We report the observation of two-photon fluorescence excitation in a continuous-wave (cw) 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 for 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 by the trapped cells. This spectral technique should prove extremely useful for monitoring cellular activity and the effects of confinement by optical tweezers.
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