Two-photon excitation in living cells induced by low-power cw laser beams

We demonstrate multi-photon excitation in optically-trapped living cells. Intracellular non-resonant two-photon excitation of endogenous and exogenous chromophores was induced by CW near infrared (NIR) trapping beams of 105 mW power. In the case of fluorescent chromophores, detection of NIR-excited visible fluorescence was achieved by imaging and spectroscopy methods. Trap-induced, two-photon excited fluorescence was employed as a novel diagnostic method to monitor intracellular redox state and cell vitality of single motile spermatozoa and Chinese hamster ovary cells. We found, that nonlinear absorption of NIR photons <800 nm may lead to oxidative stress and severe cell damage. Biological response was amplified in multimode CW lasers due to longitudinal mode-beating and partial mode- locking. As a result, we recommend the use of longwavelength-NIR, single-frequency traps ("optical tweezers") for micromanipulation of vital cells.