Diode laser-induced thermal damage evaluation on the retina with a liposome dye system

BACKGROUND AND OBJECTIVES: The aim of the study was to evaluate the feasibility of retinal thermal damage assessment in a rabbit eye model by using laser-induced release of liposome-encapsulated dye. STUDY DESIGN/MATERIALS AND METHODS: After anesthesia, thermosensitive liposomes (DiStearoyl Phosphatidyl Choline: DSPC) loaded with 5,6-carboxyfluorescein were injected intravenously to pigmented rabbits. Retinal photocoagulations were performed with a 810nm diode laser (P=100-400 mW, laser spot=500 microm, 1s) (OcuLight, IRIS Medical Instruments, Mountain View, CA). Fluorescence measurements in the area of the laser exposures were then realized with a digitized angiograph (CF-60UVi, Canon-Europe, The Netherlands; OcuLab, Life Science Resources, UK). RESULTS: Fluorescent spots were observed for power ranging from 100 +/- 5 mW to 400 +/- 5 mW. The fluorescence intensity increased linearly with the power and reached a plateau at 280 +/- 5 mW. The fluorescence intensity was correlated to the maximum temperature at the center of the laser spot with a linear increase from 42 +/- 3 degrees C to 65 +/- 3 degrees C. These results are in agreement with our two previous studies with DSPC liposomes for temperature measurements in a tissue model and then in a vascular model. CONCLUSION: This preliminary study demonstrates the possibility of a laser-induced release of liposome-encapsulated dye for a quantification of diode laser induced thermal damage in ophthalmology. Such a method could be useful for a real-time monitoring of laser photocoagulation for conditions such as choroidal neovascular membranes when a precise thermal damage is required near the foveolar area.     

Functional efficacy of glatiramer acetate treatment for laser-induced retinal damage in rats

BACKGROUND AND OBJECTIVES: To functionally evaluate the efficacy of glatiramer acetate (Cop-1) as a neuroprotective treatment for laser-induced retinal injuries in rats. STUDY DESIGN/MATERIALS AND METHODS: Using standard lasering and flash ERG techniques, we evaluated the effect of photocoagulation and of Cop-1 treatment on retinal function 3, 20, and 60 days after covering one-half of the retina with of 23 rats with argon laser lesions. RESULTS: Significant neuroprotective effects of Cop-1 treatment on functional recovery were observed 20 and 60 days after retinal photocoagulation. Two months post-lasering, the amplitude of electroretinographic signals in lasered eyes (mean+/-SEM) was 99.5+/-10.2% of that of intact eyes in the Cop-1-treated group and 85.8+/-5.5% in the untreated lasered control group (P<0.05). CONCLUSIONS: Cop-1 immunization in rats is neuroprotective against laser-induced injuries to the outer retina and improves functional recovery of the injured retina. Studies have documented effective neuroprotective treatment after laser damage to myelinated neurons, but this is the first report of neuroprotection of nonmyelinated neurons.     

Early differentiation between caries and tooth demineralization using laser-induced autofluorescence spectroscopy

BACKGROUND AND OBJECTIVES: The intrinsic fluorescence of carious human teeth, of different stages of teeth demineralization, and the correspondence of such fluorescence to the mineral and organic distribution within the lesions were investigated. STUDY DESIGN/MATERIALS AND METHODS: Fluorescence spectra of teeth excited with 337 nm nitrogen laser were recorded. Spectra were obtained from healthy enamel, dentine, demineralized areas, and different carious stages of the teeth investigated. RESULTS: Spectra obtained from sound enamel consisted of one intensive peak at 480-500 nm and one secondary peak at 430-450 nm. In dentine, this secondary component had much higher intensity. Fluorescence spectra of normal teeth were similar to those of enamel layer. A significant decrease of the intensity of the fluorescence signal was observed in both cases-in demineralized teeth and in carious lesion. The appearance of a fluorescence peak in the red spectral region was observed in the spectra of the initial carious lesions. In the teeth demineralization process, we observed an increase of the relative fluorescence peak intensity at 430-450 nm related to thinned out of enamel. CONCLUSIONS: A differentiation between initial tooth demineralization and early stages of caries could be made by the laser-induced fluorescence spectroscopy method.     

