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 (, 0.8 NA), P-DHM shows a coherent noise level typically corresponding to 1 nm at the single-pixel scale, in agreement with the -law, allowing to readily visualize the -wide thin neuronal processes with a signal-to-noise ratio of .Conclusions: Therefore, P-DHM represents a very promising label-free technique to study neuronal connectivity and its development, including neurite outgrowth, elongation, and branching.
Highlights
Digital holographic microscopy (DHM), an important approach in the field of quantitative-phase imaging techniques, has started to be efficiently used in the last decade as a label-free, highresolution, and live-imaging method for quasitransparent biological samples such as living cells.[1,2,3] Due to its quantitative phase signal (QPS), it provides accurate and noninvasive measurements of various important cellular parameters, including dry mass,[4] whole-cell stiffness,[5] membrane fluctuations,[6] transmembrane water fluxes,[7] and absolute cell volume.[8]
Following preliminary observations suggesting that the coherent noise of optical path difference (OPD) images can be reduced by taking advantage of a multispectral hologram recording,[25] we present in this letter a multiwavelength off-axis DHM, in a transmission configuration, providing quasicoherent-noise-free OPD images
While the “X” shape formed by neuronal processes highlighted in (ii) can hardly be identified in the control image, it is sharply visible in the polychromatic DHM (P-DHM) image
Summary
Digital holographic microscopy (DHM), an important approach in the field of quantitative-phase imaging techniques, has started to be efficiently used in the last decade as a label-free, highresolution, and live-imaging method for quasitransparent biological samples such as living cells.[1,2,3] Due to its quantitative phase signal (QPS), it provides accurate and noninvasive measurements of various important cellular parameters, including dry mass,[4] whole-cell stiffness,[5] membrane fluctuations,[6] transmembrane water fluxes,[7] and absolute cell volume.[8]. With neuronal activity.[7,9] coherent noise (an acknowledged source of image quality limitation in coherent optical microscopy systems10) remains an issue, when considering an off-axis DHM configuration,[11] as it requires the use of a highly or partially coherent light source.[12] Seen as granularity in the quantitative-phase (QP) images, it is caused by unwanted reflections and small defects or dirt in the optical path This precludes the possibility to explore minute cellular structures especially neuronal processes, a major part of neural circuits through which the information is integrated and conveyed between neurons. Typical coherent noise amplitude is larger than the QPS generated by a significant part of the neuronal processes, especially the thin projections including dendrites
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