Lipid oxidation in food emulsions is mediated by emulsifiers in the water phase and at the oil–water interface. To unravel the physico-chemical mechanisms and to obtain local lipid and protein oxidation rates, we used confocal laser scanning microscopy (CLSM), thereby monitoring changes in both the fluorescence emission of a lipophilic dye BODIPY 665/676 and protein auto-fluorescence. Our data show that the removal of lipid-soluble antioxidants from mayonnaises promotes lipid oxidation within oil droplets as well as protein oxidation at the oil–water interface. Furthermore, we demonstrate that ascorbic acid acts as either a lipid antioxidant or pro-oxidant depending on the presence of lipid-soluble antioxidants. The effects of antioxidant formulation on local lipid and protein oxidation rates were all statistically significant (p < 0.0001). The observed protein oxidation at the oil–water interface was spatially heterogeneous, which is in line with the heterogeneous distribution of lipoprotein granules from the egg yolk used for emulsification. The impact of the droplet size on local lipid and protein oxidation rates was significant (p < 0.0001) but minor compared to the effects of ascorbic acid addition and lipid-soluble antioxidant depletion. The presented results demonstrate that CLSM can be applied for unraveling the roles of colloidal structure and transport in mediating lipid oxidation in complex food emulsions.

Turbidity poses a major challenge for the microscopic characterization of food systems. Local mismatches in refractive indices, for example, lead to significant image deterioration along sample depth. To mitigate the issue of turbidity and to increase the accessible optical resolution in food microscopy, we added adaptive optics (AO) and flat-field illumination to our previously published open microscopy framework, the miCube. In the detection path, we implemented AO via a deformable mirror to compensate aberrations and to modulate the emission wavefront enabling the engineering of point spread functions (PSFs) for single-molecule localization microscopy (SMLM) in three dimensions. As a model system for a non-transparent food colloid such as mayonnaise, we designed an oil-in-water emulsion containing the ferric ion binding protein phosvitin commonly present in egg yolk. We targeted phosvitin with fluorescently labelled primary antibodies and used PSF engineering to obtain two- and three-dimensional images of phosvitin covered oil droplets with sub 100 nm resolution. Our data indicated that phosvitin is homogeneously distributed at the interface. With the possibility to obtain super-resolved images in depth, our work paves the way for localizing biomacromolecules at heterogeneous colloidal interfaces in food emulsions. This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 2)’.

Spatiotemporal assessment of lipid and protein oxidation is key for understanding quality deterioration in emulsified food products containing polyunsaturated fatty acids. In this work, we first mechanistically validated the use of the lipid oxidation-sensitive fluorophore BODIPY 665/676 as a semi-quantitative marker for local peroxyl radical formation. Next, we assessed the impact of microfluidic and colloid mill emulsification (respectively producing mono- and polydisperse droplets) on local protein and lipid oxidation kinetics in whey protein isolate (WPI)-stabilized emulsions. We further used BODIPY 581/591 C11 and CAMPO-AFDye 647 as colocalisation markers for lipid and protein oxidation. The polydisperse emulsions showed an inverse relation between droplet size and lipid oxidation rate. Further, we observed less protein and lipid oxidation occurring in similar sized droplets in monodisperse emulsions. This observation was linked to more heterogeneous protein packing at the droplet surface during colloid mill emulsification, resulting in larger inter-droplet heterogeneity in both protein and lipid oxidation. Our findings indicate the critical roles of emulsification methods and droplet sizes in understanding and managing lipid oxidation.

Recent development in nanofabrication technology has enabled the fabrication of plasmonic nanoapertures that can provide strong field concentrations beyond the diffraction limit. Further utilization of plasmonic nanoaperture requires the broadband tuning of the operating wavelength and precise control of aperture geometry. Here, we present a novel plasmonic coaxial aperture that can support resonant extraordinary optical transmission (EOT) with a peak transmittance of ~10% and a wide tuning range over a few hundred nanometers. Because of the shadow deposition process, we could precisely control the gap size of the coaxial aperture down to the sub–10‐nm scale. The plasmonic resonance of the SiN _x /Au disk at the center of the coaxial aperture efficiently funnels the incident light into the sub–10‐nm gap and allows strong electric field confinement for efficient second harmonic generation (SHG), as well as EOT. In addition to the experiment, we theoretically investigated the modal properties of the plasmonic coaxial aperture depending on the structural parameters and correlation between EOT and SHG through finite‐difference time‐domain simulations. We believe that our plasmonic coaxial apertures, which are readily fabricated by the nanoimprinting process, can be a versatile, practical platform for enhanced light–matter interaction and its nonlinear optical applications.

In single-molecule localization microscopy (SMLM), the use of engineered point spread functions (PSFs) provides access to three-dimensional localization information. The conventional approach of fitting PSFs with a single 2-dimensional Gaussian profile, however, often falls short in analyzing complex PSFs created by placing phase masks, deformable mirrors or spatial light modulators in the optical detection pathway. Here, we describe the integration of PSF modalities known as double-helix, saddle-point or tetra-pod into the phasor-based SMLM (pSMLM) framework enabling fast CPU based localization of single-molecule emitters with sub-pixel accuracy in three dimensions. For the double-helix PSF, pSMLM identifies the two individual lobes and uses their relative rotation for obtaining z-resolved localizations. For the analysis of saddle-point or tetra-pod PSFs, we present a novel phasor-based deconvolution approach entitled circular-tangent pSMLM. Saddle-point PSFs were experimentally realized by placing a deformable mirror in the Fourier plane and modulating the incoming wavefront with specific Zernike modes. Our pSMLM software package delivers similar precision and recall rates to the best-in-class software package (SMAP) at signal-to-noise ratios typical for organic fluorophores and achieves localization rates of up to 15 kHz (double-helix) and 250 kHz (saddle-point/tetra-pod) on a standard CPU. We further integrated pSMLM into an existing software package (SMALL-LABS) suitable for single-particle imaging and tracking in environments with obscuring backgrounds. Taken together, we provide a powerful hardware and software environment for advanced single-molecule studies.

Turbidity poses a major challenge for the microscopic characterization of many food systems. In these systems, local mismatches in refractive indices can cause reflection, absorption and scattering of incoming as well as outgoing light leading to significant image deterioration along sample depth. To mitigate the issue of turbidity and to increase the achievable optical resolution, we combined adaptive optics (AO) with single-molecule localization microscopy (SMLM). Building on our previously published open hardware microscopy framework, the miCube, we first added a deformable mirror to the detection path. This element enables both the compensation of aberrations directly from single-molecule data and, by further modulating the emission wavefront, the introduction of various point spread functions (PSFs) to enable SMLM in three dimensions. We further added a top hat beam shaper to the excitation path to obtain an even illumination profile across the field of view (FOV). As a model system for a non-transparent food colloid in which imaging in depth is challenging, we designed an oil-in-water emulsion in which phosvitin, a ferric ion binding protein present in from egg yolk, resides at the oil water interface. We targeted phosvitin with fluorescently labelled primary antibodies and used PSF engineering to obtain 2D and 3D images of phosvitin covered oil droplets with sub 100 nm resolution. Droplets with radii as low as 200 nm can be discerned, which is beyond the range of conventional confocal light microscopy. Our data indicated that in the model emulsion phosvitin is homogeneously distributed at the oil-water interface. With the possibility to obtain super-resolved images in depth of nontransparent colloids, our work paves the way for localizing biomacromolecules at colloidal interfaces in heterogeneous food emulsions.