Significance: Over the past decade, laser-based digital holographic microscopy (DHM), an important approach in the field of quantitative-phase imaging techniques, has become a significant label-free modality for live-cell imaging and used particularly in cellular neuroscience. However, coherent noise remains a major drawback for DHM, significantly limiting the possibility to visualize neuronal processes and precluding important studies on neuronal connectivity.

Aim: The goal is to develop a DHM technique able to sharply visualize thin neuronal processes.

Approach: By combining a wavelength-tunable light source with the advantages of hologram numerical reconstruction of DHM, an approach called polychromatic DHM (P-DHM), providing OPD images with drastically decreased coherent noise, was developed.

Results: When applied to cultured neuronal networks with an air microscope objective (20  ×  , 0.8 NA), P-DHM shows a coherent noise level typically corresponding to 1 nm at the single-pixel scale, in agreement with the 1  /  N-law, allowing to readily visualize the 1-μm-wide thin neuronal processes with a signal-to-noise ratio of ∼5.

Conclusions: Therefore, P-DHM represents a very promising label-free technique to study neuronal connectivity and its development, including neurite outgrowth, elongation, and branching.