Optical characterization of thin female breast biopsies based on the reduced scattering coefficient.

One of the main goals in optical characterization of biopsies is to discern between tissue types. Usually, the theory used for deriving the optical properties of such highly scattering media is based on the diffusion approximation. However, biopsies are usually small in size compared to the transport mean free path and thus cannot be treated with standard diffusion theory. To account for this, an improved theory was developed, by the authors, that can correctly describe light propagation in small geometries (Garofalakis et al 2004 J. Opt. A: Pure Appl. Opt. 6 725-35). The theory's limit was validated by both Monte Carlo simulations and experiments performed on tissue-like phantoms, and was found to be two transport mean free paths. With the aid of this theory, we have characterized 59 samples of breast tissue including cancerous samples by retrieving their reduced scattering coefficients from time-resolved transmission data. The mean values for the reduced scattering coefficients of the normal and the tumour tissue were measured to be 9.7 +/- 2.2 cm(-1) and 10.8 +/- 1.8 cm(-1), respectively. The correlation with age was also investigated.

In vitro optical characterization and discrimination of female breast tissue during near infrared femtosecond laser pulses propagation.

Ultrashort infrared laser pulses 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 ultrashort 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 reduced scattering coefficients, which are related to the optical characteristics of each type of tissue. The goal of the study was to use the theoretical values of the coefficients to discriminate different kinds of tissue.

Artificial neural networks for discriminating pathologic from normal peripheral vascular tissue.

The identification of the state of human peripheral vascular tissue by using artificial neural networks is discussed in this paper. Two different laser emission lines (He-Cd, Ar+) are used to excite the chromophores of tissue samples. The fluorescence spectrum obtained, is passed through a nonlinear filter based on a high-order (HO) neural network neural network (NN) [HONN] whose weights are updated by stable learning laws, to perform feature extraction. The values of the feature vector reveal information regarding the tissue state. Then a classical multilayer perceptron is employed to serve as a classifier of the feature vector, giving 100% successful results for the specific data set considered. Our method achieves not only the discrimination between normal and pathologic human tissue, but also the successful discrimination between the different types of pathologic tissue (fibrous, calcified). Furthermore, the small time needed to acquire and analyze the fluorescence spectra together with the high rates of success, proves our method very attractive for real-time applications.

The role of laser-induced fluorescence in myocardial tissue characterization: an experimental in vitro study.

OBJECTIVE: The fluorescence of tissue when stimulated by a laser beam is a well-known phenomenon. The resulting emission spectra depend on the biochemical and structural composition of the tissue. In this study, we examined the spectra of laser-induced fluorescence emitted by myocardial tissue. METHODS: We used an argon-ion laser to stimulate the myocardium of 20 intact sheep hearts. For each spectral emission, we calculated the intensity in specific regions in order to characterize the spectra and to reveal intercavitary and intracavitary morphologic differences. RESULTS: The statistical analysis showed significant differences in the emission spectra intensity between atria and ventricles. The intensity was higher in the atria than in the ventricles (p < 0.001). The atrial emission spectra were morphologically different from those of the ventricles. There was no difference in the intensity or morphology of emission spectra within each chamber. All measurements showed good reproducibility after a short period of time. CONCLUSIONS: Laser-induced fluorescence of myocardial tissue seems to have the characteristics necessary for tissue recognition. This might prove useful in identifying cardiomyopathies and transplant rejection, as well as for myocardial mapping, assisting electrophysiologists in discovering fibrotic arrhythmogenic foci.

Single and double wavelength excitation of laser-induced fluorescence of normal and atherosclerotic peripheral vascular tissue.

Laser-induced fluorescence spectra were recorded from the exposure of peripheral vascular tissue to both helium-cadmium and argon-ion laser radiation. Spectral analysis was based on simple algebraic expressions constructed using the intensity difference of the various spectral regions. The above methods were developed in order to determine the degree of atherosclerosis according to the laser-induced fluorescence signal. Similar results with single wavelength excitation were observed during in vivo irradiation of peripheral vessels.

