The effect of photodynamic therapy on the tumor microvasculature in the first few hours after treatment was studied at the light and electron microscopy levels. BALB/c mice with EMT-6 tumor received ip injections of hematoporphyrin derivative, chlorin, or phthalocyanine, and 24 hours later, the tumors were treated with light at 100 J/cm2 at the appropriate therapeutic wavelength for each photosensitizer. Animals were killed and their tumors removed at time 0, 30 minutes, 1 hour, and 2, 4, 6, 8, 12, 16, and 24 hours after treatment. The results indicate that for all three sensitizers the effects of photodynamic therapy leading to rapid necrosis of tumor tissue are not the result of direct tumor cell kill but are secondary to destruction of the tumor microvasculature. The first observable signs of destruction occur in the subendothelial zone of the tumor capillary wall. This zone, composed of dense collagen fibers and other connective tissue elements, is destroyed in the first few hours after phototherapy. However, the ultrastructural changes seen in this zone are different for the hematoporphyrin derivative, compared with chlorin and phthalocyanine. Binding of photosensitizers to the elements in this zone as well as altered permeability and transport through the endothelial cell layer because of the increased intraluminal pressure may be key features of tumor destruction.

The use of focused high-intensity light sources for ablative perturbation has been an important technique for cell biological and developmental studies. In targeting subcellular structures many studies have to deal with the inability to target, with certainty, an organelle or large macromolecular complex. Here we demonstrate the ability to selectively target microtubule-based structures with a laser microbeam through the use of enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) variants of green fluorescent protein fusions of tubule. Potorous tridactylus (PTK2) cell lines were generated that stably express EYFP and ECFP tagged to the alpha-subunit of tubulin. Using microtubule fluorescence as a guide, cells were irradiated with picosecond laser pulses at discrete microtubule sites in the cytoplasm and the mitotic spindle. Correlative thin-section transmission electron micrographs of cells fixed one second after irradiation demonstrated that the nature of the ultrastructural damage appeared to be different between the EYFP and the ECFP constructs suggesting different photon interaction mechanisms. We conclude that focal disruption of single cytoplasmic and spindle microtubules can be precisely controlled by combining laser microbeam irradiation with different fluorescent fusion constructs. The possible photon interaction mechanisms are discussed in detail.

The purpose of this study was to determine whether a nanosecond-pulsed, frequency-doubled Nd:YAG laser emitting at 532 nm can be used as an alternative to mechanical methods of root canal treatment or as an adjunct to conventional endodontic preparation. Laser parameters whose thermal effects did not exceed safety thresholds for adjacent periodontal tissues were selected in a preliminary study. In 27 extracted human teeth, root canals were irradiated for 30 to 60 s at fluences of 2 to 2.2 J/cm2, and 10 Hz. Samples were observed using SEM. Laser irradiation could achieve smear layer removal after minimal manual preparation. However, results were inhomogeneous, and at higher energy densities thermal damage was observed, especially in the fully manually prepared samples. Nanosecond-pulsed irradiation at 532 nm can achieve complete smear layer removal. However, mechanisms must be developed to monitor laser effects and avoid potential damage to collateral structures.

Thermal and microstructural events resulting from KTP laser use during root canal preparation were investigated in 30 extracted single-rooted human teeth. In the first section of this study, thermal events occurring on the root surfaces of 18 teeth during and after exposure of the root canal were measured using thermography. A variety of parameters were used to determine settings that would be effective without causing thermal damage to the periodontal ligament. In the second section of the study, root canals of 12 teeth exposed to KTP laser irradiation at parameters derived from section 1 were evaluated using Scanning electron microscopy. KTP laser application at a power setting of 3 W, an exposure time of 2 s, and a frequency of 5 Hz, applied five times, removed smear layer and debris from the root canal surface at temperatures below the thermal injury threshold for periodontal tissue.

The origin and process of regeneration in rabbit endometrium was evaluated following photodynamic epithelial destruction using topically applied aminolevulinic acid (ALA). Selective destruction of endometrial epithelium was performed using photodynamic therapy (PDT). ALA was diluted to 200 mg/ml dextran 70 shortly prior to administration. A volume of 1.2 ml was injected into the left uterus. Intrauterine illumination (wavelength 630 nm, light dose 40-80 J/cm2) was performed 3 h after drug administration. Tissue morphology was evaluated by light and scanning electron microscopy 1, 3, 7 and 28 days post-treatment (three animals at each time-point). Regeneration of the endometrium following epithelial ablation by PDT was fully activated after 24 h and was completed after 72 h. Endometrial surface generation occurred by proliferation, originating primarily in deeper regions of the glands. Findings from our morphological follow-up study support the origin of endometrial regeneration being mainly from undifferentiated stem cells and residual glandular epithelium.

The effects of nanosecond pulsed Nd:YAG irradiation on crown and root dentin susceptibility to artificial de- and remineralization were investigated. Laser-induced heating and structural effects were also determined. Thirty caries-free extracted human molar teeth were used. One group of 12 teeth was bisected longitudinally, and each surface covered with acid resistant varnish, leaving 4 windows on the cut dentin surface. Using a Q-switched nanosecond pulsed Nd:YAG laser (1064 nm), one half of each tooth was irradiated at one of the following parameters: 3 mm spot size, a fluence of 1.0 J/cm2. The other half of each tooth served as (non-irradiated) control. All samples were then subjected to a 4 day, thrice daily de- and remineralization cycle. Prior to and after each demineralization, microhardness measurements were taken. After 4 days, samples were again bisected, and the other sample halves used for SEM. Microhardness in the irradiated samples did not differ significantly (p < 0.01) compared with controls; SEM results showed some laser-induced alterations on crown and root dentin surfaces. Thermometric measurements on 18 teeth at energy densities of 1.0-4.1 J/cm2 revealed intrapulpal temperature increases of approximately 2°C at a dentin thickness of 2 mm during irradiation. No consistent effect of Q-switched nanosecond pulsed Nd:YAG laser irradiation on microstructure and de- and remineralization of dentin was determined (p < 0.01). but SEM results showed some surface alterations on the irradiated samples.

The clinical objective in laser treatment of hemangiomas is to photocoagulate the dilated cutaneous blood vessels, while at the same time minimizing nonspecific thermal injury to the overlying epidermis. We present an in-vivo experimental procedure, using a chicken comb animal model, and an infrared feedback system to deliver repetitive cryogen spurts during continuous Nd:YAG laser irradiation. Gross and histologic observations are consistent with calculated thicknesses of protected and damaged tissues, and demonstrate the feasibility of inducing spatially selective photocoagulation when using cryogen spray cooling in conjunction with laser irradiation. Experimental observation of epidermal protection in the chicken comb model suggests selective photocoagulation of subsurface targeted blood vessels for successful treatment of hemangiomas can be achieved by repetitive applications of a cryogen spurt during continuous Nd:YAG laser irradiation.