2-PHOTON FLUORESCENCE EXCITATION IN CONTINUOUS-WAVE INFRARED OPTICAL TWEEZERS

We report the observation of two-photon fluorescence excitation in a continuous-wave (ew) single-beam gradient force optical trap and demonstrate its use as an in situ probe to study the physiological state of an optically confined sample. In particular, a cw Nd:YAG (1064-nm) laser is used simultaneously to confine, and excite visible fluorescence from submicrometer regions of, cell specimens. Two-photon fluorescence emission spectra are presented far motile human sperm cells and immotile Chinese hamster ovary cells that have been labeled with nucleic acid (Propidium Iodide) and pH-sensitive (Snarf) fluorescent probes. The resulting spectra are correlated to Light-induced changes in the physiological state experienced try the trapped cells. This spectral technique should prove extremely useful for monitoring cellular activity and the effects of confinement by optical tweezers. (C) 1995 Optical Society of America