The usage of a GaAsP (gallium arsenide phosphide) photomultiplier for microscopical imaging allows the evaluation of low-light luminescent objects. We designed a setup for collecting a confocal microscopic image signal, which is divided into 14 equal-sized input channels. The division is achieved with a beamsplitter and two fiber bundles consisting of seven fibers each. Re-imaging the confocal pinhole by such a densely packed fiber bundle permits the utilization of a photon re-localization approach to overcome the optical resolution limit. The center fiber creates a real-time image, while the outer fibers enable a higher-resolution image via an image scanning microscope (ISM) signal calculation. The fiber bundles are enclosed in a fused silica capillary and are drawn out to create one solid fiber bundle. During the drawing process, the fiber bundles are tapered down to an outer diameter size of 400μm, with each fiber having a less than 0.3 Airy unit diameter. For the photomultiplier interface, all fibers of both fiber bundles are integrated into a v-groove array, with each fiber representing a detection input, which is followed by projection optics for imaging onto the multichannel detector. The resulting confocal super-resolution microscope is suitable for the application of time-correlated single photon counting (TCSPC) techniques such as fluorescence lifetime imaging (FLIM), time-resolved anisotropy, or F¨orster resonance energy transfer (FRET) imaging.
The human neurotensin receptor one (NTSR1) is a G protein-coupled receptor. The receptor is activated by a small peptide ligand neurotensin. NTSR1 can be expressed in HEK cells by stable transfection. Previously we used the fluorescent protein markers mRuby3 or mNeonGreen fused to NTSR1 for EMCCD-based Structured Illumination Microscopy (SIM) in living HEK cells. Ligand binding induced conformational changes in NTSR1 which triggered the intracellular signaling processes. Recent single-molecule studies revealed a dynamic monomer/dimer equilibrium of this receptor in artificial lipid bilayers. Here we report on the oligomerization state of human NTSR1 from living cells by trapping them into lipid nanodiscs. Briefly, SMALPs (styrene-maleic acid copolymer lipid nanoparticles) were produced directly from the plasma membranes of living HEK293T FlpIn cells. SMALPs with a diameter of 15 nm were soluble and stable. NTSR1 in SMALPs were analyzed by single-molecule intensity measurements one membrane patch at a time using a custom-built confocal anti-Brownian electrokinetic trap (ABEL trap) microscope. We found oligomerization changes before and after stimulation of the receptor with its ligand neurotensin.
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