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1. True FRET‐Based Sensing of pH via Separation of FRET and Photon Reabsorption

   https://doi.org/10.1002/adom.202200242  

   Corbella_Bagot, Conrad; Rappeport, Eric; Das, Ananda; Ba_Tis, Taleb; Park, Wounjhang (May 2022, Advanced Optical Materials)

   Abstract Förster resonance energy transfer (FRET)‐based devices have been extensively researched as potential biosensors due to their highly localized responsivity. In particular, dye‐conjugated upconverting nanoparticles (UCNPs) are among the most promising FRET‐based sensor candidates. UCNPs have a multi‐modal emission profile that allows for ratiometric sensing, and by conjugating a biosensitive dye to their surface, this profile can be used to measure localized variations in biological parameters. However, the complex nature of the UCNP energy profile as well as reabsorption of emitted photons must be taken into account in order to properly sense the target parameters. To the authors’ knowledge, no proposed UCNP‐based sensor has accurately taken care of these intricacies. In this article, the authors account for these complexities by creating a FRET‐based sensor that measures pH. This sensor utilizes Thulium (Tm³⁺)‐doped UCNPs and the fluorescent dye fluorescein isothiocyanate (FITC). It is first demonstrated that photon reabsorption is a serious issue for the 475 nm Tm³⁺emission, thereby limiting its use in FRET‐based sensing. It is then shown that by taking the ratio of the 646 and 800 nm emissions rather than the more popular 475 nm one, it is possible to measure pH exclusively through FRET. 

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2. Unraveling multi-state molecular dynamics in single-molecule FRET experiments. I. Theory of FRET-lines

   https://doi.org/10.1063/5.0089134  

   Barth, Anders; Opanasyuk, Oleg; Peulen, Thomas-Otavio; Felekyan, Suren; Kalinin, Stanislav; Sanabria, Hugo; Seidel, Claus A. (April 2022, The Journal of Chemical Physics)

   Conformational dynamics of biomolecules are of fundamental importance for their function. Single-molecule studies of Förster Resonance Energy Transfer (smFRET) between a tethered donor and acceptor dye pair are a powerful tool to investigate the structure and dynamics of labeled molecules. However, capturing and quantifying conformational dynamics in intensity-based smFRET experiments remains challenging when the dynamics occur on the sub-millisecond timescale. The method of multiparameter fluorescence detection addresses this challenge by simultaneously registering fluorescence intensities and lifetimes of the donor and acceptor. Together, two FRET observables, the donor fluorescence lifetime τ D and the intensity-based FRET efficiency E, inform on the width of the FRET efficiency distribution as a characteristic fingerprint for conformational dynamics. We present a general framework for analyzing dynamics that relates average fluorescence lifetimes and intensities in two-dimensional burst frequency histograms. We present parametric relations of these observables for interpreting the location of FRET populations in E–τ D diagrams, called FRET-lines. To facilitate the analysis of complex exchange equilibria, FRET-lines serve as reference curves for a graphical interpretation of experimental data to (i) identify conformational states, (ii) resolve their dynamic connectivity, (iii) compare different kinetic models, and (iv) infer polymer properties of unfolded or intrinsically disordered proteins. For a simplified graphical analysis of complex kinetic networks, we derive a moment-based representation of the experimental data that decouples the motion of the fluorescence labels from the conformational dynamics of the biomolecule. Importantly, FRET-lines facilitate exploring complex dynamic models via easily computed experimental observables. We provide extensive computational tools to facilitate applying FRET-lines. 

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3. A DRO Fret Slotter

   French, Mark (January 2018, American lutherie)

   Want a robot lutherie apprentice? It is here today and it is cheap. But it doesn’t look like something from the Jetsons. It looks like this; a digital readout connected to a lead screw. With a friendly whirr, it will move the saw guide right up to the next fret positionfor you. But get your own dang coffee. 

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4. A DRO Fret Slotter

   French, Mark (July 2018, American lutherie)

   Want a robot lutherie apprentice? It is here today and it is cheap. But it doesn’t look like something from the Jetsons. It looks like this; a digital readout connected to a lead screw. With a friendly whirr, it will move the saw guide right up to the next fret position for you. But get your own dang coffee. 

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5. Effects of tissue autofluorescence on FRET efficiency estimates

   https://doi.org/10.1117/12.2583284  

   Pleshinger, D.J.; Annamdevula, Naga; Leavesley, Silas J.; Rich, Tom C. (March 2021, Proc. SPIE 11649, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXVIII)

   Brown, Thomas G.; Wilson, Tony; Waller, Laura (Ed.)