A simple but precise method for quantitative measurement of the quality of the laser focus in a scanning optical microscope

We report a method for characterizing the focussing laser beam exiting the objective in a laser scanning microscope. This method provides the size of the optical focus, the divergence of the beam, the ellipticity and the astigmatism. We use a microscopic-scale knife-edge in the form of a simple transmission electron microscopy (TEM) 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, a beam profile in the form of an error function can be obtained and by repeating this with the knife-edge at different axial positions relative to the beam waist, the divergence and astigmatism of the post-objective laser beam can be obtained. The measured divergence can be used to quantify how much of the full numerical aperture of the lens is used in practice. We present data of the beam radius, beam divergence, ellipticity, and astigmatism obtained with low (0.15, 0.7) and high (1.3) numerical aperture lenses and lasers commonly used in confocal and multi-photon laser scanning microscopy. Our knife-edge method has several advantages over alternative knife-edge methods used in microscopy including that the knifeedge is easy to prepare, that the beam can be characterized also directly under a coverslip, as necessary to reduce spherical aberrations for objectives designed to be used with a coverslip, and it is suitable for use with commercial laser scanning microscopes where access to the laser beam can be limited.