Label-free visualization of cholesteatoma in the mastoid and tympanic membrane using CARS microscopy()

OBJECTIVE: The present study aimed to evaluate the possibility of using coherent anti-Stokes Raman spectroscopy (CARS) microscopy to determine the specific molecular morphology of cholesteatoma by detecting the natural vibrational contrast of the chemical bonds without any staining. MATERIALS AND METHODS: Specimens from the mastoid and tympanic membrane with and without cholesteatoma were analyzed using CARS microscopy, two-photon excited fluorescence (TPEF) microscopy, and the second harmonic generation (SHG) microscopy. RESULTS: In cholesteatoma tissues from the mastoid, a strong resonant signal at 2845 cm(−1) was observed by CARS, which indicated the detection of the CH(2) hydro-carbon lipid bonds that do not generate visible signals at 2940 cm(−1) suggestive of CH(3) bonds in amino acids. A strong resonant signal at 2940 cm(−1) appeared in an area of the same specimen, which also generated abundant signals by TPEF and SHG microscopy at 817 nm, which was suggestive of collagen. In the tympanic membrane specimen with cholesteatoma, a strong resonant signal with corrugated morphology was detected, which indicated the presence of lipids. A strong signal was detected in the tympanic membrane with chronic otitis media using TPEF/SHG at 817 nm, which indicated collagen enrichment. The CARS and TPEF/SHG images were in accordance with the histology results. CONCLUSION: These results suggest the need to develop a novel CARS microendoscope that can be used in combination with TPEF/SHG to distinguish cholesteatoma from inflammatory tissues.