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

Abstract

<b>Significance:</b> 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. <b>Aim</b>: The goal is to develop a DHM technique able to sharply visualize thin neuronal processes. <b>Approach</b>: 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. <b>Results</b>: When applied to cultured neuronal networks with an air microscope objective ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mn>20</mml:mn> <mml:mo>×</mml:mo></mml:mrow> </mml:math> , 0.8 NA), P-DHM shows a coherent noise level typically corresponding to 1 nm at the single-pixel scale, in agreement with the <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mn>1</mml:mn> <mml:mo>/</mml:mo> <mml:msqrt><mml:mi>N</mml:mi></mml:msqrt> </mml:mrow> </mml:math> -law, allowing to readily visualize the <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mn>1</mml:mn> <mml:mtext>-</mml:mtext> <mml:mi>μ</mml:mi> <mml:mi>m</mml:mi></mml:mrow> </mml:math> -wide thin neuronal processes with a signal-to-noise ratio of <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mo>∼</mml:mo> <mml:mn>5</mml:mn></mml:mrow> </mml:math> . <b>Conclusions</b>: Therefore, P-DHM represents a very promising label-free technique to study neuronal connectivity and its development, including neurite outgrowth, elongation, and branching.