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Quantitative evaluation of LED based optical autofocus module

Habte, S.; Kumar, S.; Lightley, J.; Garcia, E.; Neil, M.; French, P. M.

2026-04-14 bioengineering
10.64898/2026.04.10.717415 bioRxiv
Show abstract

We report an improved version of the open-source optical autofocus module ("openAF") for light microscopy using a light emitting diode (LED), together with a method to independently quantify the performance of optical autofocus systems using 2D autocorrelation analysis of astigmatic imaging of fluorescent nanobeads. We apply the latter for both the LED-based and the previous super luminescent diode (SLD) based implementations of the openAF optical autofocus approach used in conjunction with a 100x 1.4 NA oil-immersion objective lens. The new approach accounts for power variations in the autofocus light source and we demonstrate that the convenient LED-based system can provide axial stability with a standard deviation <10 nm over at least 45 minutes when switched on from cold, during which the LED power varies as it reaches thermal equilibrium.

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