Figure 3.
Multiplex SRS Imaging Cytometry Reveals the Heterogeneity of LDs in Single Cells
(A) A scatterplot of LDs outside the ER (red) and inside the ER (blue) boundaries in the phasor space.
(B) Averaged SRS spectra of LDs outside the ER (red) and inside the ER (blue) boundaries in the C-H region. Shaded area indicates the standard deviation of SRS spectral measurements from different LDs. SRS spectra of LDs outside the ER (red) and inside the ER are statistically different at 2,870 cm−1. ∗p < 0.05.
(C) Spontaneous Raman spectra of cholesteryl ester (red) and triglyceride (blue) in the C-H region.
(D) An SRS image of G3K cells.
(E) The molecular composition map of LDs in G3K cells by 2,900 cm−1/2,870 cm−1 SRS ratio image. Lower values indicate more cholesteryl ester contents, and higher values indicate more triglyceride contents. Scale bar, 100 μm.
(F and G) A zoom-in SRS image (F), and a zoom-in composition map (G) of the dashed rectangular region in (D) and (E), respectively. Red arrows indicate the directions of LDs away from the cell center.
(H–J) 2D scatterplots of two features from LDs outside the ER (red) and LDs inside the ER (blue) boundaries. The “distance in phasor domain” reflects “lipid compositions” of the LDs. The higher value of “distance in phasor domain” indicates more cholesteryl ester contents. The lower value of “distance in phasor domain” indicates more triglyceride contents. The features are (H) “lipid composition” versus “the distance of LD to the cell center,” (I) “lipid composition” versus “LD mean intensity,” and (J) “distance of LD to the cell center” versus “LD mean intensity.”
(K) A transmission image of G3K cells. Arrows indicate LDs analyzed by Raman spectroscopy.
(L) Spontaneous Raman spectra of selected LDs in (K). The gray region highlights the cholesteryl ester signature peak around 704 cm−1.