Novel autofocus technique advances high-resolution, time-lapse imaging

A team of researchers from University of North Varolina at Chapel Hill and Burnham Institute for Medical Research Inc., San Diego, California, utilized new autofocus technique for high-resolution oil-immersion microscopy using transmitted light differential interference contrast (DIC) imaging. DIC imaging was utilized to circumvent miniscus distorting phase contrast in microtiter wells and avoid losing any fluorescence emission light resulting from the objective phase ring. Selection of the most appropriate spatial frequency band involved excluding the lower frequencies that increases the sharpness of the focus measurement. A 12-bit monochrome scientific-grade digital Photometrics CCD camera interfaced with the computer through a PCI card captured images for autofocus. Tests on 225 fixed-cell samples revealed a precision of 10.3 nm, confirming that the autofocus precision is independent of thickness.