Abstract

Two-photon excitation laser scanning microscopy was used to image human, porcine, and rabbit nasal septal cartilage. TPM provides optical sections of thick tissue specimens in situ without the use of exogenous dyes or need for tissue fixation. The cartilage tissue was imaged using near-infrared light generated by a mode-locked titanium/sapphire laser that was raster-scanned and coupled to an inverted microscope. Absorption of two photons by endogenous molecules and subsequent fluorescence was filtered to specific spectral bandwidths and detected with photomultiplier tubes. Two-photon stimulated fluorescence was detected with photomultiplier tubes optimized to specific spectral bandwidths. Signal intensity corresponds to the concentration of fluorophores, principally NADH, NADPH, and flavoproteins hence providing a means of redox imaging the cellular metabolic state. Specimens were scanned from the surface to a depth of about 150 μm. Image size was 50 × 50 μm with a diffraction limited pixel size of 0.4 μm. Cell membranes, nuclei, and matrix structures were identified in human, pig, and rabbit tissues. TPM provides a means to study three dimensional chondrocyte structure and matrix organization in situ at substantial depths, and permits longitudinal examination of cultured tissue explants without the need for exogenous dyes, tissue preparation, or fixation.