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

TL;DR

This paper introduces a novel computational method that simultaneously reconstructs 3D fluorescence and refractive index images from a single dataset, enabling improved imaging of biological samples by correcting scattering effects.

Contribution

A new multi-modal epi-microscopy technique that jointly measures 3D fluorescence and RI, enhancing biological imaging capabilities.

Findings

Successfully reconstructs 3D fluorescence and RI from one dataset

Demonstrates digital correction of scattering in fluorescence images

Provides quantitative 3D RI imaging of biological samples

Abstract

Fluorescence microscopy is a powerful tool for imaging biological samples with molecular specificity. In contrast, phase microscopy provides label-free measurement of the sample's refractive index (RI), which is an intrinsic optical property that quantitatively relates to cell morphology, mass, and stiffness. Conventional imaging techniques measure either the labeled fluorescence (functional) information or the label-free RI (structural) information, though it may be valuable to have both. For example, biological tissues have heterogeneous RI distributions, causing sample-induced scattering that degrades the fluorescence image quality. When both fluorescence and 3D RI are measured, one can use the RI information to digitally correct multiple-scattering effects in the fluorescence image. Here, we develop a new computational multi-modal imaging method based on epi-mode microscopy that reconstructs both 3D fluorescence and 3D RI from a single dataset. We acquire dozens of fluorescence images, each 'illuminated' by a single fluorophore, then solve an inverse problem with a multiple-scattering forward model. We experimentally demonstrate our method for epi-mode 3D RI imaging and digital correction of multiple-scattering effects in fluorescence images.