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Holotomography-driven learning for in-silico staining of single cells in flow cytometry avoiding co-registration

Pirone, D.; Giugliano, G.; Schiavo, M.; Montella, A.; Mugnano, M.; Cerbone, V.; Raia, M.; Scalia, G.; Kurelac, I.; Medina, D. L.; Miccio, L.; Capasso, M.; Iolascon, A.; Memmolo, P.; Ferraro, P.

2025-07-26 biophysics
10.1101/2025.07.22.666145 bioRxiv
Show abstract

Virtual staining is the current state-of-the-art computational technique to cleverly enhance intracellular specificity in unstained biological samples by using convolutional neural networks (CNNs) trained on co-registered pairs of unstained/stained images. While effective, this approach suffers from unpredictable biases inherent to fluorescence microscopy and encounters challenges when applied to flow cytometry data as it would require accurate co-registration on a huge number of images. Here, we present a novel method that exploits for the first time a Holotomography-driven learning to completely eliminate the need for co-registration. We demonstrate that training a CNN on a stain-free dataset of 3D refractive index tomograms of flowing cells elegantly unlocks stain-free intracellular specificity in quantitative phase imaging flow cytometry. This breakthrough, by circumventing the critical co-registration bottleneck, opens unprecedented perspectives for label-free, high-throughput imaging flow cytometry, offering a powerful new paradigm for advanced 2D and 3D single-cell analysis.

Published in Opto-Electronic Science · not in our set (fewer than 10 published preprints to learn from) · training set

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