Bone marrow purging by photodynamic treatment in children with acute leukemia: cytoprotective action of amifostine.

In order to evaluate the combined effect of Amifostine and Merocyanine 540 during photoirradiation in neoplastic cells, bone marrow cells from children with acute leukemia (AL), age-matched controls as well as HL-60 cell line were studied. Cell suspensions were incubated with Amifostine, then with MC 540 and they were subsequently exposed to different irradiation doses by Argon Laser 514 nm. Cell survival was estimated by trypan blue supravital stain following a 24-h incubation. The leukemic cell line was studied in continuous liquid cell cultures for 4 weeks. The survival of normal bone marrow progenitors has been estimated by colony formation assay in methylcellulose cultures. Our results showed that Amifostine enhances the photokilling effect of MC 540 on leukemic cells and significantly protects bone marrow nucleated and committed progenitors (BFU-E and CFU-GM) from children with AL under chemotherapy. In conclusion, Amifostine seems to be a promising cytoprotective agent in the clinical use of purging with MC 540 mediated phototherapy.

Photon statistics of laserlike emission from polymeric scattering gain media.

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-fs pulsed laser emitting at 400 nm. Spectral and temporal features were simultaneously recorded with a spectrograph and a streak camera operating in photon-counting mode. Photon-number distributions were thus created. The temporal coherence of the laserlike emission above and below the excitation energy threshold was investigated from the photon-number distribution that was obtained.

Investigation of the laserlike behavior of polymeric scattering gain media under subpicosecond laser excitation.

The narrowing effects of scatterers on the lifetime and the spectral width of the laser-induced fluorescence of organic dyes hosted in poly(methyl methacrylate) polymer sheets were studied. The excitation source was a distributed-feedback dye laser emitting 0.5-ps pulses at 496 nm. Spectral and temporal features were recorded simultaneously on a spectrograph-streak-camera detection system. The results were then compared with those obtained from dye solutions in methanol that were recorded in previous studies. The effects of the different host environments on the fluorescence characteristics of the dye were thus investigated. These effects are currently studied when the dye is inserted into human tissue in an attempt to boost tumor detection and photodynamic-therapy efficiency. Some initial results are presented.

Corneal hydration monitored by laser-induced breakdown spectroscopy.

BACKGROUND: Corneal hydration is an important factor in laser corneal ablation. In photorefractive keratectomy (PRK), corneal ablation rate and final ablation surface quality are strongly dependent on corneal hydration. We used a spectroscopic technique for monitoring corneal hydration during PRK. METHODS: Hydroxyethlymethacrylate (HEMA) was employed for corneal hydration modeling. Hydrated HEMA samples were irradiated with a pulsed Nd:YAG laser (1064 nm, 10 mJ/pulse, pulse duration 15 nsec). Successive emission spectra corresponding to different degrees of hydration were recorded on a gated optical multichannel analyzer. The weight of the sample and hence its water content was monitored during the entire procedure with a sensitive balance. One rabbit and one human cornea were used to demonstrate the spectral analogy between the model and corneal tissue. RESULTS: The most noticeable dependence on water content of the substrate was that of atomic emission lines of Ca at 393 nm and 396 nm. CONCLUSION: Plasma emission spectra exhibited significant dependence on sample hydration. This dependence can be used for estimation of water content of irradiated model material and real cornea.

Effect of liquid-nitrogen and formalin-based conservation in the in vitro measurement of laser-induced fluorescence from peripheral vascular tissue.

In order to investigate the effects of conservation in liquid nitrogen and formalin on peripheral vascular tissue (abdominal aortic, femoral, flank, ham, fibular and tibial artery tissue), laser-induced fluorescence spectra have been recorded during the exposure of these tissues to helium-cadmium and argon ion radiation. The spectral distribution of tissue fluorescence allows the development of simple algorithms based on the intensity difference in order to discriminate the tissue samples when they are fresh and after they have been stored for 24 and 28 h in liquid nitrogen or formalin.