Laser-induced fluorescence emission: I. The spectroscopic identification of fibrotic endocardium and myocardium.

Laser-induced fluorescence has been developed as a guidance system for laser angioplasty. Laser ablation has been used for resection of arrhythmogenic ventricular scar. We have investigated the use of laser-induced fluorescence for the detection of fibrotic and ischemic changes in endocardium and myocardium. Fluorescence emission spectra from human necropsy specimens were correlated with histologic examination. Normalized fluorescence intensity detected from both the endocardial and the myocardial surfaces of the fibrotic ventricular specimens was significantly higher than that of corresponding normal specimens at 440 to 475 nm. Fibrotic endocardium could be identified by a fluorescence emission intensity ratio less than 1.5 for wavelength ratio 375/450nm. Acutely infarcted endocardium was recognizable by a ratio of 1.5 to 2.0. The specificity and sensitivity of detection of scarred endocardium was 70 and 100%, respectively. Fibrotic myocardium was also consistently identified by fluorescence spectroscopy. Conclusion: Fluorescence emission spectroscopy can differentiate normal and fibrotic endocardium and myocardium, in vitro. This technique may be useful for guidance during laser ablation of arrhythmogenic ventricular scar.     

Alteration of spectral characteristics of human artery wall caused by 476-nm laser irradiation

Fluorescence spectroscopy is a promising new technique for discrimination of normal and atherosclerotic arterial tissues. It has been suggested that this technique be used as a guidance system for laser angiosurgery catheters; however, irradiation by 476-nm light can change the spectroscopic properties of arterial tissue. We present studies that establish intensity levels and exposure times at which alterations in tissue spectral properties are minimal. We also investigate the nature of spectral alterations following exposure of normal human aorta to high intensities of 476-nm laser light. Changes in laser-induced fluorescence (LIF) are characterized by two prominent features: the peak fluorescence intensity decreases permanently, and the fluorescence lineshape changes in a largely reversible way. We relate these changes to alterations in individual tissue chromophores: permanent changes in absolute fluorescence intensity are due to irreversible changes in tissue fluorophores, reversible changes in fluorescence lineshape are due alterations in tissue absorbers. A simple kinetic model is used to describe the decrease in absolute fluorescence intensity.     

Discrimination of normal and atherosclerotic aorta by laser-induced fluorescence

Precise targeting of laser energy to atherosclerotic plaque is crucial for the safe performance of laser angioplasty. The present study was designed to evaluate whether laser-induced fluorescence could distinguish atherosclerotic from normal aorta. Segments of human aorta obtained at necropsy were classified grossly and histologically as normal aorta (n = 7), thin yellow fatty plaque (n = 5), and thick white atheromatous plaque (n = 9), and analyzed by laser-induced fluorescence spectroscopy using a pulsed nitrogen laser. Fluorescence spectra were recorded over a wavelength range from 385 to 725 nm. Normal specimens had maximal fluorescence intensity at 514 nm. A prominent fluorescence peak at 448 nm was present in specimens characterized as white atheromatous plaque and at 538 nm in specimens characterized as yellow fatty plaque. The ratios of fluorescence intensity at 448 nm/514 nm and at 538 nm/514 nm correctly classified all specimens according to their gross and histologic type (p < .001). Thus, a “smart” laser angioplasty catheter system might incorporate low-power laser radiation for arterial fluorescence spectroscopy to guide delivery of high-power laser radiation for plaque ablation.     

Laser irradiative tissue probed in situ by collagen 380-nm fluorescence imaging

BACKGROUND AND OBJECTIVE: There is no ideal method to detect molecular changes in situ of laser-irradiated tissue without removing a section of tissue for histologic examination. A new method is reported to evaluate laser-induced thermal tissue damage in situ by using 380-nm native fluorescence imaging under 340-nm excitation. STUDY DESIGN/MATERIALS AND METHODS: Native fluorescence imaging was performed on laser irradiated bovine tendon tissue and compared with light illuminated photography and histology with picrosirius red F3BA stain. RESULTS AND CONCLUSION: The results indicate that a decrease in collagen fluorescence at 380 nm was observed in laser-induced thermally damaged tissues. The injured region of tissue defined by its fluorescence image coincided with an area defined by photography and histology.     

