Integral refractive index imaging of flowing cell nuclei using quantitative phase microscopy combined with fluorescence microscopy

TL;DR

This paper introduces a novel multimodal imaging method combining quantitative phase microscopy and fluorescence microscopy to measure the integral refractive index of live cell nuclei in suspension, enabling rapid analysis suitable for flow cytometry.

Contribution

The study presents a new technique for measuring nuclear refractive index in live cells using combined microscopy modalities, improving speed and accuracy over existing methods.

Findings

Nucleus refractive index is lower than cytoplasm in cancer cells.

The method accurately localizes and measures nuclei in flowing cells.

Potential application in flow cytometry for rapid cell analysis.

Abstract

We suggest a new multimodal imaging technique for quantitatively measuring the integral (thickness-average) refractive index of the nuclei of live biological cells in suspension. For this aim, we combined quantitative phase microscopy with simultaneous 2-D fluorescence microscopy. We used 2-D fluorescence microscopy to localize the nucleus inside the quantitative phase map of the cell, as well as for measuring the nucleus radii. As verified offline by both 3-D confocal fluorescence microscopy and by 2-D fluorescence microscopy while rotating the cells during flow, the nucleus of cells in suspension that are not during division can be assumed to be an ellipsoid. The entire shape of a cell in suspension can be assumed to be a sphere. Then, the cell and nucleus 3-D shapes can be evaluated based on their in-plain radii available from the 2-D phase and fluorescent measurements, respectively. Finally, the nucleus integral refractive index profile is calculated. We demonstrate the new technique on cancer cells, obtaining nucleus refractive index values that are lower than those of the cytoplasm, coinciding with recent findings. We believe that the proposed technique has the potential to be used for flow cytometry, where full 3-D refractive index tomography is too slow to be implemented during flow.