(Brasselet and Moerner, Single Molecules 1, 15 (2000))

The fluorescence behavior of a well-known pH-reporter molecule diluted in an aqueous gel has been measured at the single-molecule level. Single copies of the pH-sensitive seminaphthorhodafluor SNARF-1-dextran were immobilized in the pores of agarose gels at different pH in the range 6-9. This probe shows separate emission wavelengths for its protonated and deprotonated forms, such that the ratio between these two emission intensities is a signature of the pH of the surrounding medium. Two-color confocal detection of the time trajectories of single chromophore emission signals allowed characterization of the behavior, molecule by molecule (see Figure). In particular the distribution of the ratio between the two emission wavelengths, measured over a large number of individual molecules, shows a broadening at pH close to pKa, which cannot be explained by the experimental noise. The observed range of average ratios appears to arise from local inhomogeneities in the environment of the fluorophore which alter the response to local pH.

Figure: Two-color confocal images of single copies of SNARF-1 dextran. The images (emission 580nm left, and 640nm right) were obtained by scanning a 10m mX10m m area at 10m m/s, resolution 100 pixels. The integration time is 10ms per pixel, and 1V signal on the scale corresponds to 100 photon counts. The images have not been corrected by the detection correction factor.