Label-Free Nucleoli Measurement by 3D Holo-Tomographic Flow Cytometry Using Biolens Phase Compensation
Pirone, D.; Schiavo, M.; Giugliano, G.; Montefusco, S.; Montella, A.; Mugnano, M.; Cerbone, V.; Raia, M.; Scalia, G.; Bianco, V.; Miccio, L.; Capasso, M.; Iolascon, A.; Medina, D. L.; Memmolo, P.; Psaltis, D.; Ferraro, P.
Show abstract
Holo-Tomographic Flow Cytometry (HTFC) holds the potential to transform cellular research and clinical screening through 3D label-free quantitative phase imaging (QPI) of flowing single cells. However, it has been limited by insufficient intracellular specificity in 3D refractive index (RI) distributions, since suspended cells act as highly aberrating spherical biolenses obscuring internal structures. Here, we show the Biolens Phase Compensation (BPC), a method that corrects phase aberrations in 2D QPI projections to transform the 3D RI tomogram. Working within this new 3D pseudo-RI space demonstrates for the first time the extraction of nucleoli in HTFC. Extensive validation against 2D fluorescence flow cytometry and 3D confocal microscopy demonstrates that BPC achieves reliable intranuclear specificity. Using statistically significant single-cell analysis, we provide multiplexed quantitative 3D measurements of nested intracellular compartments (cytoplasm, nucleoplasm, nucleoli). This approach extends label-free HTFC toward capabilities of gold-standard Fluorescence Microscopy, overcoming its well-known drawbacks in intracellular and intranuclear staining.
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