High-resolution 3D refractive index microscopy of multiple-scattering samples from intensity images

Area of Science:

• Biophysics
• Optical Microscopy
• 3D Imaging

Background:

• Standard optical diffraction tomography (ODT) provides quantitative 3D refractive index (RI) visualizations of biological samples.
• Existing ODT methods are limited by phase instabilities, complex mechanics, noise, and inability to reconstruct multiple-scattering samples.

Purpose of the Study:

• To develop and demonstrate a novel 3D RI microscopy technique capable of imaging multiple-scattering biological samples.
• To overcome the limitations of conventional ODT for complex biological specimens.

Main Methods:

• Implementation of a computational multi-slice beam propagation method for inverting optical scattering.
• Acquisition of intensity-only measurements from varying illumination angles.
• Solving a nonlinear optimization problem to reconstruct the 3D RI distribution.

Main Results:

• Successful high-resolution (NA > 1.0) 3D RI reconstruction of multiple-scattering samples.
• Demonstrated imaging of a fibroblast cell, C. elegans embryos, and a whole C. elegans worm.
• Achieved lateral resolution of ≤ 240 nm and axial resolution of ≤ 900 nm.

Conclusions:

• The developed technique enables high-contrast, quantitative 3D RI imaging of highly scattering biological organisms.
• This work provides a foundation for future optical studies of complex biological structures using RI distributions.