Total internal reflection fluorescence microscopy imaging-guided confocal single-molecule fluorescence spectroscopy.

We have developed an integrated spectroscopy system combining total internal reflection fluorescence microscopy imaging with confocal single-molecule fluorescence spectroscopy for two-dimensional interfaces. This spectroscopy approach is capable of both multiple molecules simultaneously sampling and in situ confocal fluorescence dynamics analyses of individual molecules of interest. We have demonstrated the calibration with fluorescent microspheres, and carried out single-molecule spectroscopy measurements. This integrated single-molecule spectroscopy is powerful in studies of single molecule dynamics at interfaces of biological and chemical systems.