A simple technique for measuring the M2 parameter and the optical focus of the light exiting the objective lens in a laser scanning microscope

We report a method for measuring the optical quality of the post-objective laser radiation in a laser scanning microscope. We use a microscopic-scale knife-edge in the form of a simple transmission electron microscopy grid attached to a glass microscope slide and a light-collecting optical fibre and photodiode underneath the specimen. By scanning the laser spot from a reflective to a transmitting part of the grid, an error function measuring the beam can be obtained and by repeating this with the knife-edge at different axial positions relative to the beam waist, the M2 parameter of the post-objective laser beam can be obtained, together with measurements of the size of the optical focus, the ellipticity and the astigmatism of the laser beam exiting the objective lens. This measurement is useful not only for measuring the size of the optical focus, but also for evaluating the beam divergence and thus evaluating how much of the full numerical aperture of the lens is used in practice, which impacts the imaging quality in confocal and multi-photon microscopy. We will present details of the measurement technique, as well as data of beam divergence, ellipticity, astigmatism and M2 parameters obtained with very high (1.3) and low (0.15, 0.7) numerical aperture lenses and lasers commonly used in confocal and multi-photon laser scanning microscop