Two-photon excitation laser scanning microscopy of porcine nasal septal cartilage following Nd: YAG laser mediated stress relaxation

Laser irradiation of hyaline cartilage result in stable shape changes due to temperature dependent stress relaxation. In this study, we determined the structural changes in chondrocytes within porcine nasal septal cartilage tissue over a 4-day period using a two-photon laser scanning microscope (TPM) following Nd:YAG laser irradiation (λ = 1.32 gm) using parameters that result in mechanical stress relaxation. (6.0 W, 5.4 mm spot diameter). TPM excitation (780 nm) result in induction of fluorescence from endogenous agents such as NADH, NADPH, and flavoproteins in the 400-500 nm spectral region. During laser irradiation diffuse reflectance (from a probe HeNe laser, λ = 632.8 nm), surface temperature, and stress relaxation were measured dynamically. Each specimen received one, two, or three sequential laser exposures (average irradiation times of 5, 6, and 8 seconds). The cartilage reached a peak surface temperature of about 70 °C during irradiation. Cartilage denatured in 50% EtOH (20 minutes) was used as a positive control. TPM was performed using a mode-locked 780 nm Titanium:Sapphire (Ti:Al203) beam with a, 63X, 1.2 N.A. water immersion objective (working distance of 200 mm) to detect the fluorescence emission from the chondrocytes. Images of chondrocytes were obtained at depths up to 150 microns (lateral resolution = 35 μm×35 μm). Images were obtained immediately following laser exposure, and also after 4 days in culture. In both cases, the irradiated and non-irradiated specimens do not show any discernible difference in general shape or autofluorescence. In contrast, positive controls (immersed in 50% ethanol), show markedly increased fluorescence relative to both the native and irradiated specimens, in the cytoplasmic region.