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Microrheology with optical tweezers (MOT) is an all-optical technique that allows the user to investigate a materials' viscoelastic properties at microscopic scales, and is particularly useful for those materials that feature complex microstructures, such as biological samples. MOT is increasingly being employed alongside 3D imaging systems and particle tracking methods to generate maps showing not only how properties may vary between different points in a sample but also how at a single point the viscoelastic properties may vary with direction. However, due to the diffraction limited shape of focussed beams, optical traps are inherently anisotropic in 3D. This can result in a significant overestimation of the fluids' viscosity in certain directions. As such, the rheological properties can only be accurately probed along directions parallel or perpendicular to the axis of trap beam propagation. In this work, a new analytical method is demonstrated to overcome this potential artefact. This is achieved by performing principal component analysis on 3D MOT data to characterise the trap, and then identify the frequency range over which trap anisotropy influences the data. This approach is initially applied to simulated data for a Newtonian fluid where the trap anisotropy induced maximum error in viscosity is reduced from ~ 150% to less than 6%. The effectiveness of the method is corroborated by experimental MOT measurements performed with water and gelatine solutions, thus confirming that the microrheology of a fluid can be extracted reliably across a wide frequency range and in any arbitrary direction. This work opens the door to fully spatially and angularly resolved 3D mapping of the rheological properties of soft materials over a broad frequency range. [ABSTRACT FROM AUTHOR]

Biomechanical cues from the extracellular matrix (ECM) are essential for directing many cellular processes, from normal development and repair, to disease progression. To better understand cell-matrix interactions, we have developed a new instrument named 'OptoRheo' that combines light sheet fluorescence microscopy with particle tracking microrheology. OptoRheo lets us image cells in 3D as they proliferate over several days while simultaneously sensing the mechanical properties of the surrounding extracellular and pericellular matrix at a sub-cellular length scale. OptoRheo can be used in two operational modalities (with and without an optical trap) to extend the dynamic range of microrheology measurements. We corroborated this by characterising the ECM surrounding live breast cancer cells in two distinct culture systems, cell clusters in 3D hydrogels and spheroids in suspension culture. This cutting-edge instrument will transform the exploration of drug transport through complex cell culture matrices and optimise the design of the next-generation of disease models. A new instrument named OptoRheo combines light sheet fluorescence microscopy and particle tracking microrheology for live imaging and micromechanical sensing of extracellular matrix-cell interactions. [ABSTRACT FROM AUTHOR]

A common feature of morphogenesis is the formation of three-dimensional structures from the folding of two-dimensional epithelial sheets, aided by cell shape changes at the cellular-level. Changes in cell shape must be studied in the context of cell-polarised biomechanical processes within the epithelial sheet. In epithelia with highly curved surfaces, finding single-cell alignment along a biological axis can be difficult to automate in silico. We present 'Origami', a MATLAB-based image analysis pipeline to compute direction-variant cell shape features along the epithelial apico-basal axis. Our automated method accurately computed direction vectors denoting the apico-basal axis in regions with opposing curvature in synthetic epithelia and fluorescence images of zebrafish embryos. As proof of concept, we identified different cell shape signatures in the developing zebrafish inner ear, where the epithelium deforms in opposite orientations to form different structures. Origami is designed to be user-friendly and is generally applicable to fluorescence images of curved epithelia. Author summary: During embryonic development, two-dimensional epithelial sheets bend and fold into complex three-dimensional structures–like paper in the origami art form. The genetic and biomechanical processes driving epithelial folding can be polarised in the epithelium, leading to asymmetric shape changes at the single cell level. Defects in such epithelial shaping have been linked to many developmental anomalies and diseases. It is, therefore, important not only to quantify shape change at the single cell level, but also to orientate these asymmetrical changes along an epithelial axis of polarity when studying morphogenetic processes. Origami is a MATLAB-based software that has been developed to automatically extract such single-cell asymmetrical shape features along the epithelial apico-basal axis from fluorescence microscopy images of folding epithelia. Origami provides a solution to computing directional vectors along the epithelial apico-basal axis followed by extracting direction-variant shape features of each segmented cell. It is generally applicable to epithelial structures regardless of complexity or direction of folding and is robust to imaging conditions. As proof of concept, Origami successfully differentiated between different cell shape signatures in highly curved structures at different developmental timepoints in the zebrafish inner ear. [ABSTRACT FROM AUTHOR]

Microbial activity in planktonic systems creates a dynamic and heterogeneous microscale seascape that harbors a diverse community of microorganisms and ecological interactions of global significance. In recent decades great effort has been put into understanding this complex system, particularly focusing on the role of chemical patchiness, while overlooking a physical parameter that governs microbial life and is affected by biological activity: viscosity. Here we reveal spatial heterogeneity of viscosity in planktonic systems by using microrheological techniques that allow measurement of viscosity at length scales relevant to microorganisms. We show the viscous nature and the spatial extent of the phycosphere, the region surrounding phytoplankton. In ~45% of the phytoplankton cells analyzed we detected increases in viscosity that extended up to 30 µm away from the cell with up to 40 times the viscosity of seawater. We also show how these gradients of viscosity can be amplified around a lysing phytoplankton cell as its viscous contents leak away. Finally, we report conservative estimates of viscosity inside marine aggregates, hotspots of microbial activity, more than an order of magnitude higher than in seawater. Since the diffusivities of dissolved molecules, particles, and microorganisms are inversely related to viscosity, microheterogeneity in viscosity alters the microscale distribution of microorganisms and their resources, with pervasive implications for the functioning of the planktonic ecosystem. Increasing viscosities impacts ecological interactions and processes, such as nutrient uptake, chemotaxis, and particle encounter, that occur at the microscale but influence carbon and nutrient cycles at a global scale. [ABSTRACT FROM AUTHOR]

The sperm mid-piece has traditionally been considered to be the engine that powers sperm. Larger mid-pieces have therefore been assumed to provide greater energetic capacity. However, in the zebra finch Taeniopygia guttata, a recent study showed a surprising negative relationship between mid-piece length and sperm energy content. Using a multi-dimensional approach to study mid-piece structure, we tested whether this unexpected relationship can be explained by a trade-off between mid-piece length and mid-piece thickness and/or cristae density inside the mitochondrial helix. We used selective plane illumination microscopy to study mid-piece structure from three-dimensional images of zebra finch sperm and used high-resolution transmission electron microscopy to quantifymitochondrial density. Contrary to the assumption that longer mid-pieces are larger and therefore produce or contain a greater amount of energy, our results indicate that the amount of mitochondrial material is consistent across mid-pieces of varying lengths, and longer mid-pieces are simply proportionately 'thinner'. [ABSTRACT FROM AUTHOR]

A correction notice for "A trade-off between thickness and length in the zebra finch sperm mid-piece" is presented, which is published in the July 25, 2018 issue of the periodical.

We introduce a novel 3D microrheology system that combines for the first time Optical Tweezers with Integrated Multiplane Microscopy (OpTIMuM). The system allows the 3D tracking of an optically trapped bead, with ~ 20 nm accuracy along the optical axis. This is achieved without the need for a high precision z-stage, separate calibration sample, nor a priori knowledge of either the bead size or the optical properties of the suspending medium. Instead, we have developed a simple yet effective in situ spatial calibration method using image sharpness and exploiting the fact we image at multiple planes simultaneously. These features make OpTIMuM an ideal system for microrheology measurements, and we corroborate the effectiveness of this novel microrheology tool by measuring the viscosity of water in three dimensions, simultaneously. [ABSTRACT FROM AUTHOR]