We are reporting elsewhere, the promising photodynamic effect of Hypericum perforatum L. extract (PMF) against T24, NBT-II tumor bladder cells, and HL-60 leukemic cells (using 630nm, and 530nm laser light respectively). The main advantages of the extract as a photosensitizer are its low cost, extensive availability, adequate solubility, minimal toxicity, and use with a range of wavelengths. Extraction of dry herb with methanol yields the methanolic extract (ME) in 11%, which is then fractionated using liquid / liquid extraction, yielding the polar methanolic fraction (PMF) in 9,9% overall yield. Hypericin, a photosensitizing ingredient of the herb, was found in these extracts in concentrations as low as 0,51%, and 0,57% respectively. Laser induced fluorescence spectra from the ME and PMF were recorded in order to evaluate their photodiagnostic capacity. An Argon-ion laser was employed for the excitation of the samples. It was shown that the extracts resulted in different fluorescence spectra related both to their intensity, and shape. The intensities of these spectra were only 8 times less compared to the fluorescence of pure hypericin. The dependence of the signal on the pH of the medium of pure hypericin and of PMF was also investigated in order to determine specific spectra variations. According to the results hypericin fluorescence signal fades smoothly in highly acidic medium, while it decreases sharply in highly basic environment. On the contrary PMF gives a slow decrease of fluorescence in both acidic and basic medium. These data suggest that PMF-induced fluorescence is highly sensitive in basic and acidic environment.
Second-harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) are relatively new and promising tools for the detailed imaging of biological samples and processes at the microscopic level. By exploiting these nonlinear phenomena phototoxicity and photobleaching effects on the specimens are reduced dramatically. The main target of this work was the development of a compact inexpensive and reliable experimental apparatus for nonlinear microscopy measurements. Femtosecond laser pulses were utilized for excitation. We achieved high-resolution imaging and mapping of Caenorhabditis elegans (C. elegans) neurons and muscular structures of the pharynx, at the microscopic level by performing SHG and TPEF measurements. By detecting nonlinear phenomena such as SHG and TPEF it is feasible to extract valuable information concerning the structure and the function of nematode neurons.
Laser-induced fluorescence spectra were obtained during the exposure of lamb heart (n=20) tissue to Argon-ion radiation (457.9nm). Fluorescence spectra from different heart compartments (the left and right atria and ventricles, the myocardium, the epicardium, and the aorta) were recorded. Simple algebraic algorithms based on the spectral intensity variation were constructed in order to detect spectral features and characterize the different cardiac compartments. Additionally, it was investigated whether each chamber exhibited constant spectral response. After the end of each experiment the lamb hearts were stored in formalin (10%). The samples were irradiated again after forty eight (48) hours in order to investigate the spectral differences that appear due to formalin conservation. Similar fluorescence measurements were taken from a limited number of human heart tissues (n=2) ex vivo.
Dynamic scattering of coherent light by moving particles causes a Doppler shift of the original frequency, depending on the velocity and the scattering angles. This phenomenon was used for the detection of abnormal spontaneous muscle activity caused by the denervation of muscles. Transmission measurements of low frequency modulated laser irradiation have been made in 110 denervated first dorsal interosseous who had previously been imposed to electromyography which detected abnormal activity. Measurements have also been made in 173 normal muscles. The laser used was a diode laser emitting at 830nm and a pulse generator modulated the laser radiation to a
low repetition frequency of 84Hz. While passing through the denervated muscles, the incoming laser beam gets a frequency shift due to the contraction of the denervated muscle fibers and mixes with the ballistic part of the beam. To analyze the inherent information the outcoming laser light was transformed into electric current by a photodiode and the signal after being selected by an A/D card was submitted to the Fast Fourrier transformation. The findings of our
suggested method have been compared to those of the normal muscles as well as to the electromyographic findings of each denervated muscle.
