Characterisation and correction of polarisation effects in fluorescently labelled fibres.

Many biological structures take the form of fibres and filaments, and quantitative analysis of fibre organisation is important for understanding their functions in both normal physiological conditions and disease. In order to visualise these structures, fibres can be fluorescently labelled and imaged, with specialised image analysis methods available for quantifying the degree and strength of fibre alignment. Here we show that fluorescently labelled fibres can display polarised emission, with the strength of this effect varying depending on structure and fluorophore identity. This can bias automated analysis of fibre alignment and mask the true underlying structural organisation. We present a method for quantifying and correcting these polarisation effects without requiring polarisation-resolved microscopy and demonstrate its efficacy when applied to images of fluorescently labelled collagen gels, allowing for more reliable characterisation of fibre microarchitecture.

A method for reproducible high-resolution imaging of 3D cancer cell spheroids.

Multicellular tumour cell spheroids embedded within three-dimensional (3D) hydrogels or extracellular matrices (ECM) are widely used as models to study cancer growth and invasion. Standard methods to embed spheroids in 3D matrices result in random placement in space which limits the use of inverted fluorescence microscopy techniques, and thus the resolution that can be achieved to image molecular detail within the intact spheroid. Here, we leverage UV photolithography to microfabricate PDMS (polydimethylsiloxane) stamps that allow for generation of high-content, reproducible well-like structures in multiple different imaging chambers. Addition of multicellular tumour spheroids into stamped collagen structures allows for precise positioning of spheroids in 3D space for reproducible high-/super-resolution imaging. Embedded spheroids can be imaged live or fixed and are amenable to immunostaining, allowing for greater flexibility of experimental approaches. We describe the use of these spheroid imaging chambers to analyse cell invasion, cell-ECM interaction, ECM alignment, force-dependent intracellular protein dynamics and extension of fine actin-based protrusions with a variety of commonly used inverted microscope platforms. This method enables reproducible, high-/super-resolution live imaging of multiple tumour spheroids, that can be potentially extended to visualise organoids and other more complex 3D in vitro systems.

Unconventional Biocatalytic Approaches to the Synthesis of Chiral Sulfoxides.

Sulfoxides are a class of organic compounds that find wide application in medicinal and organic chemistry. Several biocatalytic approaches have been developed to synthesise enantioenriched sulfoxides, mainly by exploiting oxidative enzymes. Recently, the use of reductive enzymes such as Msr and Dms has emerged as a new, alternative method to obtain enantiopure sulfoxides from racemic mixtures. In parallel, novel oxidative approaches, employing nonclassical solvents such as ionic liquids (ILs) and deep eutectic solvents (DESs), have been developed as greener and more sustainable biocatalytic synthetic pathways. This minireview aims highlights the recent advances made in the biocatalytic synthesis of enantioenriched sulfoxides by employing such unconventional approaches.