Autofocus for high speed scanning in image cytometry

A high-speed image collection and autofocus system has been developed for automated microscopic scanning of biological specimens. This system tracks focus and keeps the image sharply focused during continuous stage motion. A simultaneous multiplanar image acquisition, based on a dedicated volume-imaging camera, is connected to focus measurement circuits for parallel focus processing. By adjusting focus dynamically while a biological specimen is scanned, on-the-fly focusing is achieved. Focus measurement circuits were designed and implemented with analog circuitry for use in incremental and continuous image scanning. A volume camera was designed using time-delay-and-integrate (TDI) CCD sensors coupled to imaging fiber bundles. The volume camera samples the image volume produced by the microscope and is also synchronized to the constant-velocity stage movement. The parallel autofocus circuits tremendously reduced the computational load required for maintaining focus from the multi-image data stream; allowing high-speed high-resolution fluorescence image acquisition. The hardware development, the strategies for tracking focus, and the software engine that integrates the hardware elements into a real-time autofocus are described. The performance measurements and the new challenges presented by continuous autofocus-and-scanning for automated fluorescence image analysis are described.