Optical characterization of biopsies can be used to discern between tissues when performing diffuse optical tomography of the female breast. The theory used for deriving the optical properties of such highly scattering media is based on the diffusion approximation. However, focusing the study in the regime of geometries comparable to the scattering mean free path, the diffusion approximation must be rearranged. Here we present several theoretical assumptions in this direction. In order to investigate the validity of the improved theory, experiments were made involving the transmittance of laser light through turbid phantom models. After the validation of our theoretical model, we have managed to derive the optical properties of over 50 excised breast tissue samples.
New and fascinating field of highly scattering gain media, random lasers and the study of the behavior of such materials of biological significance is presented. Especially the study of the behavior of various fluorophores embedded in highly scattering matrices. The observed fluorescence signal of the fluorophore quenches in both temporal and spectral domains due to the intense scattering that the photons undergo. This happens when the excitation energy reaches and exceeds a threshold value. Above that value the narrowed fluorescence exhibits the same features as the excitation laser pulse. Lifetime in the order of 50 psec and spectral FWHM in the order of few nanometers are observed. For energies below the threshold value the amplification is negligible and the samples behave exactly as those without scatterers. As the excitation energy increases the laser action requirements are fulfilled and the sudden narrowing takes place. Similar studies are underway when various fluorophores are embedded in biological tissues. The final goal is to take advantage of this effect towards a more spatially and spectrally confined agent in Photodynamic Therapy of target tissue lesions on skin or other types of superficial lesions. Very promising in the field of skin PDT would be thin polymer sheets with various dyes, which could be applied directly on the lesion and allow the selection of different irradiation wavelengths using the same laser as excitation source. Thus, improving by far the efficiency of the destruction of different types of cancerous cells. In the biomedical arena, the materials high conversion efficiency per unit volume (≥ 50 percent in a 250 μm thickness) allows for wavelength-shifted catheters and laser creams, potentially useful for the removal of tattoos and other skin discolorations. In addition, random lasers can be used for wavelength shifting the installed base of lasers for other applications such as photodynamic therapy where narrow-band excitations are required for drugs such as benzoporphyrin derivatives (BPD). As far as detection is concerned random lasers could be implemented in various applications such as medical imaging. The combined use of these materials with chromophores that are selectively absorbed by malignant tumors and the narrowband emission along with the high-emitted intensity could boost the detection efficiency.
The coherent properties of the temporally and spectrally narrowed emission of organic dyes hosted inside artificial scattering matrices, after one- and two-photon excitation, were investigated by performing photon-counting measurements. The excitation source was a frequency doubled 200 femtosecond pulsed laser emitting at 400 nm for one- photon or the 800 nm fundamental emission for two-photon excitation. Spectral and temporal features were simultaneously recorded using a spectrograph and a streak camera operating on the photon counting mode. Photon number distributions were thus created. The temporal coherence of the laser-like emission above and bellow the excitation energy threshold has been investigated from the photon number distributions obtained.
Random lasing and second harmonic generation after two-photon excitation was observed for the first time, in both scattering and amplifying polymer samples. Pumping was performed with sub-picosecond laser pulses at 830 nm and the emission, was observed with a spectrograph streak camera detection system for simultaneous recording of spectral and temporal features. SHG was detected at 415 nm and random lasing at 470 nm. The advantages of two photons absorption are discussed because of its enhanced detection efficiency and minimized effects on the irradiated sample (e.g. healthy tissue surrounding tumors).
This study concerns the identification of the state of human peripheral vascular tissue by using Artificial Neural Networks. The fluorescence spectra, obtained by dual wavelength excitation of the tissue samples, were passed through a non-linear filter, based on a High Order Neural Network (HONN). Then a classical Multi-Layer Perceptron was employed to serve as the classifier of the feature vector. The above process resulted in the successful discrimination between normal and different types of pathological tissue.
The coherent properties of the temporally and spectrally narrowed emission of laser-induced fluorescence of organic dyes hosted inside artificial scattering matrices (random lasers) were investigated. The excitation source was a frequency doubled 200 femtosecond pulsed laser emitting at 400 nm. Spectral and temporal features were simultaneously recorded using a spectrograph and a streak camera operating on the photon counting mode. Photon number distributions were thus created. The temporal coherence of the laser-like emission above and below the excitation energy threshold has been investigated from the photon number distribution obtained.
