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 degrees, 50 degrees, 55 degrees, 60 degrees, 65 degrees, 70 degrees, 75 degrees and 80 degrees C) and normalised with respect to the corresponding values at the ambient temperature of 30 degrees 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 degrees 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 degrees 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.

Microscopical examination of the localisation patterns of two novel rhodamine derivatives in normal and neoplastic colonic mucosa.

Tissue characterisation by fluorescence imaging, using exogenous fluorophores, is a promising method for cancer detection. Histochemical alterations in the composition of mucins, when neoplastic transformations occur, could be exploited to derive more selective fluoroprobes indicative of early malignant transformation. The aim of this work was to develop and examine tumour selective fluoroprobes for colon cancer diagnosis, as well as to determine the morphological components where selective dye accumulation has occurred. Two novel fluoroprobes: rhodamine B-L-leucine amide and rhodamine B-phenylboronic acid were synthesised and examined together with Mayer's mucicarmine, alexa 350-wheat germ agglutinin (WGA) and tetramethyl rhodamine-concanavalin A (ConA). Fluorescence microscopy studies were performed with deparaffinised human colon sections, using an epifluorescence microscope equipped with a colour CCD camera. The intense accumulation of the novel fluoroprobes was localised in the amorphous material in the lumen of neoplastic crypts. To gain insight into the localisation patterns, mucicarmine, alexa 350-WGA and tetramethyl rhodamine-ConA were used. Alexa 350-WGA reacted primarily with mucin secreted in the malignant crypt lumen suggesting that this material is rich in sialic acid and N-acetylglucosaminyl residues. These derivatives clearly and consistently distinguished non-neoplastic from neoplastic human colon tissue sections. The intense accumulation at the altered mucins indicates that they could be used as fluoroprobes of biochemical alterations for carcinoma detection.

Visual observation of infrared laser speckle patterns at half their fundamental wavelength.

In the course of work aimed to examine second harmonic generation in biological macromolecules, it was discovered that 1064 nm pulsed nanosecond infrared laser radiation, after scattering on the optically rough sample surface, was perceived as double the fundamental frequency green speckle pattern by the unaided eye. The threshold of this arresting phenomenon was found to be around 60 mJ/cm2 on hyaluronic acid potassium salt in dark ambient conditions. This effect is of a second order non-linear nature. After experiments with nearsighted, farsighted and normal vision observers in combination with second harmonic generation experiments on various lamb eye tissues, the effect was mainly attributed to second harmonic generation in the cornea arising from phase interference of the incident infrared radiation. The current work could possibly have implications in the fields of speckle interferometry and biomedicine.

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.

Photochemical effects and hypericin photosensitized processes in collagen.

Emission and excitation spectra of collagen were recorded in the ultraviolet and visible regions. The existence of several types of chromophores absorbing and emitting throughout these spectral regions was observed. It was shown that laser irradiation at 355 and 532 nm caused collagen fluorescence photobleaching by 30%, when the delivered light doses were 9 and 18 J/cm2, respectively. This process of collagen fluorophores photodestruction was found to be a one-photon effect. The effect of hypericin (HYP), a polycyclic quinone, photosensitization on collagen was also studied. Addition of HYP aqueous solution to collagen produced quenching, redshift of the maximum, and broadening of the spectral form of its fluorescence. These effects became more prominent with increasing HYP concentration. The fluorescence of HYP sensitized collagen decreased in a spectrally nonproportional manner during laser irradiation at both 355 and 532 nm.

Agents facilitating the electric field-induced fusion of intact rabbit erythrocytes.

In the recent years, the role of specific membrane active agents in the electrofusion process has started to draw attention, and it has been found that the presence of various substances in the cell medium can affect the fusion process either in a positive or negative way. In this work, the effect of several proteins, bivalent cations and antibiotics was tested with respect to their ability to protect intact erythrocytes from hemolysis and facilitate the fusion process. The effect of different sugars was also studied. Among the different proteins, pronase and proteinase were found to be the most effective. With respect to bivalent cations, Ca2+ and Mn2+ were more effective while Mg2+ was less important. From the antibiotics, penicillin caused a negative effect while streptomycin acted positively. Finally, glucose medium was found to be the most effective compared to all sugars tested. The results indicated that there are strong differentiations of the induced effects caused by each substance, and some possible mechanisms of action of these agents on the erythrocyte membrane were discussed.

