Single-molecule localization microscopy (SMLM) is a powerful super-resolution technique for elucidating structure and dynamics in the life-and material sciences. Simultaneously acquiring spectral information (spectrally resolved SMLM, sSMLM) has been hampered by several challenges: an increased complexity of the optical detection pathway, lower accessible emitter densities, and compromised spatio-spectral resolution.Here we present a single-component, low-cost implementation of sSMLM that addresses these challenges. Using a low-dispersion transmission grating positioned close to the image plane, the +1 st diffraction order is minimally elongated and is analyzed using existing single-molecule localization algorithms. The distance between the 0 th and 1 st order provides accurate information on the spectral properties of individual emitters. This method enables a 5-fold higher emitter density while discriminating between fluorophores whose peak emissions are less than 15 nm apart. Our approach can find widespread use in single-molecule applications that rely on distinguishing spectrally different fluorophores under low photon conditions.
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)’.
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.
Single-molecule localization microscopy (SMLM) is a powerful technique for elucidating structure and dynamics in the life- and material sciences with sub-50 nm spatial resolution. The simultaneous acquisition of spectral information (spectrally resolved SMLM, sSMLM) enables multiplexing using spectrally distinct fluorophores or enable the probing of local chemical environments by using solvachromatic fluorophores such as Nile Red. Until now, the widespread utilisation of sSMLM was hampered by several challenges: an increased complexity of the optical detection pathway, limited software solutions for data analysis, lower accessible emitter densities or smaller field-of-views, and overall compromised spatio-spectral resolution. Here, we present a low-cost implementation of sSMLM that addresses these challenges. Using a blazed, low-dispersion transmission grating positioned close to the image plane here represented by the camera sensor, the +1st diffraction order is minimally elongated compared to the point spread function of the 0th order and can therefore be analysed using common sub-pixel single-molecule localization algorithms. The distance between both PSFs provides accurate information on the spectral properties of the emitter. The minimal excess width of 1st order PSFs enables a fivefold higher emitter density compared to other sSMLM approaches whilst achieving a spatio-spectral localization accuracy sufficient to discriminate between fluorophores whose peak emission are less than 15 nm apart as demonstrated using dSTORM, DNA-PAINT and smFRET. We provide an ImageJ/Fiji plugin (sSMLMAnalyzer) and suitable Matlab scripts for data analysis. We envision that our approach will find widespread use in super-resolution applications that rely on distinguishing spectrally different fluorophores under low photon conditions.
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