Fig. 3: Evidence for single-particle sensitivity of Rdots under SRS microscopy.

a Linear concentration dependence of Rdots with sub-nM SRS detection sensitivity. Red dashed line in the insert indicates shot-noise-limited SRS detection limit where SNR = 1. Error bars, mean ± s.d.; n = 3 measurements. Solid line shows a linear fitting (R² = 0.999), shaded area indicates 95% confidence interval of the linear fitting. arb. units: arbitrary units. b A representative image of potential single-particle spots. Scale bar: 1 µm. c Raman spectra of ‘single’ Rdots2220 acquired by SRS microscopy by sweeping the pump wavelength. The original SRS images below at three marked pump wavelengths show the signal can be tuned off by only 1.5 nm. The spectra are in consistence with bulk measurements of Rdots by spontaneous Raman spectroscopy. Scale bar, 1 µm. arb. units: arbitrary units. d SRS intensity distribution of individual spots at pH 8.5. Red dash lines indicate fitted peak positions. The quantized distribution suggests spots corresponding to single-particle and double-particle aggregates. n = 289 from 7 replicates. e SRS intensity distribution of individual spots at pH 6.0. Red dash lines indicate fitted peak positions. The quantized distribution suggests spots corresponding to single-particles, double-particle aggregates, and triple-particle aggregates. n = 595 from 6 replicates. arb. units: arbitrary units. f A representative image of spots from single-particles and multi-particle aggregates. 1, 1’, and 1”: single-particle; 2: double-particle; 3: triple-particle; 4: quadruple-particle. Scale bar: 1 μm. g Line profiles across spots in f. Quantized signal intensity is evidently observed. arb. units: arbitrary units.