In vitro evaluation of the genotoxic and clastogenic potential of photodynamic therapy.

Photodynamic therapy (PDT) was recently introduced in clinical practice for the management of cancer. As far as PDT relies on the combined action of a photosensitizer and a laser source, there is a need to evaluate the genotoxic and mutagenic potential of this treatment modality. This paper reports the effects of various photosensitizer and photo-irradiation doses on lethality to the MIA PaCa cell line using ZnPcS4 as the photosensitizer. The sister chromatid exchange (SCE) assay was used to evaluate the genotoxicity of various photosensitizer and photo-irradiation doses. Also, chromosomal aberrations at various time intervals post-irradiation were evaluated. The results showed that a combination of 3 J/cm2 irradiance with 5 microM ZnPcS4 concentration leads to the LD90 72 h post-irradiation. Eight days post-irradiation the LD90 level was achieved using a light dose of 3 J/cm2, independent of ZnPcS4 concentration. The SCE assay showed that cells treated with various light and drug doses presented no genotoxic potential, as SCE levels were not different from untreated (control) cells. Chromosomal analysis after PDT treatment at various time intervals post-irradiation showed that there was no significant chromosomal damage in cells treated photodynamically compared with untreated controls. The results show that the cell killing mechanism after PDT is not at the chromosome level, but may be at a different cellular level, such as plasma membranes, mitochondria, etc.

Morphometric characteristics of red blood cells as diagnostic factors for coronary artery disease.

Digital imaging microscopy plays an important role in computer aided cytological and histological diagnosis. In this study, digital imaging microscopy was used to measure Red Blood Cells (RBCs) morphometric characteristics such as area, perimeter, major and minor axis length, elongation, compactness and roughness in both groups: healthy individuals and Coronary Artery Disease (CAD) patients. All the measured characteristics, except elongation, showed a statistically significant difference between the two groups. In particular, patients with CAD presented greater values than healthy ones. Finally multivariate analysis was used in order to take into account the whole measured profile of each patient. Compactness, perimeter and elongation improved the diagnostic ability, whereas all the others did not show any significant improvement. These three characteristics correctly classify the 84.5% of the patients. In conclusion, RBCs morphometric characteristics could be considered as diagnostic factors for CAD.

Loading of intact rabbit erythrocytes with fluorophores and the enzyme pronase by means of electroporation.

The application of electric field pulses in cell suspensions is known to alter membrane integrity, resulting in increased membrane permeabilization. This field-induced membrane poration provides the means to load cells with a variety of external substances, useful for clinical applications. In this work, intact rabbit erythrocytes were successfully loaded with low molecular weight fluorescent probes and with the high molecular weight enzyme pronase, which has been shown to mimic the effects of insulin. Attachment of the enzyme onto the cell surface was also achieved by modifying the applied pulse parameters. Both applications were efficient and accompanied by high cell survival rates. In this way, biological carriers loaded with active substances were produced, offering the potentials for useful clinical applications, either for diagnostic or therapeutic purposes.

Findings of computerized nuclear morphometry of papillary thyroid carcinoma in correlation with the age of the patients.

The aim of this study was to evaluate the usefulness of nuclear morphometry in the pathology of papillary thyroid carcinoma by computer-aided image analysis and the statistical comparison of nuclear morphometric parameters with the age of patients. Seventy cases of papillary thyroid carcinoma diagnosed by fine needle aspiration (FNA) biopsies were classified into two groups according to the patients' age (group I: <45 years and group II: >45 years). The following six nuclear morphometric parameters were measured in a large number of randomly selected nuclei of each case: area, perimeter, major axis length, elongation, roundness, and compactness. The statistical analysis was performed by using special algorithms, and the results of the two groups were compared for each of the parameters measured using the t-test. Area, perimeter, and major axis length of papillary thyroid carcinoma nuclei demonstrate a statistically significant correlation with the age of patients, whereas elongation, roundness, and compactness did not show any correlation. Nuclear morphometry in association with the follow-up of patients could be suggested as an important prognostic index for papillary thyroid carcinoma.

