Extended Data Fig. 6. Observation of multiple single-molecule isotopologues within the same diffraction-limited spot.

a, Colour-coded fluorescence imaging for a mixture of single-molecule BF1 isotopologues as shown in Fig. 4i. Red: BF1-¹³C ≡ ¹⁴N (2170 cm⁻¹); green: BF1-¹²C ≡ ¹⁵N (2195 cm⁻¹); and blue: BF1-¹²C ≡ ¹⁴N (2220 cm⁻¹). b, In-situ BonFIRE spectra obtained from three spots indicated by the corresponding numbered arrowheads in (a). Gaussian fittings (and a linear background) are shown as solid curves. Spectra of spots 1 (orange), 2 (grey), and 3 (blue) contain all three vibrational peaks. Slight variations in peak positions are possibly due to local interactions41. c.p.ms, counts per millisecond. These results demonstrate BonFIRE can resolve multiple vibrational colours within the same diffraction-limited spot at the single-molecule level.