Spectral variations of laser-induced tissue emission during in vivo detection of malignancies in the female genital tract.

A laser-based fluorescence-guided biopsy system has been developed for the screening and early detection of malignancies in the female inner/outer genital tract. Fluorescence spectra were recorded during in vivo exposure of normal and malignant tissue to He-Cd laser (442 nm) radiation. Spectral distribution of tissue natural fluorescence allowed for the development of simple algorithms, based on spectral intensity variations. A subsequent index of discrimination between normal and various malignant tissues has been calculated. This study focuses on the variability of the experimental data and the possible sources of spectral variations. These results suggest that monitoring of this index during colposcopy could enhance selective detection of the malignant tissue, reducing the risk of leaving pathologic tissue untreated during standard exploratory surgical procedures.

Merocyanine 540 mediated photolysis of normal bone marrow, committed hemopoietic progenitors and neoplastic cells. implications for bone marrow purging.

The effect of merocyanine 540 (Mc 540) mediated photoirradiation on both neoplastic and normal hemopoietic progenitor cells was studied. Bone marrow (BM) cells from children with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) at initial diagnosis, ALL in remission, neuroblastoma and normal children as well as cells of Reh-6 and HL-60 cell lines were incubated with Mc 540 in the presence of human albumin (HA) and exposed to different argon laser 514 nm doses. Cell survival was estimated using Trypan Blue supravital stain following a 24-h incubation and leukemic cell lines were studied in continuous cell cultures of 4 weeks duration. Our results showed that HA protects normal BM cells from Mc 540 mediated phototoxicity. A 99.9999% inhibition of Reh-6 and HL-60 was noted at irradiation doses where the corresponding mean survival of normal BM cells was 77.4 +/- 12 and 70.3 +/- 10%, respectively. BM leukemic cells from children with ALL and AML were also very sensitive to Mc 540 photoirradiation in contrast to neuroblastoma cells where only a three-fold reduction was observed. Finally, the survival of normal BM progenitors was 38% for colony forming unit erythroid CFU-E, 37% for burst forming unit erythroid BFU-E, 55% for CFU-GM and 29% for CFU-GEMM. In conclusion it seems that Mc 540 mediated photoirradiation in neoplastic cells exerts selective cytotoxicity and can be used in ex vivo purging of malignant cells in BM.

Laser-induced fluorescence detection of malignancies in the female genital tract via their natural emission and hypocrellin (HA) probing.

A laser-induced fluorescence-guided biopsy system has been developed for the screening and early detection of malignancies in the female inner/outer genital tract. Fluorescence spectra were recorded during exposure of normal and malignant tissue to He-Cd laser (442 nm) radiation. A characteristic increase in the fluorescence signal at 600 nm for malignant tissue was observed after treatment of the samples with hypocrellin (HA). This, combined with the spectral distribution of tissue natural fluorescence, allowed for the development of simple algorithms, based on the intensity difference. A subsequent index of discrimination between normal and various malignant tissues has been calculated. These results suggest that monitoring of this index during colposcopy could enhance selective detection of the malignant tissue, reducing the risk of leaving pathologic tissue untreated during standard exploratory surgical procedures.

Merocyanine 540 mediated photoirradiation of leukemic cells. In vitro inference on cell survival.

In order to evaluate the selective killing of merocyanine 540 (MC 540) mediated photoirradiation in neoplastic cells, bone narrow cells from children with leukaemia or neuroblastoma and normal children as well as peripheral blood cells and Reh-6 and HL-60 cell lines were studied. Cell suspensions were incubated with MC 540 and exposed to various argon laser 514 nm doses. Cell survival was estimated with trypan blue supravital stain following a 24 h incubation and has been followed in continuous cell cultures of 4 weeks duration. Our results showed that the inhibition of survival of neoplastic haemopoietic cells by laser in the presence of MC 540 is proportional to the MC 540 and photoirradiation doses. A 99.9999% inhibition of Reh-6 and HL-60 was noted at irradiation doses where the corresponding mean survival of normal bone narrow cells was (33.6 +/- 15.5)% and (50.6 +/- 10.7)% respectively. Peripheral blood mononuclear cells were not sensitive to MC 540 mediated photoirradiation. The inhibition of survival of bone marrow metastatic neuroblastoma cells was (69.9 +/- 4.1)%. In conclusion, it seems that MC 540 mediated photoirradiation in neoplastic cells exerts selective cytotoxicity and can be used in ex vivo purging of malignant cells in the bone marrow.