Ultra-short IR laser pluses were transmitted through excised female breast tissue. The resulted signal was recorded by a streak camera with a time resolution of the order of a few ps. Experimental data of the temporal spread of the ultra- short pulse during the transmission through the tissue have been analyzed using the Patterson analytical expression derived from the diffusion theory. This resulted in the calculation of the absorption and scattering coefficients, which are related to the optical characteristics of each type of tissue. A streak camera has detected early arriving photons of 100 fs laser pulses transmitted through highly scattering media. Due to their partial spatial coherence they are affected by diffraction caused by small hidden discontinuities. Experimental data of the patterns are analyzed using Fresnel diffraction theory and then corrected accordingly. Sum-mm hidden objects were scanned and imaged. Diffraction correction resulted in a significantly improved contrast of the hidden object's image.
In order to investigate the effects of conservation in liquid nitrogen and formalin on peripheral vascular tissue (abdominal aortic, femoral and flank artery tissue) laser-induced fluorescence spectra were recorded during the exposure of these tissues to He-Cd radiation (442 nm). The spectral distribution of tissue fluorescence allowed the development of simple algorithms based on the intensity difference in order to discriminate the tissue samples when they were fresh and after they were stored for 24 and 48 hours in liquid nitrogen or formalin.
This paper describes an innovative approach to tissue fluorescence as a phenomenon of inelastic scattering of electromagnetic radiation. A general formulation of the polarization vector of the medium is used in order to describe the fluorescence inelastic scattering process. The general method described, is applied to two different tissue models. In the first model, tissue is represented as a single infinitely thick, homogeneous layer, under plane wave excitation. The second model used is based on a picture of tissue as a single dielectric layer, under pulse excitation. In both cases, fluorophores and absorbing species are assumed to be homogeneously distributed inside tissue. The mathematical techniques used together with the fully evaluated theoretical results are presented. Additionally, experimental measurements were performed on collagen gels which contained various dyes in order to evaluate and validate our mathematical modeling.
Diffusely scattered light models have been extensively used by research groups on their effort to predict laser light's propagation through biologic tissue. This has provided useful results in the case of homogeneous isotropic materials. Biologic tissue is a highly complex medium consisting of single cells and various scattering and absorbing multi-cellular structures. The effect of light diffraction should not be ignored when the size and shape of hidden objects in tissue is predicted via laser transmission measurements.
A major problem in clinical laser therapy and laser based diagnosis is the prediction of the light distribution in tissue. Phantom models with homogenous and reproducible optical properties have been used in the past in order to investigate methods of measuring optical properties.
Laser induced fluorescence spectroscopy (LIFS) was used to detect the presence of PHOTOFRINR porfimer sodium and Benzoporphyrin derivative-monoacid, ring A (BPD-MA) in various tissues. Lobund Wistar rats (n equals 49) inoculated with rat prostatic adenocarcinoma (PA-III) were injected with PHOTOFRINR porfimer sodium (7.5 - 0.25 mg/kg) and BPD (0.50 - 25 mg/kg) intravenously. A Helium-Cadmium laser (442 nm) was used as an excitation source. Our study showed that the amount of PHOTOFRINR porfimer sodium and BPD-MA which localizes in the metastatic lymph nodes is higher than in tumor and all other healthy tissues. Laser induced fluorescence spectroscopy may be a feasible method to detect the distribution of photosensitizers or other fluorescent compounds in vivo.
We investigated the ability of fluorescence spectroscopy, and photosensitizers to differentiate normal, hyperplastic and atherosclerotic arterial wall lesions in vivo. Hyperplastic lesions were induced in the abdominal aorta (AB) of 24 rabbits by balloon injury (BI). Atherosclerotic arterial wall lesions were induced by BI and diet. Fluorescence signals from thoracic n equals 16 and AB n equals 15 sites were analyzed by computer. A ratio was used as an index of drug presence. Use of PPS or BPD and LIFS may be a feasible, in vivo method for the differentiation between normal, hyperplastic and atherosclerotic arterial wall lesions.
