Polychromatic digital holographic microscopy: a quasicoherent-noise-free imaging technique to explore the connectivity of living neuronal networks

Abstract

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∕ ffiffiffiffi N p-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.

Flipping Interferometric Module for Simultaneous Dual-Wavelength Unwrapping of Quantitative Phase Maps of Biological Cells

“…This is possible by controlling the seeding of the cells, by cleaning cells from the culture before imaging, or by using square chambers and locating the beam at one of the corners of the square. In addition to two-wavelength phase unwrapping, the proposed module can be used for simultaneous interferometric acquisition of two-wavelength channels for other applications, such as interferometric spectroscopy [16] and reduction of coherenceinduced disturbances [17,18]. We expect that in the future, this module will be used for quantitative phase imaging of highly dynamic samples, such as for flow cytometry, while enabling both faster acquisition and processing.…”

Autofocusing based on cosine similarity in dual-wavelength digital holographic microscopy

“…Furthermore, it is well known that multi-wavelength technology will cause noise amplification. Combining the proposed method with low-coherence multi-wavelength digital holographic microscopy will be a very meaningful application [42,43]. Moreover, the proposed method is a non-sharpness metric-based method, which is dependent on the diffraction difference between different wavelengths.…”

Ultra-parallel label-free optophysiology of neural activity

“…Most of these techniques are restricted to imaging a very limited FOV (single pixel-to-single-cell scale) or imaging slower dynamics. Full-field interferometry, quantitative phase imaging, and digital holographic microscopy have also been used to balance the spatiotemporal range of the measured scattered optical field ( Hu et al., 2019 ; Larivière-Loiselle et al., 2020 ; Ling et al., 2018 ), with specific attention toward improving the phase stability and utilizing high-speed cameras.…”