Recent developments in phase contrast microscopy have enabled the label‐free visualization of certain organelles due to their distinct morphological features, making this method an attractive alternative in the study of cellular dynamics. However tubular structures such as endoplasmic reticulum (ER) networks and complex dynamics such as the fusion and fission of mitochondria, due to their low phase contrast, still need fluorescent labeling to be adequately imaged. In this article, we report a quantitative phase microscope with ultra‐oblique illumination that enables us to see those structures and their dynamics with high contrast for the first time without labeling. The imaging capability was validated through comparison to the fluorescence images with the same field‐of‐view. The high image resolution (~270 nm) was validated using both beads and cellular structures. Furthermore, we were able to record the vibration of ER networks at a frame rate of 250 Hz. We additionally show complex cellular processes such as remodeling of the mitochondria networks through fusion and fission and vesicle transportation along the ER without labels. Our high spatial and temporal resolution allowed us to observe mitochondria “spinning”, which has not been reported before, further demonstrating the advantages of the proposed method.

Mitochondria are delicate organelles that play a key role in cell fate. Current research methods rely on fluorescence labeling that introduces stress due to photobleaching and phototoxicity. Here we propose a new, gentle method to study mitochondrial dynamics, where organelle-specific three-dimensional information is obtained in a label-free manner at high resolution, high specificity, and without detrimental effects associated with staining. A mitochondria cleavage experiment demonstrates that not only do the label-free mitochondria-specific images have the required resolution and precision, but also fairly include all cells and mitochondria in downstream morphological analysis, while fluorescence images omit dim cells and mitochondria. The robustness of the method was tested on samples of different cell lines and on data collected from multiple systems. Thus, we have demonstrated that our method is an attractive alternative to study mitochondrial dynamics, connecting behavior and function in a simpler and more robust way than traditional fluorescence imaging.

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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 mesoscale 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.dark-field microscope, label free, optical sectioning, small organisms | INTRODUCTIONWith the advent of modern gene-editing techniques, human physiological and pathological processes can be simulated and studied in model organisms with small and tractable genomes [1,2]. In order to study and connect behavior and disease with the underlying physiological and pathological pathways, often multi-modal, multiscale information including both labeled and nonlabeled images at both micro-and meso-scales are desired.