Sensitivity of laser-induced fluorescence spectroscopy (LIFS) in detecting a change in tissue pH, and blood perfusion was determined. Rabbits were anesthetized, paralyzed, and mechanically ventilated. The arterial and venous blood supplies of the kidney were isolated and ligated to alter the perfusion. The femoral artery was cannulated to extract samples for blood gas analysis. A 308-nm XeCl was used as an excitation source. A 600 micrometers core diameter fiber was used for fluorescence acquisition, and the spectra analyzed by an optical multichannel analyzer (EG & G, OMA III). the corresponding intensity ratio R equals INADH / ICOLL was used as an index for respiratory acidosis. Blood perfusion was assessed using the following algorithm: (IELAS minus ICOLL) divided by (INADH minus ICOLL). The intensity ratio linearly decreased with the reduction of blood perfusion. When we totally occluded the artery the ratio decreased tenfold when compared to the ratio of a fully perfused kidney. Results of monitoring blood acidosis by laser-induced fluorescence spectroscopy shows a significant trend between pH and intensity ratio. Since all the slopes were negative, there is an obvious significant correlation between the pH and NADH.COLLAGEN RATIO. Blue-light-induced fluorescence measurements and ratio fluorometry is a sensitive method for monitoring blood perfusion and acidity or alkalinity of an organ.
The diagnostic capability of an optical biopsy probe combined with a tumor selective fluorescence drug (Photofrin IIR) was tested in an in vivo animal model. The optical biopsy system is comprised of an arc lamp, an optical contact probe, and low cost detectors. We illuminated tissue with blue (405 nm) light and detected the diffusely reflected excitation light, the tissue autofluorescence (500 - 540 nm), and the drug fluorescence near 630 nm. By using the appropriate signal ratios, normal, inflamed and malignant lymph nodes could be distinguished with a high specificity and sensitivity (85 - 95%).
In this paper we evaluate whether argon-ion laser induced fluorescence could help to discriminate atheromatous from normal tissue stained with tetracarboxylate ion dyes. Eighteen cadaveric abdominal aortic samples (9 normal, 9 atherosclerotic) from 4 autopsies were obtained in an unfixed state within 12 hours of death. They were incubated in the dye solution for two hours and then they were rinsed in saline for another two hours to ensure that only dye bound to the tissue was present during the experiment. An argon ion laser (476.5 nm) was used for excitation. Fluorescence was collected by a 600 micrometers core diameter step-index UV-visible fiber. The fiber was butt-connected to the 20 micrometers slit of a 0.25 m spectrograph. Data acquisition and analysis were performed via an optical multichannel analyzer. A characteristic increase of the fluorescence signal at the 520 to 560 nm spectral region for atheromatous tissue was observed after treatment of the samples with the dye. This allowed the development of a simple discriminatory index (spectrum integral from 520 to 560 nm vs. spectrum integral from 580 to 620 nm).
The goal of this study was to detect the presence of benzoporphyrin derivative-monoacid (BPD-MA) in tissues of a tumor bearing animal model. Eighty one Lobund-Wistar rats, inoculated with Pollard rat adenocarcinoma cells, were used. This animal model exhibits unique predictable, unilateral, metastatic spread. The animals were injected intravenously with 0.75 mg/kg of BPD-MA. A Helium-Cadmium (He-Cd) laser (442 nm, 17 mW) was used as an excitation source and coupled to a 400 micrometers core diameter fiber. Following laparotomy, exploration of the abdominal and inguinal area was performed with laser induced fluorescence. Fluorescence spectra of the primary tumor, bilateral lymph nodes, and various organs were recorded. Fluorescence measurements were conducted four hours post injection. The spectra obtained were characterized by a broadband autofluorescence (approximately 540 nm) and a characteristic peak of BPD-MA (approximately 690 nm). Overall, the BPD-MA concentration was higher in lymph nodes than in the skin, kidney, large bowel, muscle or spleen. Skin exhibited the lowest fluorescence intensity ratio, indicative of a lower drug concentration in this tissue. In summary, our results suggest that laser induced fluorescence spectroscopy may provide an alternative way of assessing the biodistribution of BPD-MA or other photosensitizers.