Detection and diagnosis of oral cancer by light-induced fluorescence

BACKGROUND AND OBJECTIVE: New techniques for non-invasive early detection and diagnosis of oral dysplasia and carcinoma are required. Our objective was to determine in the hamster cheek pouch model whether differentiation between the healthy tissue and the different stages of oral premalignancy and malignancy is possible using laser-induced fluorescence after tissue exposure to 5-Aminolevulinic acid (ALA). STUDY DESIGN/MATERIALS AND METHODS: DMBA carcinogenesis was applied to one cheek pouch in 18 hamsters for 0-20 weeks. Prior to sacrifice, 20% ALA was applied to the cheek tissues. Excised cheek tissues were cryosectioned and imaged using fluorescence microscopy with excitation at 405 nm, detection at 635 nm. After fluorescence measurement, H&E staining and histopathological evaluation were performed. RESULTS: Fluorescence intensity was significantly lower in healthy tissue than in pathological tissues. Significantly higher intensities and more "fluorescence hot spots" occurred in severe dysplasia and carcinoma than in healthy tissue, hyperkeratosis, mild and moderate dysplasia. CONCLUSIONS: Light-induced fluorescence after ALA exposure can differentiate between the different stages of premalignancy and malignancy. Its ability to differentiate between healthy tissue and early pathology is particularly interesting     

Analysis of laser effects on chromosomal structure by fluorescence in situ hybridization and image cytometry: Methodological approach for precise microdissection

Laser effects on human chromosomes have been studied with an image cytometer using an argon ion laser emitted beam at 488 nm, with a 50 mW power. The smallest laser spot was of 0.2m. Aiming of the laser beam was controlled by a computer. Irradiated and non-irradiated chromosomal fragments were analysed by measuring the fluorescence intensity of propidium iodide and by fluorescence in situ hybridization (FISH) with fluorescein biotinylated Alu polymerase chain reaction products. No propidium iodide staining or FISH could be observed on irradiated chromosome fragments indicating total elimination of DNA by the laser beam. The non-irradiated fragments of chromosome showed Alu hybridization similar to control metaphase spreads, suggesting that the DNA structure remained intact. This methodological approach could be used to carry out precise and rapid microdissection of chromosomes.     

Photodynamic therapy for esophageal lesions: selectivity depends on wavelength, power, and light dose

BACKGROUND: Photodynamic therapy with 5-aminolevulinic acid-induced photosensitization could selectively eliminate esophageal epithelial lesions. This study aimed at optimizing laser parameters for 5-aminolevulinic acid photodynamic therapy of the normal rat esophagus. METHODS: Sixty rats received 200 mg/kg 5-aminolevulinic acid orally and were illuminated 3 hours later with either 633 or 532 nm light (n = 30 for each group) through an endoesophageal balloon catheter. Rats received either 8.3 or 25 J/cm diffuser, applied with a 33, 100, or 300 mW/cm diffuser. During illumination, tissue fluorescence measurements and light dosimetry were done. Rats were sacrificed at 48 hours after photodynamic therapy. RESULTS: During illumination, protoporphyrin IX fluorescence declined faster when a higher power output was used. Fluence rate at the esophageal surface was highest for 633-nm light. At 532 nm, light caused less damage to the epithelium and muscle than 633-nm light. Illumination with 33 mW resulted in selective epithelial ablation, whereas illumination with 300 mW caused muscle damage with minor epithelial damage. CONCLUSIONS: The assumed selective epithelial damage of 5-aminolevulinic acid photodynamic therapy in the esophagus largely depends on the combination of wavelength, power, and light dose applied. Most selective epithelial damage was found when low-power 633-nm light was used.     

