Laser tweezers are sources of two-photon excitation

Abstract

The most important application of continuous wave (cw) near infrared (NIR) microbeams in cellular and molecular biology are single-beam gradient force optical traps, also called "laser tweezers". Laser tweezers have been used for optical picoNewton force determination as well as for 3D cellular and intracellular micromanipulation, such as optical spermatozoa transportation in laser-assisted in vitro fertilization. Light intensities in the MW/cm2 range are necessary to confine motile spermatozoa in the optical trap. The enormous photon concentration in space and time results in non-resonant two-photon excitation of endogenous and exogenous absorbers with electronic transitions in the ultraviolet and visible spectral range. Trap-induced two-photon excitation of intracellular flurorophores can be used to study metabolism and vitality of motile cells without additional fluorescence excitation sources. Therefore, laser tweezers as sources of two-photon excitation may act as novel non-linear tools in cell diagnostics. The far red/NIR trapping radiation, in particular <800 nm, may also excite endogenous absorbers such as NAD(P)H, flavins, porphyrins and cytochromes. Excitation of these cellular absorbers may result in oxidative stress via type I and type II photooxidation processes. Severe non-linear-induced cell damage in a variety of cells confined in <800 nm traps was found. Two-photon induced destructive effects are enhanced in multimode traps due to longitudinal mode-beating phenomena. Pulsed laser sources are not suitable for safe optical trapping of living cells. The use of single frequency long-wavelength NIR traps (800 nm-1200 nm) for vital cell handling is recommended.