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1. Genetic encoding of a highly photostable, long lifetime fluorescent amino acid for imaging in mammalian cells

   https://doi.org/10.1039/D1SC01914G  

   Jones, Chloe M.; Robkis, D. Miklos; Blizzard, Robert J.; Munari, Mika; Venkatesh, Yarra; Mihaila, Tiberiu S.; Eddins, Alex J.; Mehl, Ryan A.; Zagotta, William N.; Gordon, Sharona E.; et al (September 2021, Chemical Science)

   Acridonylalanine (Acd) is a fluorescent amino acid that is highly photostable, with a high quantum yield and long fluorescence lifetime in water. These properties make it superior to existing genetically encodable fluorescent amino acids for monitoring protein interactions and conformational changes through fluorescence polarization or lifetime experiments, including fluorescence lifetime imaging microscopy (FLIM). Here, we report the genetic incorporation of Acd using engineered pyrrolysine tRNA synthetase (RS) mutants that allow for efficient Acd incorporation in both E. coli and mammalian cells. We compare protein yields and amino acid specificity for these Acd RSs to identify an optimal construct. We also demonstrate the use of Acd in FLIM, where its long lifetime provides strong contrast compared to endogenous fluorophores and engineered fluorescent proteins, which have lifetimes less than 5 ns. 

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2. Fluorescence-based Methods for the Study of Protein-Protein Interactions Modulated by Ligand Binding

   https://doi.org/10.2174/1381612826666201116120934  

   Stoneman, Michael R.; Raicu, Naomi; Biener, Gabriel; Raicu, Valerică (December 2020, Current Pharmaceutical Design)

   null (Ed.)

   Background: The growing evidence that G protein-coupled receptors (GPCRs) not only form oligomersbut that the oligomers also may modulate the receptor function provides a promising avenue in the area ofdrug design. Highly selective drugs targeting distinct oligomeric sub-states offer the potential to increase efficacywhile reducing side effects. In this regard, determining the various oligomeric configurations and geometricsub-states of a membrane receptor is of utmost importance. Methods: In this report, we have reviewed two techniques that have proven to be valuable in monitoring thequaternary structure of proteins in vivo: Fӧrster resonance energy transfer (FRET) spectrometry and fluorescenceintensity fluctuation (FIF) spectrometry. In FRET spectrometry, distributions of pixel-level FRET efficiencyare analyzed using theoretical models of various quaternary structures to determine the geometry andstoichiometry of protein oligomers. In FIF spectrometry, spatial fluctuations of fluorescent molecule intensitiesare analyzed to reveal quantitative information on the size and stability of protein oligomers. Results: We demonstrate the application of these techniques to a number of different fluorescence-based studiesof cells expressing fluorescently labeled membrane receptors, both in the presence and absence of variousligands. The results show the effectiveness of using FRET spectrometry to determine detailed information regardingthe quaternary structure receptors form, as well as FIF and FRET for determining the relative abundanceof different-sized oligomers when an equilibrium forms between such structures. Conclusion: FRET and FIF spectrometry are valuable techniques for characterizing membrane receptor oligomers,which are of great benefit to structure‐based drug design. 

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3. Comparison of spectral FRET microscopy approaches for single-cell analysis

   https://doi.org/10.1117/12.2546308  

   Deal, Joshua; Annamdevula, Naga S.; Pleshinger, D J.; Griswold, John Robert; Odom, Aliyah; Tayara, Alia; Lall, Malvika; Browning, Craig M.; Parker, Marina; Rich, Thomas C.; et al (February 2020, Proc. SPIE 11243, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVIII)

