Epi‐illumination dark‐field microscopy enables direct visualization of unlabeled small organisms with high spatial and temporal resolution.

Dark‐field microscopy is known to offer both high resolution and direct visualization of thin samples. However, its performance and optimization on thick samples is under‐explored and so far, only meso‐scale information from whole organisms has been demonstrated. In this work, we carefully investigate the difference between trans‐ and epi‐illumination configurations. Our findings suggest that the epi‐illumination configuration is superior in both contrast and fidelity compared to trans‐illumination, while having the added advantage of experimental simplicity and an "open top" for experimental intervention. Guided by the theoretical analysis, we constructed an epi‐illumination dark‐field microscope with measured lateral and axial resolutions of 260 nm and 520 nm, respectively. Subcellular structures in whole organisms were directly visualized without the need for image reconstruction, and further confirmed via simultaneous fluorescence imaging. With an imaging speed of 20 to 50 fps, we visualize fast dynamic processes such as cell division and pharyngeal pumping in Caenorhabditis elegans. [ABSTRACT FROM AUTHOR]