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Raman spectroscopy is a powerful tool to generate a characteristic signature of specific tissues and operates by detecting energy with the molecular bond vibration of incident photons. The process results in non-elastically scattered light, also known as Raman scattering [137]. Raman spectroscopy is capable of discerning molecular pathology of differential proliferative middle ear lesions and may help in the assessment of borders of the pathological process to improve the surgical outcome of the middle ear diseases [7, 138–141]. CARS occurs when a target molecule is irradiated using 2 laser beams simultaneously at different frequencies, a pump beam and a Stokes beam. When the difference between the higher frequency (pump beam) and the lower frequency (Stokes beam) equals the vibrational frequency of the target bond of the molecule, a CARS signal is generated [139–141]. Zou et al. [7] has recently demonstrated the feasibility of using CARS microscopy to display the specific molecular morphology of cholesteatoma that has the potential to be integrated in a novel endoscope for cholesteatoma imaging in the clinic. There are reports on developing CARS endoscopes, although the system needs further improvement [142, 143].