Confocal Fluorescence Lifetime Imaging Microscopy (FLIM) at the single molecule level

We report on confocal fluorescence lifetime imaging microscopy (CFLIM) of single dye molecules adsorbed on glass surface. Applying a short‐pulse diode laser emitting at 635 nm with a repetition rate of 64 MHz we studied the time‐resolved identification of individual carbocyanine and oxazine dyes via their characteristic fluorescence lifetimes of 2.06±0.37 ns (Cy5) and 3.89±0.91 ns (JA242). Fluctuations in fluorescence intensity and lifetime of individual adsorbed molecules were investigated with millisecond time resolution. These jumps exhibit short off‐states τ_off of 0.5 ms which can be ascribed to the triplet state lifetime under dry conditions with an intersystem crossing yield Y_ISC of ∼0.2 %. Besides triplet states, other quantum jumps into longer lived states (several milliseconds) with lower transition probability were observed. The correlation of rotational and spectral jumps of single and coupled fluorophores with changes in the observed fluorescence lifetime are discussed.