In this Letter we propose a fast off-axis hologram autofocusing (AF) approach that is based on the redundant data elimination by the critical resampling of the contained complex field. Implementation of the proposed methodology enables the real-time AF with up to 12x speed-up factors in comparison to the classical approach. The method is further extended for single-shot physical autofocus of the fluorescence imaging channel of multimodal imaging instruments capable of off-axis hologram acquisition. (C) 2012 Optical Society of America