Time-resolved fluorescence of human aortic wall: use for improved identification of atherosclerotic lesions

BACKGROUND AND OBJECTIVE:This study characterized aortic time-resolved fluorescence spectra for stratified levels of atherosclerosis and proposed interpretation of spectrotemporal variations in terms of histologic changes. STUDY DESIGN/MATERIALS AND METHODS: Fluorescence emission transients were measured at 370-510 nm (337 nm excitation) on 94 excised human aortic samples, ranging from normal to advanced fibrous atherosclerotic lesion. Global analysis yielded a three-exponential approximation of the time-resolved spectra from which average lifetime and decay-associated spectra were derived. RESULTS: Average lifetime at 390 nm gradually increased from 2.4+/-0.1 nsec (normal aorta) to 3.9+/-0.1 nsec (advanced lesion). Fluorescence intensity was markedly decreased above 430 nm in intermediate and advanced lesions. Spectral intensity associated with the intermediate decay increased at 470-490 nm for early and intermediate lipid-rich lesions. CONCLUSION: Time-resolved fluorescence spectra of aortic samples presented distinctive features for each atherosclerotic lesion type, which could serve as characteristic markers for optical analysis of the aortic wall.     

Detection of squamous cell carcinomas and pre-cancerous lesions in the oral cavity by quantification of 5-aminolevulinic acid induced fluorescence endoscopic images

BACKGROUND AND OBJECTIVES: Studies of 5-aminolevulinic acid-induced protoporphyrin IX fluorescence have shown a sensitivity of 95-100% for oral cancer diagnosis, but the specificity is only about 50-60%. Here, we explore the applicability of quantifying PPIX fluorescence images to improve the diagnostic specificity and detect early oral lesions. STUDY DESIGN/MATERIALS AND METHODS: PPIX Fluorescence endoscopy and imaging were performed on 28 patients with a known or suspected premalignant or malignant oral cavity lesion. A total of 70 biopsies were taken from the tissue sites imaged for histological analysis. The red-to-blue and red-to-green intensity ratios were calculated from the fluorescence images to correlate with histology. RESULTS: Suspicious lesions display bright reddish fluorescence, while normal mucosas exhibit blue color background in the fluorescence images. The red-to-blue and red-to-green intensity ratios of malignant tissues are larger than those of benign tissues. Combining the two ratio diagnostic algorithms yields a sensitivity and specificity of 95% and 97%, respectively, exceeding each diagnostic algorithm alone for discriminating malignant tissue from benign tissue. CONCLUSIONS: Quantifying PPIX fluorescence endoscopic images combined with the ratio diagnostic algorithms developed in this study has the potential to significantly improve the noninvasive diagnosis of oral cavity lesions in vivo.     

Continuous laser radiation effect at 1.06 microns on gastrointestinal tract

The thermal effect of 1.06 microns YAG:Nd laser irradiation at temperature conditions up to 100 degrees C without crater formation on gastrointestinal (GI) tissue samples was investigated. The theoretical and experimental data show that at an intensity of 160-400 W/cm2 laser-induced heating of the tissue with an initial temperature of 20 degrees C leads to coagulation lesions at a temperature no less than 60 degrees C and at a depth of 1.7-2.1 mm.     

Using the laser-induced fluorescence spectroscopy in the differentiation between normal and neoplastichuman breast tissue

This article reports results of the in vitro study for potential evaluation of the laser-induced fluorescence spectroscopy in the differentiation between normal and neoplastic human breast tissue. A coumarine dye laser pumped by nitrogen laser generated an excitation light centered at 458 nm. In order to collect the fluorescence signal was used an optical fiber catheter coupled to a spectrometer and CCD detector. Fluorescence spectra were recorded from normal and neoplastic (benign and malignant) human breast tissue, adding up 94 different areas. The discrimination between normal and neoplasm groups reach a sensitivity and specificity of 100%.     

