Fig. S2.
Fluorescence lifetime of GFP is unperturbed by the optical trap. (A) Time-correlated single-photon counting (TCSPC) was used to measure the fluorescence lifetime of a GFP-DNA construct tethered in the optical trapping assay (12 acquisitions, 3.5–10 s duration each). The time resolution was 150 ps and limited by the pulse duration of the excitation laser. The time axis includes an arbitrary offset (11 ns) due to the delay between the laser trigger pulse and photon measurement. The early time data (0–1.5 ns from the emission peak) are contaminated by fluorescence impurities from the glass sample chamber, which bleach rapidly but peak when the laser is first switched on. These points were excluded from all fits. (B) TCSPC measurement of the same construct free in solution (60 s acquisition time) is compared with averaged data from the optical trap. Measurement of the GFP-DNA construct in solution represents an average of many single molecules that diffuse within the confocal excitation spot. These data show less contamination from the glass sample chamber, because the measurement is sufficiently long compared with the time it takes for the fluorescence impurities in the glass to bleach. All data shown fit to a fluorescence lifetime of 2.46 0.03 ns.