Effect of diffraction on early-arriving photons during femtosecond laser transillumination of highly scattering media of biological significance.

Early-arriving photons of 100-fs laser pulses transmitted through highly scattering media have been detected by a streak camera. Because of their partial spatial coherence, they are affected by diffraction from small hidden discontinuities. The experimental data of the patterns are analyzed with Fresnel diffraction theory and then corrected accordingly. Submillimeter hidden objects were scanned and imaged. Diffraction correction resulted in a significantly improved contrast in the hidden object's image.

Malignancies and atherosclerotic plaque diagnosis--is laser induced fluorescence spectroscopy the ultimate solution?

A non-invasive diagnostic tool that can identify diseased tissue sites in situ and in real time could have a major impact on the detection and treatment of cancer and atherosclerosis. A review of the research performed on the utilization of laser induced fluorescence spectroscopy (LIFS) as a means of diseased tissue diagnosis is presented. Special emphasis is given to problems which were raised during clinical trials and recent experimental studies. The common origin and possible solution of these problems are shown to be related to, firstly, the identification of the fluorescent chemical species, secondly, the determination of the excitation/collection geometry and its effect to the method and, finally, the further elaboration on the laser-tissue interaction.

Detection of cardiovascular calcified deposits via tetracarboxylate ion dye (BTC) probing and laser-induced fluorescence spectroscopy.

In order to develop a reliable, laser-induced, fluorescence-guided, angioplasty system, fluorescence spectra were recorded during exposure of normal and atherosclerotic cadaveric aortic tissue to argon ion laser irradiation. A characteristic increase in the fluorescence signal in the 520-560 nm spectral region for atheromatous tissue was observed after treatment of the samples with a tetracarboxylate ion dye (BTC). This allowed the development of a simple algorithm (based on the intensity difference) and a subsequent index of discrimination between normal and atheromatous tissue. Our results suggest that monitoring of this index through the catheter could enhance selective ablation.

Laser-induced fluorescence detection of cardiovascular atherosclerotic deposits via their natural emission and hypocrellin (HA) probing.

In order to develop a reliable laser-induced-fluorescence-guided angioplasty system, fluorescence spectra were recorded during exposure of normal and atherosclerotic cadaveric aortic tissue to He-Cd laser radiation (442 nm). A characteristic increase in the fluorescence signal at 600 nm for atheromatous tissue was observed after treatment of the samples with hypocrellin (HA). This, combined with the spectral distribution of tissue natural fluorescence, allowed the development of simple algorithms, based on the intensity difference and the full width at half-maximum (FWHM), and a subsequent index of discrimination between normal and various atheromatous tissues. Our results suggest that monitoring of this index through the catheter could enhance selective ablation, reducing the risk of normal vessel perforation.

Photophysical characterization of hematoporphyrin incorporated within collagen gels.

Physical properties of hematoporphyrin-enriched collagen gels relevant to the photodynamic treatment of cancer are characterized. The incorporation of the sensitizer within the gels does not affect either the structure of the gel or the absorption and fluorescence spectra of the sensitizer. The gel-embedded sensitizer photodegrades efficiently with the formation of a product emitting near 635 nm. Differences in the collagen scattering are observed following sensitization vs. prolonged irradiation with Ar+ laser, indicating contrasting structural modifications effected to the biopolymer matrix in the two cases. The results correspond well to in vivo observations, suggesting that hematoporphyrin-enriched collagen gels may be appropriate systems for modeling the influence of the semisolid nature of tissues, and in particular of the tumour stroma collagen, on the photodynamic phenomenon.