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

Philip Tinnefeld, Volker Buschmann, Dirk-Peter Herten, Kyung-Tae Han, Markus Sauer

Research output: Contribution to journalArticlepeer-review

65 Citations (Scopus)


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 τoff of 0.5 ms which can be ascribed to the triplet state lifetime under dry conditions with an intersystem crossing yield YISC 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.
Original languageEnglish
Pages (from-to)215-223
Number of pages9
JournalSingle Molecules
Issue number3
Publication statusPublished - 28 Sept 2000


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