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z-STED Imaging and Spectroscopy to Investigate Nanoscale Membrane Structure and Dynamics
Super-resolution stimulated emission depletion (STED) microcopy provides optical resolution beyond the diffraction limit. The resolution can be increased laterally (xy) or axially (z). Two-dimensional STED has been extensively used to elucidate the nanoscale membrane structure and dynamics via imaging or combined with spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and spectral imaging. On the contrary, z-STED has not been used in this context. Here, we show that a combination of z-STED with FCS or spectral imaging enables us to see previously unobservable aspects of cellular membranes. We show that thanks to an axial resolution of ∼100 nm, z-STED can be used to distinguish axially close-by membranes, early endocytic vesicles, or tubular membrane structures. Combination of z-STED with FCS and spectral imaging showed diffusion dynamics and lipid organization in these structures, respectively. © 2020 Biophysical Society
Aggregation and mobility of membrane proteins interplay with local lipid order in the plasma membrane of T cells
To disentangle the elusive lipid-protein interactions in T-cell activation, we investigate how externally imposed variations in mobility of key membrane proteins (T-cell receptor [TCR], kinase Lck, and phosphatase CD45) affect the local lipid order and protein colocalisation. Using spectral imaging with polarity-sensitive membrane probes in model membranes and live Jurkat T cells, we find that partial immobilisation of proteins (including TCR) by aggregation or ligand binding changes their preference towards a more ordered lipid environment, which can recruit Lck. Our data suggest that the cellular membrane is poised to modulate the frequency of protein encounters upon alterations of their mobility, for example in ligand binding, which offers new mechanistic insight into the involvement of lipid-mediated interactions in membrane-hosted signalling events.
Spectral imaging based on 2D diffraction patterns and a regularization model
We present a variational approach to reconstruct a multispectral image from an array of diffraction patterns captured in one monochromatic image. The mathematical model relies on the superposition of wavelength dependent calibration diffraction patterns and a spectral regularization with second order differences. The necessary preprocessing steps which influence the mathematical setting are discussed in detail. For computing a minimizer of our model we propose an active set method with model specific modifications. We validate our approach by experimental results for spectra within the range of 400 nm to 700 nm.