Digital differential interference contrast autofocus for high-resolution oil-immersion microscopy

Continued advances in cellular fluorescent biosensors enable studying intracellular protein dynamics in individual, living cells. Autofocus is valuable in such studies to compensate for temperature drift, uneven substrate over multiple fields of view, and cell growth during long-term high-resolution time-lapse studies of hours to days. Observing cellular dynamics with the highest possible resolution and sensitivity motivates the use of high numerical aperture (NA) oil-immersion objectives, and control of fluorescence exposure to minimize phototoxicity. To limit phototoxicity, to maximize light throughput of the objective for biosensor studies, and because phase contrast is distorted by the meniscus in microtiter plates, we studied autofocus in differential interference contrast (DIC) microscopy with a 60X 1.45 NA oil objective after removing the analyzer from the fluorescent light path. Based on a study of the experimental DIC modulation transfer function, we designed a new bandpass digital filter for measuring image sharpness. Repeated tests of DIC autofocus with this digital filter on 225 fields-of-view resulted in a precision of 8.6 nm (standard deviation). Autofocus trials on specimens with thicknesses from 9.47 to 33.20 μm, controlled by cell plating density, showed that autofocus precision was independent of specimen thickness. The results demonstrated that the selected spatial frequencies enabled very high-precision auto-focus for high NA DIC automated microscopy, thereby potentially removing the problems of meniscus distortion in phase contrast imaging of microtiter plates and rendering the toxicity of additional fluorescence exposure unnecessary.