Simultaneous measurement of one- and two-photon excited fluorescence from a single sample: a detection method for...

A new method has been developed that is based on the use of a single-excitation wavelength from a cw laser to excite simultaneously one-photon and two-photon fluorescence (TPF). Fluorescence bands of a sample containing two oligonucleotides, one labeled with a one-photon fluorescence dye and the other with a TPF dye, can be measured simultaneously. The two fluorescence bands are well separated, because the one-photon excited fluorescence band is redshifted, whereas the TPF band is blueshifted from the excitation wavelength. The spectral separation was found to be as large as 200 nm when ADS 840NCS was used to label one oligonucleotide for one-photon fluorescence and Rhodamine Red-X dye was used for TPF. Spectral overlapping problem that plagues current DNA sequencing techniques can be eliminated effectively with this method.