Bjf. Wong et al., Two-photon excitation laser scanning microscopy of human, porcine, and rabbit nasal septal cartilage, TISSUE ENG, 7(5), 2001, pp. 599-606
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.