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Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
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Chromatically Corrected Multicolor Multiphoton Microscopy.

Hugo Blanc1, Gabriel Kaddour2, Nicolas B David1

  • 1Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS, INSERM, Institut Polytechnique de Paris, 91128 Palaiseau, France.

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|December 25, 2023
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Summary
This summary is machine-generated.

This study introduces an adaptive strategy to correct chromatic aberration in multiphoton microscopy. This technique enables clear, simultaneous multicolor imaging of biological samples, enhancing the study of complex processes.

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Area of Science:

  • Biomedical Optics
  • Microscopy
  • Cell Biology

Background:

  • Multiphoton microscopy enables deep tissue imaging and multicolor visualization of biological processes.
  • Chromatic aberration, a spatial shift of different wavelengths, degrades image quality in multicolor multiphoton microscopy.
  • Existing methods struggle with comprehensive chromatic aberration correction across the field of view.

Purpose of the Study:

  • To develop and validate an adaptive strategy for correcting axial and lateral chromatic aberration in two-beam multiphoton microscopy.
  • To improve the performance and effective area for beam-mixing in multicolor multiphoton imaging.
  • To enable robust, high-resolution, simultaneous multicolor imaging of biological specimens.

Main Methods:

  • Implemented a refractive phase mask to correct axial chromatic aberration by introducing defocus.
  • Utilized a piezoelectric mirror for dynamic compensation of lateral chromatic aberration during scanning.
  • Tested the system with simultaneous three-color, two-photon imaging of zebrafish embryos and mouse brain slices.

Main Results:

  • Achieved seamless chromatic correction across the entire field of view for various multiphoton objectives.
  • Maintained spatial and temporal resolution without compromising image quality.
  • Increased the effective area for beam-mixing processes by over an order of magnitude.

Conclusions:

  • The proposed adaptive strategy effectively corrects chromatic aberration in multiphoton microscopy.
  • This method provides a robust and efficient solution for high-quality, simultaneous multicolor imaging.
  • Enables advanced visualization of complex biological structures and processes in vivo and in fixed tissues.