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.
KEYWORDS: Molecular interactions, Molecules, Biological imaging, Nanodomains, Time correlated single photon counting, Molecular spectroscopy, Cancer detection, Single molecule spectroscopy, Quantum detection, Lung cancer
Fluorescence lifetime imaging (FLIM) is a sensitive tool to investigate protein-protein interactions in biology and may have some niche applications for early detection of cancer in liquid biospies. The gold-standard for such a technique is time-correlated single photon counting (TCSPC). For imaging, this has largely been exploited in laser scanning microscopy techniques. However, these imaging modalities are limited primarily by their particularly slow acquisition speeds compared to conventional imaging methodologies especially when the sample is sparse. We have developed a simple optical platform to exploit the sparseness of single molecule imaging and apply this to preliminary study of the heterodimer status of EGFR and HER3, in cell secreted vesicles (exosomes). In parallel we perform single EV trapping experiments with an ABEL trap to extend our spectroscopic analysis. We perform both fluorescence lifetime spectroscopy and SMLM on single exosomes in vitro to assess changes in the proclivity of heterodimer formation following Osimertinib treatment in lung cancer cells (H1975), which may elude to a possible functional role in cancer treatment resistance.
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