   Förster resonance energy transfer (FRET) is a valuable tool for measuring molecular distances and the effects of biological processes such as cyclic nucleotide messenger signaling and protein localization. Most FRET techniques require two fluorescent proteins with overlapping excitation/emission spectral pairing to maximize detection sensitivity and FRET efficiency. FRET microscopy often utilizes differing peak intensities of the selected fluorophores measured through different optical filter sets to estimate the FRET index or efficiency. Microscopy platforms used to make these measurements include wide-field, laser scanning confocal, and fluorescence lifetime imaging. Each platform has associated advantages and disadvantages, such as speed, sensitivity, specificity, out-of-focus fluorescence, and Zresolution. In this study, we report comparisons among multiple microscopy and spectral filtering platforms such as standard 2-filter FRET, emission-scanning hyperspectral imaging, and excitation-scanning hyperspectral imaging. Samples of human embryonic kidney (HEK293) cells were grown on laminin-coated 28 mm round gridded glass coverslips (10816, Ibidi, Fitchburg, Wisconsin) and transfected with adenovirus encoding a cAMP-sensing FRET probe composed of a FRET donor (Turquoise) and acceptor (Venus). Additionally, 3 FRET “controls” with fixed linker lengths between Turquoise and Venus proteins were used for inter-platform validation. Grid locations were logged, recorded with light micrographs, and used to ensure that whole-cell FRET was compared on a cell-by-cell basis among the different microscopy platforms. FRET efficiencies were also calculated and compared for each method. Preliminary results indicate that hyperspectral methods increase the signal-to-noise ratio compared to a standard 2-filter approach. 

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4. High-speed compressed-sensing fluorescence lifetime imaging microscopy of live cells

   https://doi.org/10.1073/pnas.2004176118  

   Ma, Yayao; Lee, Youngjae; Best-Popescu, Catherine; Gao, Liang (January 2021, Proceedings of the National Academy of Sciences)

   We present high-resolution, high-speed fluorescence lifetime imaging microscopy (FLIM) of live cells based on a compressed sensing scheme. By leveraging the compressibility of biological scenes in a specific domain, we simultaneously record the time-lapse fluorescence decay upon pulsed laser excitation within a large field of view. The resultant system, referred to as compressed FLIM, can acquire a widefield fluorescence lifetime image within a single camera exposure, eliminating the motion artifact and minimizing the photobleaching and phototoxicity. The imaging speed, limited only by the readout speed of the camera, is up to 100 Hz. We demonstrated the utility of compressed FLIM in imaging various transient dynamics at the microscopic scale. 

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5. Enhanced detection of oral dysplasia by structured illumination fluorescence lifetime imaging microscopy

   https://doi.org/10.1038/s41598-021-84552-8  

   Hinsdale, Taylor A.; Malik, Bilal H.; Cheng, Shuna; Benavides, Oscar R.; Giger, Maryellen L.; Wright, John M.; Patel, Paras B.; Jo, Javier A.; Maitland, Kristen C. (December 2021, Scientific Reports)

   null (Ed.)

   Abstract We demonstrate that structured illumination microscopy has the potential to enhance fluorescence lifetime imaging microscopy (FLIM) as an early detection method for oral squamous cell carcinoma. FLIM can be used to monitor or detect changes in the fluorescence lifetime of metabolic cofactors (e.g. NADH and FAD) associated with the onset of carcinogenesis. However, out of focus fluorescence often interferes with this lifetime measurement. Structured illumination fluorescence lifetime imaging (SI-FLIM) addresses this by providing depth-resolved lifetime measurements, and applied to oral mucosa, can localize the collected signal to the epithelium. In this study, the hamster model of oral carcinogenesis was used to evaluate SI-FLIM in premalignant and malignant oral mucosa. Cheek pouches were imaged in vivo and correlated to histopathological diagnoses. The potential of NADH fluorescence signal and lifetime, as measured by widefield FLIM and SI-FLIM, to differentiate dysplasia (pre-malignancy) from normal tissue was evaluated. ROC analysis was carried out with the task of discriminating between normal tissue and mild dysplasia, when changes in fluorescence characteristics are localized to the epithelium only. The results demonstrate that SI-FLIM (AUC = 0.83) is a significantly better (p-value = 0.031) marker for mild dysplasia when compared to widefield FLIM (AUC = 0.63). 

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