Fig. 3

Spectral unmixing of D-labeled lipids, proteins, and DNA. a Separation of CD protein and DNA signals via unmixing in dividing cells (see Methods for details). Dashed outlines enclose the nuclei. In all the figures, CD_L, CD_P, CD_DNA, CH_L, CH_P, and CH_DNA channels show signals collected at 2135, 2185, 2210, 2846, 2940, and 2973 cm⁻¹, respectively, and are color-coded in green, red, pink, yellow, blue, and gold, respectively. b SRS microscopic images, collected from the CD_L and CD_P channels, of COS-7 cells grown in 70% D₂O DMEM for 24 h and images of the same cells after methanol wash, with or without the application of unmixing algorithm. c, d Quantification of the mean SRS microscopic intensity (mean ± s.d.) from CD_L and CD_P channels of COS-7 cells under various conditions before and after unmixing (N = 12 for each condition). Double asterisk indicates p < 0.01 in an unpaired t test. e–g Example sets of images before and after the application of CD_L/CD_P unmixing for COS-7 cells grown in 70% D₂O-containing DMEM for 24 h (e), xenograft colon tumor tissues from mice drinking 25% D₂O for 15 days (f), and sebaceous gland tissues from mice drinking 25% D₂O for 3 days (g). CH_L/CH_P unmixing was performed according to previous studies²². Scale bar = 20 μm. Tissue-specific unmixing parameters can be found in Methods