Figure 2

(A) Brightfield image focused on non-treated OvCa429 cells on the surface of the multicellular aggregate is in good agreement with (B) CARS image of −2930 cm⁻¹ peak area. The comparison of (C) the maximum intensity CARS spectrum and average spectrum of the map show similar spectral structure across the cell surface of the sample. The spontaneous Raman spectrum obtained from OvCa429 cells is plotted over the equivalent Raman shifts for comparison (See Fig. S3 for more details). (D) The CARS intensity (black) is fit (orange) to Eq. 1 to ascertain the nonresonant background (green) and two resonant contributions from the CH₂ and CH₃ vibrational bands at −2850 cm⁻¹ (red) and −2930 cm⁻¹ (blue), respectively. (E) The frequency scatter plot distinguishes two spectral clusters with 95% confidence ellipses indicative of lipids (red) and proteins (blue). The spectral separation is described by the separation angle, φ, from the ratios of CH₂/CH₃ signal. (F) The reconstructed map shows the chemical distribution of the segmented lipid (red) and protein (blue) pixels. Experimental parameters: Pump = 720 nm, 26.50 mW; Supercontinuum = 785–945 nm, 3.70 mW; Acquisition time = 500 ms/pixel; Map size = 51 × 51 pixels; Steps = 300 nm; Objective = 100x, 0.9 NA.