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1. 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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2. [n]Cycloparaphenylenes as Compatible Fluorophores for Melt Electrowriting

   https://doi.org/10.1002/smll.202400882  

   Hall, Patrick C; Reid, Harrison W; Liashenko, Ievgenii; Tandon, Biranche; O'Neill, Kelly L; Paxton, Naomi C; Lindberg, Gabriella_C J; Jasti, Ramesh; Dalton, Paul D (June 2024, Small)

   Abstract Fluorescent probes are an indispensable tool in the realm of bioimaging technologies, providing valuable insights into the assessment of biomaterial integrity and structural properties. However, incorporating fluorophores into scaffolds made from melt electrowriting (MEW) poses a challenge due to the sustained, elevated temperatures that this processing technique requires. In this context, [n]cycloparaphenylenes ([n]CPPs) serve as excellent fluorophores for MEW processing with the additional benefit of customizable emissions profiles with the same excitation wavelength. Three fluorescent blends are used with distinct [n]CPPs with emission wavelengths of either 466, 494, or 533 nm, identifying 0.01 wt% as the preferred concentration. It is discovered that [n]CPPs disperse well within poly(ε‐caprolactone) (PCL) and maintain their fluorescence even after a week of continuous heating at 80 °C. The [n]CPP‐PCL blends show no cytotoxicity and support counterstaining with commonly used DAPI (Ex/Em: 359 nm/457 nm), rhodamine‐ (Ex/Em: 542/565 nm), and fluorescein‐tagged (Ex/Em: 490/515 nm) phalloidin stains. Using different color [n]CPP‐PCL blends, different MEW fibers are sequentially deposited into a semi‐woven scaffold and onto a solution electrospun membrane composed of [8]CPP‐PCL as a contrasting substrate for the [10]CPP‐PCL MEW fibers. In general, [n]CPPs are potent fluorophores for MEW, providing new imaging options for this technology. 

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3. 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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4. Multiplexed imaging in live cells using pulsed interleaved excitation spectral FLIM

   https://doi.org/10.1364/OE.505667  

   Nguyen, Trung_Duc; Chen, Yuan-I; Nguyen, Anh-Thu; Chen, Limin_H; Yonas, Siem; Litvinov, Mitchell; He, Yujie; Kuo, Yu-An; Hong, Soonwoo; Rylander, H_Grady; et al (January 2024, Optics Express)

   Multiplexed fluorescence detection has become increasingly important in the fields of biosensing and bioimaging. Although a variety of excitation/detection optical designs and fluorescence unmixing schemes have been proposed to allow for multiplexed imaging, rapid and reliable differentiation and quantification of multiple fluorescent species at each imaging pixel is still challenging. Here we present a pulsed interleaved excitation spectral fluorescence lifetime microscopic (PIE-sFLIM) system that can simultaneously image six fluorescent tags in live cells in a single hyperspectral snapshot. Using an alternating pulsed laser excitation scheme at two different wavelengths and a synchronized 16-channel time-resolved spectral detector, our PIE-sFLIM system can effectively excite multiple fluorophores and collect their emission over a broad spectrum for analysis. Combining our system with the advanced live-cell labeling techniques and the lifetime/spectral phasor analysis, our PIE-sFLIM approach can well unmix the fluorescence of six fluorophores acquired in a single measurement, thus improving the imaging speed in live-specimen investigation. 

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5. 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)

   Abstract MotivationSpectral 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. ResultsWe 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. Availability and implementationThe 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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