Single-frame rapid autofocusing for brightfield and fluorescence whole slide imaging

A critical consideration for whole slide imaging (WSI) platform is to perform accurate autofocusing at high speed. Typical WSI systems acquire a z-stack of sample images and determine the best focal position by maximizing a figure of merit. This strategy, however, has suffered from several limitations, including low speed due to multiple image acquisitions, relatively low accuracy of focal plane estimation, short axial range for autofocusing, and difficulties in handling transparent samples. By exploring the autocorrelation property of the tissue sections, we report a novel single-frame autofocusing scheme to address the above challenges. In this approach, we place a two-pinhole-modulated camera at the epiillumination arm. The captured image contains two copies of the sample separated by a certain distance. By identifying this distance, we can recover the defocus distance of the sample over a long z-range without z-scanning. To handle transparent samples, we set an offset distance to the autofocusing camera for generating out-of-focus contrast in the captured image. The single-frame nature of our scheme allows autofocusing even when the stage is in continuous motion. We demonstrate the use of the our autofocusing scheme for fluorescence WSI and quantify the focusing performance on 1550 different tissue tiles. The average autofocusing error is ~0.11 depth-of-field, 3 folds better than that of conventional methods. We report an autofocusing speed of 0.037 s per tile, which is much faster than that of conventional methods. The autofocusing range is ~80 μm, 8 folds longer than that of conventional methods. The reported scheme is able to solve the autofocusing challenges in WSI systems and may find applications in high-throughput brightfield/fluorescence WSI.