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We analyzed the effects of a hypo-osmotic environment on rat Schwann cells, a type of glial cell surrounding neurons, using ultra-broadband multiplex coherent anti-Stokes Raman scattering (CARS) microscopy. After hypo-osmotic treatment, we detected vacuole-like components in the cytoplasm using both bright-field and CARS spectroscopic imaging. An approach integrating both morphological examination and a spectroscopic analysis based on multiple vibrational bands revealed that these structures are predominantly water-filled, and their characteristics closely resembled those of the vacuoles observed in plant cells.