Characterization of microscopic laser beams by 2-D scanning of fluorescence emission

We describe a fast, accurate, and reproducible method for calibrating microscopic laser beams. Fundamental mode (TEM(00)) output from an argon-ion laser is focused on a thin (<0.5-microm) aqueous fluorescein solution located at the sample plane of a fluorescence microscope. The fluorescent image of the focused beam is scanned in two dimensions at the emission diaphragm of the microscope by a computer-controlled X-Y scanning mirror. Plots of fluorescence intensity vs position demonstrate that the beam profile is Gaussian in two dimensions. Both the 1/e(2) beam radius and the coordinates of peak intensity are obtained by nonlinear least-squares analysis. An adaptation of this method is used to characterize microscopic elliptical Gaussian beams. We present experimental and theoretical evidence, the latter based on the optical transfer function of an objective lens, that Gaussian beams used in fluorescence photobleaching recovery experiments are not significantly distorted by diffraction or defocusing effects.