A simple method for checking the illumination profile in a laser scanning microscope and the dependence of resolution on this profile

One of the conditions for a laser scanning microscope to reach its optimal performance is for it to operate at its full numerical aperture (NA). In most commonly used systems, the illumination intensity at the back focal plane of the objective lens is apodized. This paper presents a simple method using a photodiode for checking the actual illumination intensity profile. We show as an example the measured profiles of a laser beam when working with two high-NA immersion objectives in two different confocal systems, and also show that in theoretical studies of the point-spread function, the assumption of a flat compared with a truncated Gaussian beam profile gives rise to severe discrepancies. The measured profiles also serve as an indication of the necessity of a realignment of the optical system.