143 research outputs found
3D correlative single-cell imaging utilizing fluorescence and refractive index tomography
Cells alter the path of light, a fact that leads to well-known aberrations in
single cell or tissue imaging. Optical diffraction tomography (ODT) measures
the biophysical property that causes these aberrations, the refractive index
(RI). ODT is complementary to fluorescence imaging and does not require any
markers. The present study introduces RI and fluorescence tomography with
optofluidic rotation (RAFTOR) of suspended cells, quantifying the intracellular
RI distribution and colocalizing it with fluorescence in 3D. The technique is
validated with cell phantoms and used to confirm a lower nuclear RI for HL60
cells. Furthermore, the nuclear inversion of adult mouse photoreceptor cells is
observed in the RI distribution. The applications shown confirm predictions of
previous studies and illustrate the potential of RAFTOR to improve our
understanding of cells and tissues.Comment: 15 pages, 5 figure
Single-cell diffraction tomography with optofluidic rotation about a tilted axis
Optical diffraction tomography (ODT) is a tomographic technique that can be
used to measure the three-dimensional (3D) refractive index distribution within
living cells without the requirement of any marker. In principle, ODT can be
regarded as a generalization of optical projection tomography which is
equivalent to computerized tomography (CT). Both optical tomographic techniques
require projection-phase images of cells measured at multiple angles. However,
the reconstruction of the 3D refractive index distribution post-measurement
differs for the two techniques. It is known that ODT yields better results than
projection tomography, because it takes into account diffraction of the imaging
light due to the refractive index structure of the sample. Here, we apply ODT
to biological cells in a microfluidic chip which combines optical trapping and
microfluidic flow to achieve an optofluidic single-cell rotation. In
particular, we address the problem that arises when the trapped cell is not
rotating about an axis perpendicular to the imaging plane, but instead about an
arbitrarily tilted axis. In this paper we show that the 3D reconstruction can
be improved by taking into account such a tilted rotational axis in the
reconstruction process.Comment: 7 pages, 3 figure
Accurate evaluation of size and refractive index for spherical objects in quantitative phase imaging
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 future studies
in cell biology.Comment: 14 pages, 10 figures, 1 tabl
Materials and technologies for soft implantable neuroprostheses
Implantable neuroprostheses are engineered systems designed to restore or substitute function for individuals with neurological deficits or disabilities. These systems involve at least one uni-or bidirectional interface between a living neural tissue and a synthetic structure, through which information in the form of electrons, ions or photons flows. Despite a few notable exceptions, the clinical dissemination of implantable neuroprostheses remains limited, because many implants display inconsistent long-term stability and performance, and are ultimately rejected by the body. Intensive research is currently being conducted to untangle the complex interplay of failure mechanisms. In this Review, we emphasize the importance of minimizing the physical and mechanical mismatch between neural tissues and implantable interfaces. We explore possible materials solutions to design and manufacture neurointegrated prostheses, and outline their immense therapeutic potential
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Colloidal crystals of compliant microgel beads to study cell migration and mechanosensitivity in 3D
Tissues are defined not only by their biochemical composition, but also by their distinct mechanical properties. It is now widely accepted that cells sense their mechanical environment and respond to it. However, studying the effects of mechanics in in vitro 3D environments is challenging since current 3D hydrogel assays convolve mechanics with gel porosity and adhesion. Here, we present novel colloidal crystals as modular 3D scaffolds where these parameters are principally decoupled by using monodisperse, protein-coated PAAm microgel beads as building blocks, so that variable stiffness regions can be achieved within one 3D colloidal crystal. Characterization of the colloidal crystal and oxygen diffusion simulations suggested the suitability of the scaffold to support cell survival and growth. This was confirmed by live-cell imaging and fibroblast culture over a period of four days. Moreover, we demonstrate unambiguous durotactic fibroblast migration and mechanosensitive neurite outgrowth of dorsal root ganglion neurons in 3D. This modular approach of assembling 3D scaffolds from mechanically and biochemically well-defined building blocks allows the spatial patterning of stiffness decoupled from porosity and adhesion sites in principle and provides a platform to investigate mechanosensitivity in 3D environments approximating tissues in vitro
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