One- and two-photon induced fluorescence

For DNA sequencing. One common method of sequencing DNA is to have a free-floating short strand of DNA with a known sequence of bases— called an oligonucleotide—find its complementary sequence on the full strand. If the oligonucleotide also has a fluorescent dye attached, researchers can then easily locate that sequence in the main DNA. As many as four dyed oligonucleotides have been used at once to speed the sequencing, but the dyes’ broad emission peaks can overlap and confuse identification of the short strands. Now, Chieu Tran and Troy Alexander at Marquette University in Milwaukee have come up with a novel approach: They tagged two oligonucleotides with special dyes that absorb the same wavelength of light very differently. One of the dyes conventionally absorbs one photon and emits another slightly redshifted one. The other dye is activated only when two photons are absorbed at once, a low-probability event; it then emits a greatly blue-shifted photon. Eschewing the high-intensity pulsed laser that is generally needed for two-photon fluorescence, the researchers showed that a continuous-wave laser and an acousto-optic tunable filter can do the job. Using a single wavelength of exciting light, they successfully detected the fluorescence, separated by 200 nm, of two different oligonucleotides in a single sample. Tran expects to develop another similar pair of dyes to increase the multiplexing capability of their scheme. (T. Alexander, C. D. Tran, Appl. Opt. 41 , 2285, 2002 http://dx.doi.org/10.1364/AO.41.002285 .)