# Computational Microscopy Reveals Compound-Specific Flickering Phenotypes of Red Blood Cells Under Flavonoid Exposure

## TL;DR

A new computational microscopy method reveals how different flavonoids affect red blood cell mechanics by analyzing their flickering behavior.

## Contribution

Introduces a framework combining bright-field imaging and flickering spectroscopy to phenotype RBC mechanics under flavonoid exposure.

## Key findings

- Flavonoid treatment alters RBC flickering spectra and mechanical parameters in a compound-specific manner.
- Aglycone flavonoids show distinct modulation patterns compared to glycosylated ones, indicating different membrane interactions.
- Combining static and dynamic analysis improves discrimination of mechanical phenotypes compared to using either alone.

## Abstract

Red blood cell (RBC) membrane flickering arises from the interplay between thermal fluctuations, cytoskeletal elasticity, and metabolically driven non-equilibrium processes, making it a sensitive reporter of membrane mechanical state. Here, we introduce a computational microscopy framework that integrates bright-field morphometry with high-speed flickering spectroscopy to phenotype single-cell RBC mechanics under flavonoid exposure. As a proof of concept, human erythrocytes from a single donor were incubated with structurally distinct flavonoids (quercetin, apigenin, and rutin) prepared at sub-hemolytic concentrations, ensuring preservation of membrane integrity. Static shape descriptors and dynamic fluctuation spectra were extracted from segmented cell contours and analyzed through Fourier-mode decomposition to obtain compound-specific mechanical signatures. While gross morphology remained largely discocytic across conditions, flavonoid treatment induced reproducible alterations in flickering spectra and effective mechanical parameters, revealing distinct dynamical phenotypes that depend on flavonoid structure. In particular, aglycone flavonoids exhibited modulation patterns that differed from the glycosylated compound, consistent with differential membrane interactions. The combined analysis of geometry and dynamics provided enhanced discriminative power compared to either modality alone. These results establish computational microscopy as a sensitive, label-free approach to map compound-specific perturbations of RBC membrane mechanics and flickering, with potential applications in membrane biophysics, drug–membrane interaction screening, and single-cell mechanical phenotyping.