Quantitative evaluation of LED-based optical autofocus module

We report an improved version of the open-source optical autofocus (OAF) module (openAF) for light microscopy, replacing the superluminescent diode (SLD) with a multimode-optical fibre-coupled LED as a more accessible AF module light source. We also present a method for independently quantifying AF system performance using 2D autocorrelation analysis of astigmatic fluorescent nanobead images. We applied this method to evaluate both this new LED-based openAF module and our previous SLD-based module implemented on a microscope with a 100 × 1.4 numerical aperture oil-immersion objective lens. Our new approach compensates for AF light source power variation, and we demonstrate that the LED-based system can provide axial stability with a standard deviation below 10 nm for at least 45 min from a cold start as the LED power varies, whereas it reaches thermal equilibrium.