Non-mechanical refocusing in brightfield microscopy via amplified longitudinal chromatic aberration and wavelength tuning

A simple, cost-effective system is presented for non-mechanical axial refocusing in conventional brightfield microscopy. The standard achromatic tube lens is replaced with a singlet lens of short focal length ( f = 100 mm) and a standard plan achromat objective (10 × /0.25 NA), deliberately amplifying the system’s longitudinal chromatic aberration. Axial refocusing is achieved by tuning the wavelength of a liquid crystal tunable filter (LCTF) in white-light illumination, enabling virtual z-scanning without any mechanical movement. Precise experimental calibration of the spectral response and axial displacement curve is performed, and the method is demonstrated on a resolution target and a three-dimensional lens tissue sample under brightfield illumination. This minimal-modification technique provides vibration-free, rapid axial exploration of unstained three-dimensional microscopic samples.