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.
KEYWORDS: Fluorescence lifetime imaging, Microscopes, Confocal microscopy, Luminescence, Live cell imaging, Surface plasmons, In vivo imaging, Single photon, Resonance energy transfer, Imaging systems
We report the development of a novel confocal line-scanning microscope capable of acquiring video-frame rate TCSPC-based FLIM. The system consists of a one-dimensional laser beam, which is optically conjugated to a 1024×16 single photon avalanche diode(SPAD) based line-imaging CMOS(1), with 23.78 μm pixel pitch at 49.31% fill factor. Incorporation of on-chip histogramming on the line-sensor facilitates the acquisition of up to 16.5 Giga-photon counts/s, enabling operation 66 times faster than our previously reported bespoke high speed FLIM platforms. We will demonstrate its use in live-cell imaging investigating the roles that PAK proteins play in regulation of cytoskeletal dynamics.
Precisely characterising and quantifying interactions between tumour cells and their environment to understand metastatic mechanisms requires a multi-dimensional, high-speed imaging system. To this end, we report on the development of a compressive full spectrum fluorescence lifetime microscope that exploits a novel SPAD line sensor and a DMD to enable monitoring of dynamic sub-cellular interactions. At no cost to its temporal performance, the hyperspectral nature of the system helps to improve unmixing and, crucially, can detect the small spectral changes in the emission of fluorescent probes and intrinsic fluorophores that can occur in complex environments.
A tension-sensitive biosensor was used to detect changes in applied force across the mechanosensitive focal adhesion protein vinculin. When the biosensor is under tension, two fluorescent proteins separate, decreasing the amount of Förster Resonance Energy Transfer (FRET) observed. By measuring FRET using time-correlated single-photon counting fluorescence lifetime imaging microscopy (TCSPC-FLIM) we observe the loss of FRET, as a direct consequence of an applied intracellular force across the biosensor. Mouse embryonic fibroblasts (MEFs) transfected with a vinculin construct encoding the Tension Sensing Module (TSM), demonstrate how force-transduction changes within maturing adhesions in both fixed and live cells.
We report the development of a novel massively-parallelised high-speed multifocal FLIM platform with the ability to acquire data 1024 times faster than a conventional TCSPC system. We demonstrate the system performanceFRET imaging of the fluorescent protein biosensor PercivalHR in iPSC derived neurons to measure the dynamic concentration of ADP/ATP in live cells. The advantages and performance envelope of the system will be shown and the potential for further applications explored.
Time-domain microfluidic fluorescence lifetime flow cytometry enables observation of fluorescence decay of particles or cells over time using time-correlated single photon counting (TCSPC). This method requires the fluorescence lifetime measured from a limited number of photons and in a short amount of time. In current implementations of the technique, the low throughput of state of the art detectors and lack of real-time statistical analysis of the current technology, the timedomain approaches are usually coupled with off-line analysis which impedes its use in flow cell sorting, tracking and capturing. In this work, we apply a 32×32 CMOS SPAD array (MegaFrame camera) for real-time imaging flow cytometry analysis. This technology is integrated into a 1024-beam multifocal fluorescence microscope and incorporating a microfluidic chip at the sample plane enables imaging of cell flow and identification. Furthermore, the 1.5% native pixel fill-factor of the MegaFrame camera is overcome using beamlet reprojection with <10 μW laser power at 490 nm for each beam. Novel hardware algorithms incorporating the center-of-mass method (CMM) with real-time background subtraction and division are implemented within the firmware, allowing lossless recording of TCSPC events at a 500 kHz frame rate with 1024 histogram bins at 52 ps time resolution. Live calculation of background compensated CMM-based fluorescence lifetime is realized at a user-defined frame rate (typically 0.001 ~ 27 kHz) for each SPAD pixel. The work in this paper considers the application of the SPAD array to confocal fluorescence lifetime imaging of multiple coincident particles flowing within a microfluidic channel. Compared to previous flow systems based on single-point detectors, the multi-beam flow system enables visualization, detection and categorization of multiple groups of cells or particles according to their fluorescence lifetime.
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