Abstract
Measuring the average refractive index (RI) of spherical objects, such as suspended cells, in quantitative phase imaging (QPI) requires a decoupling of RI and size from the QPI data. This has been commonly achieved by determining the object's radius with geometrical approaches, neglecting light-scattering. Here, we present a novel QPI fitting algorithm that reliably uncouples the RI using Mie theory and a semi-analytical, corrected Rytov approach. We assess the range of validity of this algorithm in silico and experimentally investigate various objects (oil and protein droplets, microgel beads, cells) and noise conditions. In addition, we provide important practical cues for the analysis of spherical objects in QPI.
Highlights
Quantitative phase imaging (QPI) is a collective term for interferometric techniques that quantify the phase retardation of otherwise transparent objects
The image was recorded with quadriwave lateral shearing interferometry (QLSI) [35] using a commercial QPI camera (SID4Bio, Phasics S.A.) attached to an inverted microscope (AxioObserver Z1, Zeiss) with a 40× objective (NA 0.65, 421060-9900, Zeiss)
The resulting relative error of 0.04% is small which is reflected by residuals below 5% of the maximum optical path difference (OPD) shown in figure 4c
Summary
Quantitative phase imaging (QPI) is a collective term for interferometric techniques that quantify the phase retardation of otherwise transparent objects. Besides characterizing cells based on RI and the associated local protein concentration [1, 2, 10], knowing the RI is essential for related applications such as the optical stretcher to quantify optical forces [11, 12, 13], or for Brillouin microscopy to compute cell elasticity [14, 15]. The accurate determination of the cellular RI with QPI is difficult, because the optical path difference (OPD) measured in QPI needs to be separated into integral RI and cell thickness. The integral RI can only be computed if the cell thickness is measured, which has been achieved using scanning probe microscopy [16, 17], confocal microscopy [18], deliberate variation of the OPD [3, 19], or spatial confinement [20, 21]. State-of-the-art ODT techniques approximate light propagation with the Rytov approximation [24, 25], which can cause an underestimation of the intracellular RI for large RI gradients [26, 27]
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