Correlation of photodynamic activity and fluorescence signaling for free and pegylated mTHPC in mesothelioma xenografts

BACKGROUND/OBJECTIVES: Correlation of photodynamic activity (PDT) and fluorescence signaling for free and pegylated meta-tetrahydroxyphenylchlorin (mTHPC) in nude mice with mesothelioma xenografts. STUDY DESIGN/MATERIALS AND METHODS: Twelve animals received light delivery (20 J/cm(2), 150 mW/cm(2), spot size 1.2 cm) on the tumor and the hind leg 3 days after sensitization with 0.15 mg/kg free mTHPC (n = 6) or equimolar-dosed pegylated mTHPC (n = 6). Groups of three animals each were sensitized with 0.15 and 0.5 mg/kg free mTHPC or equimolar dosed pegylated mTHPC followed after 3 days by fluorescence microscopy measurements. RESULTS: Pegylated mTHPC resulted in a similar extent of PDT-related tumor necrosis but in lower skin phototoxicity than free mTHPC. Both mTHPC formulations were heterogeneously distributed in the tumor and were mainly localized in perivascular areas. Pegylated mTHPC revealed a higher tumor to skin fluorescence intensity ratio than free mTHPC (P<0.001). CONCLUSIONS: Fluorescence signaling measurement has the potential to predict the photodynamic activity for both mTHPC formulations in mesothelioma xenografts.     

Selective occlusion of choroidal neovascularization by photodynamic therapy with a water-soluble photosensitizer, ATX-S10

BACKGROUND AND OBJECTIVE: To determine the optimal treatment parameters for selective occlusion of choroidal neovascularization (CNV) by photodynamic therapy (PDT) by using the photosensitizer ATX-S10 and a diode laser (wavelength = 670 nm). MATERIALS AND METHODS: Experimental CNV was induced in rat fundi by argon laser photocoagulation. The distribution of ATX-S10 in the chorioretina was analyzed by fluorescence microscopy, and the optimal treatment parameters for selective occlusion of CNV were investigated by changing the dosage and timing of laser irradiation. CNV closure and resulting damage of the surrounding tissue were documented by fluorescein angiography and light and electron microscopies. RESULTS: Fluorescence of ATX-S10 was observed to be localized in the vascular lumen of the retina and choroid within 5 min after dye injection and increased in intensity in CNV up to 2-6 h and decreased rapidly in normal tissue. Laser irradiation with radiant exposures of 7.4 J/cm2 applied immediately after dye injection or with 22.0 J/cm2 at 2-4 h later effectively occluded the induced CNV without causing significant damage to normal retinal capillaries and large choroidal vessels. CONCLUSIONS: PDT using ATX-S10 can selectively occlude CNV. ATX-S10 is a potentially useful photosensitizer for the treatment of CNV.     

Localization of experimental submucosal esophageal tumor in rabbits by using mono-L-aspartyl chlorin e6 and long-wavelength photodynamic excitation

BACKGROUND AND OBJECTIVE: To increase the applicability of photodynamic diagnosis with regard to deep-seated tumor, we illuminated tumors with a long-wavelength laser beam after photosensitization with mono-L-aspartyl chlorin e6 (NPe6). STUDY DESIGN/MATERIALS AND METHODS: Rabbits with VX2 esophageal tumors were divided into four groups. The control group was not treated, and the other three groups were injected with 1, 2.5, and 5 mg/kg mono-L-aspartyl chlorin e6 (NPe6), respectively. After excitation with a 664-nm laser beam (10 mW, 10 seconds), the fluorescence image and the relative fluorescence intensity (tumor/normal tissue) were recorded every 2 hours up to 8 hours by a newly developed diode laser endoscopic fluorescence imaging system. The tissue concentration of NPe6 was examined by high performance liquid chromatography at 2, 4, and 6 hours after injection with 1 and 5 mg/kg NPe6. RESULTS: The diode laser endoscopic fluorescence imaging system was able to selectively detect fluorescence from submucosal tumor by comparison with the surrounding normal mucosa after NPe6 injection. The fluorescence intensity correlated with NPe6 dose, selectively accumulated in the tumor tissue and relative intensity peaked at 6 hours after injection. No fluorescent images were detected in controls. CONCLUSION: Given intravenously, NPe6 at a dose of 5 mg/kg and excited with a 664-nm wavelength laser beam 6 hours later can define experimentally induced deep-seated esophageal carcinoma in rabbits, by using an endoscopic fluorescence imaging system.     

