Abstract
Biophysical properties of cells such as intracellular mass density and cell mechanics are known to be involved in a wide range of homeostatic functions and pathological alterations. An optical readout that can be used to quantify such properties is the refractive index (RI) distribution. It has been recently reported that the nucleus, initially presumed to be the organelle with the highest dry mass density (ρ) within the cell, has in fact a lower RI and ρ than its surrounding cytoplasm. These studies have either been conducted in suspended cells, or cells adhered on 2D substrates, neither of which reflects the situation in vivo where cells are surrounded by the extracellular matrix (ECM). To better approximate the 3D situation, we encapsulated cells in 3D covalently-crosslinked alginate hydrogels with varying stiffness, and imaged the 3D RI distribution of cells, using a combined optical diffraction tomography (ODT)-epifluorescence microscope. Unexpectedly, the nuclei of cells in 3D displayed a higher ρ than the cytoplasm, in contrast to 2D cultures. Using a Brillouin-epifluorescence microscope we subsequently showed that in addition to higher ρ, the nuclei also had a higher longitudinal modulus (M) and viscosity (η) compared to the cytoplasm. Furthermore, increasing the stiffness of the hydrogel resulted in higher M for both the nuclei and cytoplasm of cells in stiff 3D alginate compared to cells in compliant 3D alginate. The ability to quantify intracellular biophysical properties with non-invasive techniques will improve our understanding of biological processes such as dormancy, apoptosis, cell growth or stem cell differentiation.
Highlights
Earlier optical studies on two-dimensional (2D) cell cultures reported higher refractive index (RI) for the nucleus compared to the cytoplasm.[19,20,21,22] Interestingly, recent studies using a variety of microscopy techniques such as quantitative phase imaging,[23,24] orientation-independent differential interference microscopy,[25] plasmon resonance microscopy[26] and transport-of-intensity microscopy,[27] have shown the opposite
We show that the nuclei of MDA-MB-231 cells in 3D hydrogels display higher RI and r compared to the cytoplasm, differently to what is commonly found on 2D substrates
Using a combined optical diffraction tomography (ODT)-epifluorescence microscope, to our surprise, we found that human breast cancer cells MDA-MB231 encapsulated in 3D hydrogels displayed a significantly
Summary
Earlier optical studies on two-dimensional (2D) cell cultures reported higher RI for the nucleus compared to the cytoplasm.[19,20,21,22] Interestingly, recent studies using a variety of microscopy techniques such as quantitative phase imaging,[23,24] orientation-independent differential interference microscopy,[25] plasmon resonance microscopy[26] and transport-of-intensity microscopy,[27] have shown the opposite. Changes in the ECM have been associated to phenotypic and genotypic alterations in cell properties such as morphology,[29] differentiation,[30] signal transduction,[31] migration and proliferation.[32] To our knowledge, no study has investigated the effect of the dimensionality provided by the ECM on sub-cellular mass distribution For this purpose, we employed a covalently-crosslinked 3D alginate hydrogel, which allows for versatile and independent tuning of various biophysical properties such as adhesion,[33] stiffness,[33,34] viscoelasticity[35,36,37] and degradation.[38,39] Using a custom-made combined ODTepifluorescence microscope, we quantitatively mapped the 3D RI distribution of human breast cancer cells MDA-MB-231, encapsulated in 3D hydrogels with different stiffness, and compared the resulting r with cells on 2D substrates
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