Findings of computerised nuclear morphometry of papillary thyroid carcinoma in correlation with known prognostic factors.

The aim of this study was the evaluation of the usefulness of nuclear morphometry in the pathology of papillary thyroid carcinoma by computer-aided image analysis and the statistical comparison of nuclear morphometric parameters with age of patients, tumor size and the presence or not of thyroid capsule invasion. Thirty three cases of papillary thyroid carcinoma were classified in two groups according to patients' age (group I: < 45 years and group II: > 45 years), according to tumor's size (group I: < 2.5 cm, group II: > 2.5 cm) and to the presence or not of thyroid capsule invasion (group I: invasion (+), group II: no invasion (-)). The following six nuclear morphometric parameters were measured in a large number of randomly selected nuclei of each case: area, perimeter, major axis length, minor axis length, elongation and roundness. The statistical analysis was performed using special algorithms and the results of the two groups of each prognostic factor examined, were compared for each of the measured parameter using the t-test. When the classification was associated with age it was shown that younger people (group I) have lower area, perimeter, major axis length, elongation and roundness means of papillary thyroid carcinoma nuclei than older ones (group II) whereas minor axis length did not show any difference between the two groups. When the classification was associated with tumor size it was shown that younger people (group I) have lower area, perimeter, major axis length means of papillary thyroid carcinoma nuclei than older ones (group II) whereas minor axis length, elongation and roundness did not show any difference between the two groups. Finally, when the cases were classified according to the presence or not of the capsule invasion it was shown that younger people (group I) have lower area, perimeter, major axis length means of papillary thyroid carcinoma nuclei than older ones (group II) whereas roundness did not showed any difference between the two groups. On the other hand younger people (group I) have higher elongation and roundness means than the older ones (group II). Nuclear morphometry in association with the patients' follow-up could represent an important prognostic index for papillary thyroid carcinoma.

The application of image analysis in the measurement of the myocardial infarct area in an experimental model.

BACKGROUND: Thrombolytic therapy improves left ventricular ejection fraction and survival. Using the image analysis method, the study was undertaken to evaluate the effects of intraaortic balloon pump used in conjunction with reperfusion in reducing infarct size by measuring the infarct area. Also, the usefulness of image analysis as an objective method for the measurement of myocardial infarct area is evaluated. METHODS: 22 dogs were subjected to proximal left anterior descending coronary artery occlusion. The dogs were classified into 3 groups. In group I (n = 7), occlusion lasted for 6 hours. In group II (n = 6), 2 hours of occlusion were followed by reperfusion. In group III, (n = 9) after 2 hours of occlusion, the dogs were assisted with the intraaortic balloon pump throughout the 4 hours of reperfusion. The measurement of the infarcted area components (fragmentation, edema, hemorrhage and polymorphonuclear infiltrations) was followed by a semiautomatic method including in connection a microscope photographic camera, a scanner and a computer with the appropriate software. The results were analyzed statistically using the t-test. RESULT: In group I, the mean value of fragmentation was 28.2%, in group II, 10.1% (p < 0.01 versus group I) and in group III, 3.9% (p < 0.01 versus I and p < 0.05 versus group II). CONCLUSION: Reperfusion and intraaortic balloon pump increased the salvage of the ischemic myocardium over that achieved by reperfusion alone in a canine occlusion - reperfusion model. Image analysis could be considered as an accurate and objective method for the measurement of the myocardial infarct area in the experimental model of our study and it could be used in any other experimental study in which the accurate measurement of myocardial infarct area is needed.