This paper reports on the application of angioscopic technology to the endoscopy of previously inaccessible body cavities. Necessary instruments including endoscopes, light sources, cameras, video recorders, monitors, and other accessories are described. Practical considerations for effective instrumentation are discussed. An overview of our clinical microendoscopic applications in more than 630 patients is presented.
The authors studied the ability of Laser Induced Fluorescence Spectroscopy (LIFS) for the intraoperative identification of metastases using a photosensitizing agent Photofrin IIr to enhance spectroscopic detection. A He-Cd laser source (442 nm) was used to produce low-power illumination of tissue via a hand-held 400 micrometers fiberoptic probe. Through the same fiber, reflected and emitted light was returned to an optical multi-channel analyzer (OMA III) for analysis. Spectroscopic signals were displayed on a screen for immediate examination. Lobund Wistar rats, inoculated with Pollard rat adenocarcinoma cells, were used as an animal model. Photofrin IIr was administered intraperitoneal 24 or 48 hours prior to surgical exploration in doses varying from 0.75-7.5 mg/kg. Metastases detection was performed during abdominal exploration directed to ipsilateral and contralateral inguinal, iliac, para-aortic and renal lymph nodes. Nineteen tissue samples, identified as abnormal by LIFS, were removed for histologic analysis; 11 of these samples were larger than 5mm and histologic examination revealed malignancy in all cases. While LIFS signals showed malignancy in 8 tissue samples with dimensions less than 5mm, histology confirmed this in only 3. However, serial histologic sections were not performed. From the initial results, it was concluded that LIFS detection of malignant tissue is feasible and enhanced by the addition of Photofrin IIr. LIFS may be a promising technique for the intraoperative detection of primary malignant and metastatic tissue.
The goal of this study was to investigate laser induced fluorescence spectroscopy (LIFS) as a method of localization of atherosclerotic lesions not visible by angiography using Photofrin IIr enhanced fluorescence. Twenty-four New Zealand White rabbits divided into six groups varying in type of arterial wall lesion and Photofrin IIr administration time (i.v.) were used. Aortic wall fluorescence signals were acquired from the aortic arch to iliac bifurcation. The output of a He-Cd laser (442 nm, 17 mW) was directed at the arterial wall through a 400 micron fiber. The fluorescence signal created in the arterial wall was collected via the same fiber and analyzed by an optical multi-channel analyzer (OMA). The ratio of fluorescence intensities at 630 nm (Photofrin IIr) and 540 nm (autofluorescence of artery wall) was analyzed (I630nm/I540nm). Intensity ratio values 24 hours after administration of Photofrin IIr were found to be as follows: in normal artery wall of 0.30 +/- 0.14 (n equals 3), in mechanically damaged wall of 0.91 +/- 0.65 (n equals 2) and, in atheromatous tissue, 0.88 +/- 0.54 (n equals 4). The intensity ratio of atheromatous tissue without Photofrin IIr was 0.23 +/- 0.04 (n equals 7). These results suggest that the use of Photofrin IIr allows in vivo atheroma detection by LIFS because of its ability to accumulate in atheroma. In addition, accumulation of Photofrin IIr was found in artery walls traumatized by balloon catheter intervention. Using this method, a catheter-based LIFS system may be developed for atheroma detection.
The goal ofihis sludy was o develop a reliable laser inducedfluorescence specira analysis system using the
308nm Excimer Laser as an ablaiion andfluorescence inducing source. During our analysis we also aflempled
lo determine whether exogenous chloroeiracycline hydrocloride (CTC) increased the discrimination capacity
of the LIFS system. We then assessed the ability of CTC to improve the detection of the boundary between
atheroma and normal media.
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