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.