Oblique Back-Illumination Microscopy Enables Phase-Gradient Optical Sensing of Malaria-Induced Intracellular Heterogeneity
- 1Johns Hopkins University , , , ,
- 2Georgia Institute of Technology and Emory University , , , ,
- 3Johns Hopkins University School of Medicine , , , ,
Abstract
Malaria remains a major global health burden, with diagnosis often relying on ex vivo analysis of infected red blood cells (RBCs), which incompletely samples key stages of parasite development. In particular, early ring-stage parasites exhibit subtle structural perturbations, while a significant fraction of parasite burden─including immature gametocytes─is sequestered within the microvasculature and is not readily captured in peripheral blood smears. Here, we present a label-free imaging and analysis framework that integrates oblique back-illumination microscopy (OBM) with data-driven classification to detect malaria infection by sensing parasite-induced intracellular heterogeneity. Unlike stained brightfield microscopy and quantitative phase imaging (QPI) methods that depend on interferometric acquisition and phase reconstruction, OBM directly converts intracellular refractive-index gradients into measurable intensity variations through noninterferometric phase-gradient contrast. By exploiting multiply scattered illumination and asymmetric detection, OBM enhances sensitivity to parasite-induced refractive-index gradients associated with Plasmodium falciparum development, capturing morphological complexity across the intraerythrocytic lifecycle. This enables the discrimination of uninfected cells, early ring-stage infections, and mature gametocytes. Fourier-domain analysis further reveals infection-stage-dependent redistribution of spatial-frequency content, providing a physically interpretable description of progressive intracellular remodeling during parasite development. Using synchronized in vitro cultures with brightfield validation, we demonstrate automated classification of infected RBCs with accuracies of 95% for binary classification and 82% for multiclass classification, with consistent precision, recall, and F1-scores across repeated randomized data splits. Together, these results establish OBM as a scalable, label-free sensing strategy that leverages intracellular heterogeneity as a diagnostic signal while providing access to stage-resolved optical phenotypes that remain difficult to interrogate with existing approaches. By building on prior studies of in vivo phase-gradient imaging, this framework provides a pathway toward integrating OBM with real-time analysis for noninvasive and field-deployable malaria diagnostics, particularly where conventional ex vivo measurements are limited by parasite sequestration.
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BibTeX
@article{lee2026oblique,
title = {{Oblique Back-Illumination Microscopy Enables Phase-Gradient Optical Sensing of Malaria-Induced Intracellular Heterogeneity}},
author = {Michael Ka Ho Lee and Yilin Guan and Caoze Huang and Ayan Guha and Ishan Barman},
journal = {ACS Omega},
year = {2026},
month = oct,
doi = {10.1021/acsomega.6c11324},
url = {https://pubs.acs.org/acsodf/article/doi/10.1021/acsomega.6c11324/5454864/Oblique-Back-Illumination-Microscopy-Enables-Phase},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - JOUR
TI - Oblique Back-Illumination Microscopy Enables Phase-Gradient Optical Sensing of Malaria-Induced Intracellular Heterogeneity
AU - Michael Ka Ho Lee
AU - Yilin Guan
AU - Caoze Huang
AU - Ayan Guha
AU - Ishan Barman
PY - 2026
DA - 2026/10/21
JO - ACS Omega
DO - 10.1021/acsomega.6c11324
UR - https://pubs.acs.org/acsodf/article/doi/10.1021/acsomega.6c11324/5454864/Oblique-Back-Illumination-Microscopy-Enables-Phase
AB - Malaria remains a major global health burden, with diagnosis often relying on ex vivo analysis of infected red blood cells (RBCs), which incompletely samples key stages of parasite development. In particular, early ring-stage parasites exhibit subtle structural perturbations, while a significant fraction of parasite burden─including immature gametocytes─is sequestered within the microvasculature and is not readily captured in peripheral blood smears. Here, we present a label-free imaging and analysis framework that integrates oblique back-illumination microscopy (OBM) with data-driven classification to detect malaria infection by sensing parasite-induced intracellular heterogeneity. Unlike stained brightfield microscopy and quantitative phase imaging (QPI) methods that depend on interferometric acquisition and phase reconstruction, OBM directly converts intracellular refractive-index gradients into measurable intensity variations through noninterferometric phase-gradient contrast. By exploiting multiply scattered illumination and asymmetric detection, OBM enhances sensitivity to parasite-induced refractive-index gradients associated with Plasmodium falciparum development, capturing morphological complexity across the intraerythrocytic lifecycle. This enables the discrimination of uninfected cells, early ring-stage infections, and mature gametocytes. Fourier-domain analysis further reveals infection-stage-dependent redistribution of spatial-frequency content, providing a physically interpretable description of progressive intracellular remodeling during parasite development. Using synchronized in vitro cultures with brightfield validation, we demonstrate automated classification of infected RBCs with accuracies of 95% for binary classification and 82% for multiclass classification, with consistent precision, recall, and F1-scores across repeated randomized data splits. Together, these results establish OBM as a scalable, label-free sensing strategy that leverages intracellular heterogeneity as a diagnostic signal while providing access to stage-resolved optical phenotypes that remain difficult to interrogate with existing approaches. By building on prior studies of in vivo phase-gradient imaging, this framework provides a pathway toward integrating OBM with real-time analysis for noninvasive and field-deployable malaria diagnostics, particularly where conventional ex vivo measurements are limited by parasite sequestration.
SN - 2470-1343
ER -