Fig. 4: Immunostaining and imaging by Rdots with correlative fluorescence and SRS microscopy.

a–b Non-specific binding of Rdots. Imaging of C–H stretching mode (a) confirms the existence of the cell. No significant non-specific binding of Rdots2220 is observed (b). Scale bar: 20 µm. c–d Immunostaining and imaging of α-tubulin in fixed HeLa cells with Rdots2220 (c). The off-resonance image at 3 nm away shows negligible background (d). The brightness of c and d is set to be the same. Scale bar: 20 µm. e–g Correlative fluorescence and SRS immunostaining and imaging of α-tubulin in fixed Cos-7 cells with Rdots2220. e Fluorescence channel, f SRS channel, and g composite view of the two channels. Scale bar: 20 μm. h–j Correlative fluorescence and SRS immunostaining and imaging of vimentin in fixed Cos-7 cells with Rdots2177. h Fluorescence channel, i SRS channel, and j composite view of the two channels. Scale bar: 20 μm. k–m Magnified view of e–g. n–p Magnified view of h–j. q Colocalization analysis indicates good correlation between immunofluorescence and Rdots staining for α-tubulin (Pearson’s r = 0.88). r Line profiles of the fluorescence channel (blue) and SRS channel (red) of microtubule filament shown in m. arb. units: arbitrary units. s Colocalization analysis indicates good correlation between immunofluorescence and Rdots staining for vimentin (Pearson’s r = 0.82). t Line profiles of the fluorescence channel (blue) and SRS channel (red) of intermediate filament shown in p. arb. units: arbitrary units. u Immunofluorescence imaging of e-cadherin in the mouse frozen tissue section. v Immunostaining and imaging of e-cadherin with Rdots2220 in the mouse frozen tissue section. Scale bar: 400 μm. w Magnified view of u, the signals are only observed on the epithelial cell membrane. Scale bar: 50 μm.