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1. Semi-blind sparse affine spectral unmixing of autofluorescence-contaminated micrographs

   https://doi.org/10.1093/bioinformatics/btz674  

   Rossetti, Blair J. ; Wilbert, Steven A. ; Mark Welch, Jessica L. ; Borisy, Gary G. ; Nagy, James G. ; Murphy, ed., Robert ( August 2019 , Bioinformatics)

   Spectral unmixing methods attempt to determine the concentrations of different fluorophores present at each pixel location in an image by analyzing a set of measured emission spectra. Unmixing algorithms have shown great promise for applications where samples contain many fluorescent labels; however, existing methods perform poorly when confronted with autofluorescence-contaminated images.

   We propose an unmixing algorithm designed to separate fluorophores with overlapping emission spectra from contamination by autofluorescence and background fluorescence. First, we formally define a generalization of the linear mixing model, called the affine mixture model (AMM), that specifically accounts for background fluorescence. Second, we use the AMM to derive an affine nonnegative matrix factorization method for estimating fluorophore endmember spectra from reference images. Lastly, we propose a semi-blind sparse affine spectral unmixing (SSASU) algorithm that uses knowledge of the estimated endmembers to learn the autofluorescence and background fluorescence spectra on a per-image basis. When unmixing real-world spectral images contaminated by autofluorescence, SSASU greatly improved proportion indeterminacy as compared to existing methods for a given relative reconstruction error.

   The source code used for this paper was written in Julia and is available with the test data at https://github.com/brossetti/ssasu.

    

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2. Expanding the potential of standard flow cytometry by extracting fluorescence lifetimes from cytometric pulse shifts

   https://doi.org/10.1002/cyto.a.22574  

   Cao, Ruofan ; Naivar, Mark A. ; Wilder, Mark ; Houston, Jessica P. ( October 2014 , Cytometry Part A)

   Fluorescence lifetime measurements provide information about the fluorescence relaxation, or intensity decay, of organic fluorophores, fluorescent proteins, and other inorganic molecules that fluoresce. The fluorescence lifetime is emerging in flow cytometry and is helpful in a variety of multiparametric, single cell measurements because it is not impacted by nonlinearity that can occur with fluorescence intensity measurements. Yet time‐resolved cytometry systems rely on major hardware modifications making the methodology difficult to reproduce. The motivation of this work is, by taking advantage of the dynamic nature of flow cytometry sample detection and applying digital signal processing methods, to measure fluorescence lifetimes using an unmodified flow cytometer. We collect a new lifetime‐dependent parameter, referred to herein as the fluorescence‐pulse‐delay (FPD), and prove it is a valid representation of the average fluorescence lifetime. To verify we generated cytometric pulses in simulation, with light emitting diode (LED) pulsation, and with true fluorescence measurements of cells and microspheres. Each pulse is digitized and used in algorithms to extract an average fluorescence lifetime inherent in the signal. A range of fluorescence lifetimes is measurable with this approach including standard organic fluorophore lifetimes (∼1 to 22 ns) as well as small, simulated shifts (0.1 ns) under standard conditions (reported herein). This contribution demonstrates how digital data acquisition and signal processing can reveal time‐dependent information foreshadowing the exploitation of full waveform analysis for quantification of similar photo‐physical events within single cells. © 2014 The Authors. Published by Wiley Periodicals, Inc.

    

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3. M13 Virus‐Based Framework for High Fluorescence Enhancement

   https://doi.org/10.1002/smll.201901233  

   Huang, Shengnan ; Qi, Jifa ; deQuilettes, Dane W. ; Huang, Mantao ; Lin, Ching‐Wei ; Bardhan, Neelkanth M. ; Dang, Xiangnan ; Bulović, Vladimir ; Belcher, Angela M. ( May 2019 , Small)

   Fluorescence imaging is a powerful tool for studying biologically relevant macromolecules, but its applicability is often limited by the fluorescent probe, which must demonstrate both high site‐specificity and emission efficiency. In this regard, M13 virus, a versatile biological scaffold, has previously been used to both assemble fluorophores on its viral capsid with molecular precision and to also target a variety of cells. Although M13‐fluorophore systems are highly selective, these complexes typically suffer from poor molecular detection limits due to low absorption cross‐sections and moderate quantum yields. To overcome these challenges, a coassembly of the M13 virus, cyanine 3 dye, and silver nanoparticles is developed to create a fluorescent tag capable of binding with molecular precision with high emissivity. Enhanced emission of cyanine 3 of up to 24‐fold is achieved by varying nanoparticle size and particle‐fluorophore separation. In addition, it is found that the fluorescence enhancement increases with increasing dye surface density on the viral capsid. Finally, this highly fluorescent probe is applied for in vitro staining ofE. coli. These results demonstrate an inexpensive framework for achieving tuned fluorescence enhancements. The methodology developed in this work is potentially amendable to fluorescent detection of a wide range of M13/cell combinations.

    

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4. Characterization of a Bio‐sourced, Fluorescent, Ratiometric pH Indicator with Alkaline pK a

   https://doi.org/10.1111/php.13299  

   Alheety, Samar ; Valenti, Domenic ; Mujumdar, Nirvani ; Ellis, Nakita ; Campiglia, Andres D. ; Harper, James K. ; Heider, Emily C. ( July 2020 , Photochemistry and Photobiology)

   Utilizing organisms as sources of fluorophores relieves the demand for petroleum feedstock in organic synthesis of fluorescent products, and endophytic fungi provide a promising vein for natural fluorescent products. We report the characterization of a pH‐responsive fluorophore from an endophytic fungus isolated from sand pine. The endogenous fluorescence of the live organism was measured using fluorescence microscopy. Computational interpretation of the spectra was accomplished with time‐dependent density functional theory methods. The combined use of experimental and theoretically predicted spectra revealed the pH equilibria and photoexcited tautomerization of the natural product, 5‐methylmellein. This product shows promise both as a stand‐alone pH‐indicating fluorophore, with alkaline pK_a, and as green feedstock for synthesis of custom fluorophores.

    

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5. Linear dendronized polyols as a multifunctional platform for a versatile and efficient fluorophore design

   https://doi.org/10.1039/c8py00193f  

   Li, Ying ; Huth, Katharina ; Garcia, Edzna S. ; Pedretti, Benjamin J. ; Bai, Yugang ; Vincil, Gretchen A. ; Haag, Rainer ; Zimmerman, Steven C. ( January 2018 , Polymer Chemistry)

   null (Ed.)

   Fluorescent linear dendronized polyols (LDPs) were prepared in two steps involving a ring-opening metathesis polymerization (ROMP) followed by acid-catalyzed deprotection. The resulting water-soluble fluorophores are compact in size (<6 nm) and show similar photostability compared to previously reported crosslinked dendronized polyols (CDPs) and significantly improved photostability compared to the free fluorophores. In contrast to the synthesis of CDPs, the production of LDPs requires less preparation time, synthetic effort, and significantly less Grubbs catalyst. The photophysical properties, including the photostability and emission wavelength of LDPs, can be further fine-tuned by incorporating different combinations of dendronized monomers and fluorophores. Interestingly, fluorescence resonance energy transfer (FRET) was observed when two different kinds of fluorophores were incorporated into the LDPs. This provides a new type of fluorophore with a large Stokes shift allowing fluorescence detection with reduced background overlap. Cytotoxicity and fluorescence imaging studies confirmed the biocompatibility of these LDPs, which make them potential candidates for biological applications. 

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