Abstract:We report the experimental implementation of optical diffraction tomography for quantitative 3D mapping of refractive index in live biological cells. Using a heterodyne Mach-Zehnder interferometer, we record complex field images of light transmitted through a sample with varying directions of illumination. To quantitatively reconstruct the 3D map of complex refractive index in live cells, we apply optical diffraction tomography based on the Rytov approximation. In this way, the effect of diffraction is taken into account in the reconstruction process and diffraction-free high resolution 3D images are obtained throughout the entire sample volume. The quantitative refractive index map can potentially serve as an intrinsic assay to provide the molecular concentrations without the addition of exogenous agents and also to provide a method for studying the light scattering properties of single cells.
Even a casual pass through the great halls of mammals in the world’s natural history museums highlights the wide diversity of skeletal proportions that allow us to distinguish between species even when reduced to their calcified components. Similarly each individual is comprised of a variety of bones of differing lengths. The largest contribution to the lengthening of a skeletal element, and to the differential elongation of elements, comes from a dramatic increase in the volume of hypertrophic chondrocytes in the growth plate as they undergo terminal differentiation1–7. Despite this recognized importance, the mechanisms of chondrocyte volume enlargement have remained a mystery8–11. Here we use quantitative phase microscopy12 to show that chondrocytes undergo three distinct phases of volume increase, including a phase of massive cell swelling in which the cellular dry mass is significantly diluted. In light of the tight fluid regulatory mechanisms known to control volume in many cell types13, this stands as a remarkable mechanism for increasing cell size and regulating growth rate. It is, however, the duration of the final phase of volume enlargement by proportional dry mass increase at low density that varies most between rapidly and slowly elongating growth plates. Moreover, we find that this third phase is locally regulated through an Insulin-like Growth Factor-dependent mechanism. This study provides a framework for understanding how skeletal size is regulated and for exploring how cells sense, modify, and establish a volume set point.
The coupling of the rate of cell growth to the rate of cell division determines cell size, a defining characteristic that is central to cell function and, ultimately, to tissue architecture. The physiology of size homeostasis has fascinated generations of biologists, but the mechanism, challenged by experimental limitations, remains largely unknown. In this paper, we propose a unique optical method that can measure the dry mass of thick live cells as accurately as that for thin cells with high computational efficiency. With this technique, we quantify, with unprecedented accuracy, the asymmetry of division in lymphoblasts and epithelial cells. We can then use the Collins-Richmond model of conservation to compute the relationship between growth rate and cell mass. In attached epithelial cells, we find that due to the asymmetry in cell division and sizedependent growth rate, there is active regulation of cell size. Thus, like nonadherent cells, size homeostasis requires feedback control.cell division asymmetry | cell growth | cell dry mass | interferometry | synthetic phase microscopy
Upon infection and development within human erythrocytes, P. falciparum induces alterations to the infected RBC morphology and bio-mechanical properties to eventually rupture the host cells through parasitic and host derived proteases of cysteine and serine families. We used previously reported broad-spectrum inhibitors (E64d, EGTA-AM and chymostatin) to inhibit these proteases and impede rupture to analyze mechanical signatures associated with parasite escape. Treatment of late-stage iRBCs with E64d and EGTA-AM prevented rupture, resulted in no major RBC cytoskeletal reconfiguration but altered schizont morphology followed by dramatic re-distribution of three-dimensional refractive index (3D-RI) within the iRBC. These phenotypes demonstrated several-fold increased iRBC membrane flickering. In contrast, chymostatin treatment showed no 3D-RI changes and caused elevated fluctuations solely within the parasitophorous vacuole. We show that E64d and EGTA-AM supported PV breakdown and the resulting elevated fluctuations followed non-Gaussian pattern that resulted from direct merozoite impingement against the iRBC membrane. Optical trapping experiments highlighted reduced deformability of the iRBC membranes upon rupture-arrest, more specifically in the treatments that facilitated PV breakdown. Taken together, our experiments provide novel mechanistic interpretations on the role of parasitophorous vacuole in maintaining the spherical schizont morphology, the impact of PV breakdown on iRBC membrane fluctuations leading to eventual parasite escape and the evolution of membrane stiffness properties of host cells in which merozoites were irreversibly trapped, recourse to protease inhibitors. These findings provide a comprehensive, previously unavailable, body of information on the combined effects of biochemical and biophysical factors on parasite egress from iRBCs.
Refractive index imaging is a label-free technique that enables long-term monitoring of the internal structures and molecular composition in living cells with minimal perturbation. Existing tomographic methods for the refractive index imaging lack 3-D resolution and result in artifacts that prevent accurate refractive index quantification. To overcome these limitations without compromising the capability to observe a sample in its most native condition, we have developed a regularized tomographic phase microscope (RTPM) enabling accurate refractive index imaging of organelles inside intact cells. With the enhanced accuracy, we quantify the mass of chromosomes in intact living cells, and differentiate two human colon cancer lines, HT-29 and T84 cells, solely based on the non-aqueous (dry) mass of chromosomes. In addition, we demonstrate chromosomal imaging using a dual-wavelength RTPM, which shows its potential to determine the molecular composition of cellular organelles in live cells.
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