Two-photon excitation laser scanning microscopy of human, porcine, and rabbit nasal septal cartilage

Two-photon excitation laser scanning microscopy (TPM) was used to image hum an, porcine, and rabbit nasal septal cartilage. TPM provides optical sectio ns of thick tissue specimens in situ without the use of exogenous dyes or n eed for tissue fixation. The cartilage tissue was imaged using near-infrare d 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 sti mulated fluorescence was detected with photomultiplier tubes optimized to s pecific spectral bandwidths. Signal intensity corresponds to the concentrat ion of fluorophores, principally NADH, NADPH, and flavoproteins hence provi ding a means of redox imaging the cellular metabolic state. Specimens were scanned from the surface to a depth of about 150 mum. Image size was 50 x 5 0 mum with a diffraction limited pixel size of 0.4 mum. Cell membranes, nuc lei, and matrix structures were identified in human, pig, and rabbit tissue s. TPM provides a means to study three dimensional chondrocyte structure an d matrix organization in situ at substantial depths, and permits longitudin al examination of cultured tissue explants without the need for exogenous d yes, tissue preparation, or fixation.