Detection of near-infrared porphyrin fluorescence excited in experimental animal tumour by the HeNe laser

The red emission (632 nm) of a low-power (about 10 mW) HeNe laser is used to excite liposome-bound haematoporphyrin dimethylester localized in a solid tumour implanted in the mouse leg (MS-2 sarcoma). Fluorescence from the target area, filtered at >710 nm, is converted into visible green light by means of a near-infrared-visible image converter. The photographically recorded images clearly reveal differences in fluorescence intensity of tumour tissues with respect to normal tissues. Clinical application of the procedure is proposed, and the expected advantage with respect to current fluorescence endoscopy are discussed.     

Comparison between laser-induced photoemissions and phototransmission of hard tissues using fibre-coupled Nd:YAG and Er3+-doped fibre lasers

During pulsed laser irradiation of dental enamel, laser-induced photoemissions result from the laser-tissue interaction through mechanisms including fluorescence and plasma formation. Fluorescence induced by non-ablative laser light interaction has been used in tissue diagnosis, but the photoemission signal accompanying higher power ablative processes may also be used to provide real-time monitoring of the laser-tissue interaction. The spectral characteristics of the photoemission signals from normal and carious tooth enamel induced by two different pulsed lasers were examined. The radiation sources compared were a high-power extra-long Q-switched Nd:YAG laser operating at a wavelength of 1,066 nm giving pulses (with pulse durations in the range 200-250 μs) in the near infrared and a free-running Er(3+)-doped ZBLAN fibre laser operating at a wavelength near 3 μm with similar pulse durations in the mid-infrared region. The photoemission spectra produced during pulsed laser irradiation of enamel samples were recorded using a high-resolution spectrometer with a CCD array detector that enabled an optical resolution as high as 0.02 nm (FWHM). The spectral and time-dependence of the laser-induced photoemission due to thermal emission and plasma formation were detected during pulsed laser irradiation of hard tissues and were used to distinguish between normal and carious teeth. The use of these effects to distinguish between hard and soft biological tissues during photothermal ablation with a pulsed Nd:YAG laser or an Er fibre laser appears feasible. The real-time spectrally resolved phototransmission spectrum produced during pulsed Nd:YAG laser irradiation of human tooth enamel samples was recorded, with a (normalized) relative transmission coefficient of 1 (100%) for normal teeth and 0.6 (60%) for the carious teeth. The photoemission signal accompanying ablative events may also be used to provide real-time monitoring of the laser-tissue interaction.     

Quantification of 5-aminolevulinic acid induced fluorescence improves the specificity of bladder cancer detection

PURPOSE: 5-Aminolevulinic acid induced fluorescence endoscopy has outstanding sensitivity for detecting early stage bladder cancer. Nevertheless, a third of the lesions that show specific fluorescence are histologically benign. We decreased the false-positive rate of 5-aminolevulinic acid induced fluorescence endoscopy by incorporating protoporphyrin IX fluorescence quantification into the standard cystoscopy procedure. MATERIALS AND METHODS: In 25 cases (53 biopsies) of a history of or suspicion for bladder cancer 5-aminolevulinic acid induced fluorescence endoscopy and fluorescence image quantification were performed. For fluorescence image quantification images obtained with a target integrating color charge-coupled device camera were digitized and stored in a personal computer. Red-to-blue ratios were calculated from fluorescence positive lesions and results were correlated with hematoxylin and eosin histology. RESULTS: Malignant fluorescence positive lesions showed significantly stronger fluorescence intensity than fluorescing lesions with benign histology. A threshold was established that decreased the false-positive rate by 30% without affecting sensitivity. CONCLUSIONS: Fluorescence image quantification is a new endoscopic method for objectively selecting multicolor fluorescence bladder lesion images for biopsy. It has the potential of eliminating human error by different surgeons with variable experience in fluorescence endoscopy.