Chromatically Corrected Multicolor Multiphoton Microscopy - Archive ouverte HAL
Article Dans Une Revue ACS photonics Année : 2023

Chromatically Corrected Multicolor Multiphoton Microscopy

Gabriel Kaddour
Nicolas B David
Willy Supatto
Jean Livet
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Pierre Mahou

Résumé

Simultaneous imaging of multiple labels in tissues is key to studying complex biological processes. Although strategies for color multiphoton excitation have been established, chromatic aberration remains a major problem when multiple excitation wavelengths are used in a scanning microscope. Chromatic aberration introduces a spatial shift between the foci of beams of different wavelengths that varies across the field of view, severely degrading the performance of color imaging. In this work, we propose an adaptive correction strategy that solves this problem in two-beam microscopy techniques. Axial chromatic aberration is corrected by a refractive phase mask that introduces pure defocus into one beam, while lateral chromatic aberration is corrected by a piezoelectric mirror that dynamically compensates for lateral shifts during scanning. We show that this lightefficient approach allows seamless chromatic correction over the entire field of view of different multiphoton objectives without compromising spatial and temporal resolution and that the effective area for beam-mixing processes can be increased by more than 1 order of magnitude. We illustrate this approach with simultaneous three-color, two-photon imaging of developing zebrafish embryos and fixed Brainbow mouse brain slices over large areas. These results establish a robust and efficient method for chromatically corrected multiphoton imaging.
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Dates et versions

hal-04299952 , version 1 (22-11-2023)

Identifiants

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Hugo Blanc, Gabriel Kaddour, Nicolas B David, Willy Supatto, Jean Livet, et al.. Chromatically Corrected Multicolor Multiphoton Microscopy. ACS photonics, 2023, ⟨10.1021/acsphotonics.3c01104⟩. ⟨hal-04299952⟩
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