Two-Photon Excitation Laser Scanning Microscopy of Human, Porcine, and Rabbit Nasal Septal Cartilage

Abstract

Two-photon excitation laser scanning microscopy (TPM) 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 mm. Image size was 50 3 50 mm with a diffraction limited pixel size of 0.4 mm. 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.

Quantitative second harmonic generation imaging of cartilage damage

“…Both natural and tissue-engineered cartilage have previously been studied using conventional histology and biochemical assays, as well as novel visualization techniques including magnetic resonance imaging (MRI) (Gray et al 2008), contrast-enhanced microcomputed tomography (lCT) (Palmer et al 2006), Fourier transform infrared imaging spectroscopy (FT-IRIS) (Bi et al 2007), and multiphoton microscopy (Wong et al 2001;Yeh et al 2005;Mansfield et al 2006;Lee et al 2006). Histology and biochemical assays have been traditionally used to either monitor the spatial distribution of ECM or to quantify the amount and type of matrix macromolecules in cartilage; however, those methods are rather timeconsuming and subject to distortion artifacts and tissue damage.…”

Epithelial-derived TGF-β2 modulates basal and wound-healing subepithelial matrix homeostasis

“…The density of collagen fibers was studied in the extracellular matrix of the fibroblast tissue model using multiphoton microscopy (MPM) as previously described (1,57). Briefly, collagen-embedded NHLF matrices were lifted onto microscope slides and covered with full-length coverslips, thinness 1 (Fisher Scientific, Pittsburgh, PA), and excited with a 100-fs Titanium: Sapphire laser as the multiphoton excitation source.…”

Morphometric characterization of murine articular cartilage—Novel application of confocal laser scanning microscopy

“…Pyridinoline is one such compound, as well as aromatic groups in the proteins and nucleic acids, such as NADH (reduced form of nicotinamide adenine dinucleotide) NADPH (reduced form of nicotinamide adenine dinucleotide phosphate), and flavoproteins (Wong et al, 2001). A complete understanding of the exact sources and nature of fluorophores in cartilage is not fully established (Tadrous et al, 2003), making it difficult to quantify fluorescence using suggested standards, such as the molecules of equivalent soluble fluorochrome (MESF; Schwartz et al, 2004).…”