Integral equation model of light scattering by an oriented monodisperse system of triaxial dielectric ellipsoids: application in ectacytometry.

A novel mathematical model of light scattering by an oriented monodisperse system of triaxial dielectric ellipsoids of complex index of refraction is presented. It is based on an integral equation solution to the scattering of a plane electromagnetic wave by a single triaxial dielectric ellipsoid. Both the position and the orientation of a single representative scatterer in a given coordinate system are considered arbitrary. A Monte Carlo simulation is developed to reproduce the diffraction pattern of a population of aligned ellipsoids. As an example of practical importance, light scattering by a population of erythrocytes subjected to intense shear stress is modeled. Agreement with experimental observations and the anomalous diffraction theory is illustrated. Thus a novel check of the electromagnetic basis of ektacytometry is provided. Furthermore, the versatility of the integral equation method, particularly in the advent of parallel processing systems, is demonstrated.

Computerized nuclear morphometry of renal cell carcinomas.

The aim of this study was the evaluation of the usefulness of nuclear morphometry in the pathology of renal cell carcinoma by computer-aided image analysis and the statistical comparison of nuclear morphometric parameters with the histologic grade of the tumors. 44 cases of renal cell carcinoma, classified by two independent pathologists into 4 histologic grades (I-IV), were examined. The following 5 nuclear morphometric parameters were measured in a large number of randomly selected nuclei of each case: Major axis length, area, elongation, roundness and compactness. The statistical evaluation was performed using one-way analysis of variance between the four groups of the histologic grades (I-IV). Between them, there was a statistically very significant difference of the mean value of all the evaluated parameters. The values of the estimated parameters, with the exception of roundness and compactness, showed a strong tendency to increase in proportion to histologic grade. Our results suggest that image analysis is a reproducible and objective method for the grading of renal cell carcinoma, and it can be helpful in the unbiased evaluation of such tumors.

Interpretation of diagnostic implications of fluorescence parameters for atherosclerosis in fibrous, calcified and normal arteries.

Fluorescence spectroscopy presents great interest for the diagnosis of atherosclerosis. Nevertheless there are some difficulties in the interpretation of diagnostic information. This could be overcome by precise methods of extraction of the diagnostic parameters and convenient statistical analysis, which are the subject of this work. Fluorescence excitation-emission matrices from different categories of coronary arteries were developed and used to derive the optimum excitation wavelength on the one hand and to assign the spectra to specific chromophores on the other hand. Simple dimensionless functions (Fi) were formed by the ratio of the intensities at selected wavelength and the logistic model was used for statistical analysis. Decision surfaces were drawn and it was estimated that the probability of correct classification is 88%. The algorithm correctly diagnoses 97% of healthy from diseased samples and 80% of fibrous from calcified coronary arteries.

A comparative study of pulsed Nd:YAG and CO2 laser effect on aortic valves.

Laser angioplasty is a promising new technology, which sometimes offers the surgeon an alternative tool to conventional therapy. One of the main prerequisites of using powerful pulsed lasers in angioplasty is the optimum choice of the irradiation parameters (the wavelength, the pulse shape and duration, the energy parameters). Infrared lasers have numerous applications in surgery. Among them the pulsed CO2 laser emitting at 10.6 microns and the pulsed Nd:YAG laser emitting at 1.064 microns are the most widely used, since the delivery of laser energy in short pulses of high peak pulse power limits distant thermal effects. The CO2 laser wavelength is greatly absorbed by tissue water, while the Nd:YAG laser wavelength is poorly absorbed by water or other tissue chromophores, affecting deeper the tissue. The CO2 laser incises effectively the tissue, but presents inadequate coagulation, while the Nd:YAG laser seems to present better coagulation properties. In this work, a comparative study of pulsed Nd:YAG and CO2 lasers in ablating cardiovascular tissue, is presented. The irradiation regime of the two lasers is determined, for the specific tissue and/or pathology. Also the depth of thermally induced coagulation is measured. The results are presented according to the efficacy of each laser for incision and/or coagulation.