Fig. 4

Single-molecule sensitivity in PESRS. a The ensemble PESRS measurements of pure ¹⁴NA, pure ¹⁵NA and their equimolar mixture. b Three representative single-pixel PESRS spectra showing a pure ¹⁵NA SM event (1), a mix event (2), and a pure ¹⁴NA SM event (3), obtained from the 50 nM mixture sample. The vertical dash lines indicate the position of 730 cm⁻¹. c Concentration matrix coefficients obtained from MCR analysis of the hyperspectral imaging result sample (including 40000 single-pixel spectra) of the mixture. d Histogram of relative contribution of ¹⁴NA from the hyperspectral imaging result of the mixture sample. Selected 796 single-pixel spectra with a desired Raman peak and above an intensity threshold were used. SM: single molecule. e–f Representative time traces of PESRS intensity collected of 50 nM adenine sample showing digital intensity fluctuation (e) and single-step photodamage (or photobleach) processes (f). The inside labels in e and f show the X–Y pixel coordinate where the spectra were recorded in Supplementary Movie 2.