Unspecified Revathi Manoharaan Profile

Revathi Manoharaan

at Univ of Illinois

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Publications (1)

SPIE Journal Paper | 16 July 2024 Open Access

Capturing cell morphology dynamics with high temporal resolution using single-shot quantitative phase gradient imaging

Sun Woong Hur, MinSung Kwon, Revathi Manoharaan, Melika Haji Mohammadi, Ashok Zachariah Samuel, Michael Mulligan, Paul Hergenrother, Rohit Bhargava

JBO, Vol. 29, Issue S2, S22712, (July 2024) https://doi.org/10.1117/12.10.1117/1.JBO.29.S2.S22712

KEYWORDS: Cell death, Polarization, Microscopes, Tunable filters, Temporal resolution, Fluorescence, Phase reconstruction, Image restoration, Fluorescence imaging, Digital image correlation

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SignificanceLabel-free quantitative phase imaging can potentially measure cellular dynamics with minimal perturbation, motivating efforts to develop faster and more sensitive instrumentation. We characterize fast, single-shot quantitative phase gradient microscopy (ss-QPGM) that simultaneously acquires multiple polarization components required to reconstruct phase images. We integrate a computationally efficient least squares algorithm to provide real-time, video-rate imaging (up to 75 frames/s). The developed instrument was used to observe changes in cellular morphology and correlate these to molecular measures commonly obtained by staining.AimWe aim to characterize a fast approach to ss-QPGM and record morphological changes in single-cell phase images. We also correlate these with biochemical changes indicating cell death using concurrently acquired fluorescence images.ApproachHere, we examine nutrient deprivation and anticancer drug-induced cell death in two different breast cell lines, viz., M2 and MCF7. Our approach involves in-line measurements of ss-QPGM and fluorescence imaging of the cells biochemically labeled for viability.ResultsWe validate the accuracy of the phase measurement using a USAF1951 pattern phase target. The ss-QPGM system resolves 912.3 lp/mm, and our analysis scheme accurately retrieves the phase with a high correlation coefficient (∼0.99), as measured by calibrated sample thicknesses. Analyzing the contrast in phase, we estimate the spatial resolution achievable to be 0.55 μm for this microscope. ss-QPGM time-lapse live-cell imaging reveals multiple intracellular and morphological changes during biochemically induced cell death. Inferences from co-registered images of quantitative phase and fluorescence suggest the possibility of necrosis, which agrees with previous findings.ConclusionsLabel-free ss-QPGM with high-temporal resolution and high spatial fidelity is demonstrated. Its application for monitoring dynamic changes in live cells offers promising prospects.

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