Integral refractive index imaging of flowing cell nuclei using quantitative phase microscopy combined with fluorescence microscopy

Abstract

Abstract:We suggest a new multimodal imaging technique for quantitatively measuring the integral (thickness-average) refractive index of the nuclei of live biological cells in suspension. For this aim, we combined quantitative phase microscopy with simultaneous 2-D fluorescence microscopy. We used 2-D fluorescence microscopy to localize the nucleus inside the quantitative phase map of the cell, as well as for measuring the nucleus radii. As verified offline by both 3-D confocal fluorescence microscopy and 2-D fluorescence microscopy while rotating the cells during flow, the nucleus of cells in suspension that are not during division can be assumed to be an ellipsoid. The entire shape of a cell in suspension can be assumed to be a sphere. Then, the cell and nucleus 3-D shapes can be evaluated based on their in-plain radii available from the 2-D phase and fluorescent measurements, respectively. Finally, the nucleus integral refractive index profile is calculated. We demonstrate the new technique on cancer cells, obtaining nucleus refractive index values that are lower than those of the cytoplasm, coinciding with recent findings. We believe that the proposed technique has the potential to be used for flow cytometry, where full 3-D refractive index tomography is too slow to be implemented during flow. References and links 1. C. M. Vest, Holographic Interferometry (Wiley, New York, 1979). 2. N. T. Shaked, "Quantitative phase microscopy of biological samples using a portable interferometer," Opt. Lett. Marquet, "Cell morphology and intracellular ionic homeostasis explored with a multimodal approach combining epifluorescence and digital holographic microscopy,"

Cell and nucleus refractive‐index mapping by interferometric phase microscopy and rapid confocal fluorescence microscopy

“…In contrast to methods that allow the acquisition of the whole cell integral RI, without access to the cell organelles, other methods have evaluated the nucleus RI. For that purpose, nucleus thickness evaluation was done by actual isolation of the nucleus and then evaluation of its thickness by assuming a spherical shape [33,34], or by fluorescent labeling of the nucleus inside the cell and then estimation of the nucleus thickness by assuming an ellipsoidal shape [23]. Nucleus thickness evaluation was also done by using a confocal fluorescence microscope, while labeling the whole cell and assuming negligible cytoplasmic thickness at a single estimated nucleus location [26], or by using a confocal reflectance microscope in order to detect the contour of the nucleus inside the cell [1].…”

Response to Comment on “Is the nuclear refractive index lower than cytoplasm? Validation of phase measurements and implications for light scattering technologies”

“…Novel methods aside from TPM have reached similar conclusions. Dardikman et al recently developed a multimodal method to measure the integral RI distributions in cell suspensions, finding the RI of the nucleus to be lower than cytoplasm in regions other than the nucleolus . An analogous method to TPM, optical diffraction tomography (ODT) uses quantitative phase images at various angles to solve an inverse scattering problem, creating diffraction‐free 3‐dimensional RI tomograms.…”

Review on methods of solving the refractive index–thickness coupling problem in digital holographic microscopy of biological cells

“…Numerous recent DHM studies have overturned the former consensus that the nucleus is more dense than the cytoplasm in light of its biological function, by proving that the RI of the cell nucleus is in fact lower than that of cytoplasm [26][27][28]42].…”

“…Another group of methods solves the RI-thickness coupling problem by evaluating the thickness of the sample at each spatial location, which allows the isolation of the average RI in that pixel (also called integral RI). The simplest and fastest method is approximating the local thickness based on the fact that cells in suspension assume a spherical shape [21][22][23][24][25][26][27][28], yielding the integral RI 2-D profile of the cell from its quantitative phase profile with no prior knowledge other than the RI of the suspension medium. Another option is using a different imaging method to directly measure the geometrical thickness, enabling the isolation of the integral RI [29][30][31][32].…”