Simultaneous multiplanar image acquisition in light microscopy

There has been great interest in automated image cytometry because of the increased information afforded by a native 2D data set (as opposed to 1D in flow cytometry). High resolution scanning cytometry, however, has been severely hampered by cell analysis rates three orders of magnitude slower than that of flow cytometry. Thus far, a two-pass strategy of scanning at very low resolution followed by high resolution imaging of a few selected fields has been most commonly employed to avoid this problem and achieve higher rates of analysis. Fast scanning can be performed at high resolution using a time-delay-and-integrate (TDI) CCD camera synchronized to constant-velocity stage movement, but images collected by this method are degraded by inability to track focus during continuous stage movement. Here we introduce simultaneous multiplanar image acquisition and focus measurement for tracking focus. This volume-scanning, autofocus system combines multiplanar fiberoptic image coupling, an array of TDI CCD cameras, and an array of focus measurement circuits. This design will also enable future high speed 3D scanning cytometry applications.