Three-dimensional bi-functional refractive index and fluorescence microscopy (BRIEF)

Area of Science:

• Biomedical Optics
• Microscopy
• Computational Imaging

Background:

• Fluorescence microscopy offers molecular specificity but lacks structural information.
• Phase microscopy provides label-free refractive index (RI) for structural insights.
• Conventional methods capture either fluorescence or RI, not both simultaneously.

Purpose of the Study:

• To develop a computational multi-modal imaging method for simultaneous 3D fluorescence and 3D RI reconstruction.
• To enable digital correction of multiple-scattering effects in fluorescence imaging using RI data.
• To advance label-free imaging capabilities in biological samples.

Main Methods:

• Developed a novel computational multi-modal imaging method based on epi-mode microscopy.
• Acquired multiple fluorescence images illuminated by single fluorophores.
• Solved an inverse problem using a multiple-scattering forward model to reconstruct 3D RI and fluorescence.

Main Results:

• Successfully reconstructed both 3D fluorescence and 3D RI from a single dataset.
• Demonstrated the method's efficacy for epi-mode 3D RI imaging.
• Showcased digital correction of multiple-scattering effects in fluorescence images.

Conclusions:

• The developed method enables simultaneous acquisition of functional (fluorescence) and structural (RI) information.
• This technique can digitally correct scattering-induced image degradation in biological tissues.
• Offers a powerful new tool for advanced biological imaging and analysis.