via a dual beam diffuse reflectance spectrometer. Inverse Kubelka-Munk Model was applied to calculate the diffusion scattering and absorption coefficients. Diffuse absorption coefficients varied from ~200 cm-1 at 300 nm to ~3 cm-1 at 1100 nm. Kubelka-Munk scattering coefficients ranged from ~100 cm-1 at 200 nm to ~6 cm-1 at 1100 nm. Appropriate discrimination algorithms were developed in order to characterize a specimen as healthy or atherosclerotic. The first algorithm was based on the ratio of diffuse reflectance to the reflectance in infinity. The gradient of this ratio at 390 and 440 nm effectively separated healthy from atherosclerotic aorta. The discrimination between the two groups was succeeded using multivariate statistical analysis and verified by histopathology. The second discrimination algorithm was based on the ratio of diffuse reflectance to the baseline reflectance of each specimen. Effective discrimination of healthy and atherosclerotic aorta was achieved at 370 and 500 nm.
Oxidative stress has been implicated in several biological and pathological aspects. Reactive oxygen species (ROS) have been proposed to act as signal transduction molecules activating reactions leading to cell rescue or to cell apoptosis/necrosis. In the present study, oxidative stress was induced by photosensitization of zinc phthalocyanine (ZnPc) in human fibroblasts using a photodynamic dose that did not lead to apoptosis or necrosis. The induction of oxidative stress was performed at the microscope stage in preassigned time. The cascade of phenomena evoked was studied in real time and at the single cell level using confocal laser scanning microscopy. Using specific vital fluorescent probes, alterations induced by oxidative stress in mitochondria membrane potential, in intracellular pH and in calcium concentration were recorded. Image processing and analysis techniques were used to quantify the observed changes. Subcellular localization of the photosensitizer was studied in order to determine the primary and immediate ROS target. It was found that ZnPc is mainly localized in the mitochondria region.
Confocal microscopy was used for irradiation and observation of the same area of interest, allowing the imaging of low power laser effects in subcellular components and functions, at the single cell level. Coverslips cultures of human fetal foreskin fibroblasts (HFFF2) were placed in a small incubation chamber for in vivo microscopic observation. Cells were stimulated by the 647 nm line of the Argon- Krypton laser of the confocal microscope (0.1 mW/cm2). Membrane permeability, mitochondrial membrane potential ((delta) Psim), intracellular pHi, calcium alterations and nuclear chromatin accessibility were monitored, at different times after irradiation, using specific fluorescent vital probes. Images were stored to the computer and quantitative evaluation was performed using image- processing software. After irradiation, influx and efflux of the appropriate dyes monitored changes in cell membrane permeability. Laser irradiation caused alkalizatoin of the cytosolic pHi and increase of the mitochondrial membrane potential ((delta) Psim). Temporary global Ca2+ responses were also observed. No such effects were noted in microscopic fields other than the irradiated ones. No toxic effects were observed, during time course of the experiment.
We have made a detailed study of the response of LSO detectors to 511 keV (gamma) -rays. The LSO, discovered recently, has a density greater than BGO, small decay time, and high light output. As such, it should have an overall behavior better than that of BGO. We have modeled a (gamma) -ray detector using an LSO crystal of rectangular cross-section attached to a photomultiplier tube (PMT). We used our PET simulation package to study the energy resolution, efficiency, and timing resolution for various crystal sizes and various energy thresholds. The simulation takes into account the interactions of (gamma) -rays in the crystal via Compton and photoelectric effects, the production and transport of scintillation photons, the productions of photoelectrons in the PMT and the anode signal formation. We have estimated the efficiency versus energy threshold for various lengths of the LSO crystal and we find the for 400 keV threshold this efficiency is large even for 2 cm crystals and comparable to that of BGO. We also estimated the timing resolution (FWHM) versus crystal length for various energy thresholds. The timing resolution is comparable to that of CeF3 detectors. The energy resolution is about 10% (FWHM), which allows one to set the energy threshold fairly high.
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