Abstract
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.
Highlights
Focusing a continuous-wave laser beam to its diffraction limit with a high-numerical-aperture objective lens can produce a large gradient in the beam intensity profile, whereby gradient forces dominate over scattering forces and an optical laser trap is created
In this Letter we report the observation of twophoton-excited f luorescence from exogenous probes in cw infrared optical tweezers
At the wavelength of 1064 nm, a single focused Gaussian laser beam is used simultaneously to conf ine, and excite visible f luorescence from, a human sperm cell that has been tagged with Propidium Iodide, a f luorescent dye that functions as an assay of cellular physiological state
Summary
At the wavelength of 1064 nm, a single focused Gaussian laser beam is used simultaneously to conf ine, and excite visible f luorescence from, a human sperm cell that has been tagged with Propidium Iodide, a f luorescent dye that functions as an assay (live/dead monitor) of cellular physiological state. The intensity at the dye peak emission wavelength of 620 nm exhibits a nearsquare-law dependence on incident trapping-beam photon laser power, a behavior consistent with a two-photon absorption process.
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