Video-rate remote refocusing through continuous oscillation of a membrane deformable mirror

This paper presents the use of a deformable mirror (DM) configured to rapidly refocus a microscope employing a high numerical aperture (NA) objective lens. An Alpao DM97-15 membrane DM was used to refocus a 40x/0.80 NA water-immersion objective through a defocus range of -50-50 mu m at 26.3 sweeps s(-1). We achieved imaging with a mean Strehl metric of >0.6 over a field of view in the sample of 200 x 200 mu m(2) over a defocus range of 77 mu m. We describe an optimisation procedure where the mirror is swept continuously in order to avoid known problems of hysteresis associated with the membrane DM employed. This work demonstrates that a DM-based refocusing system could in the future be used in light-sheet fluorescence microscopes to achieve video-rate volumetric imaging.