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  1. Two-photon excited fluorescence (TPEF) is a powerful technique that enables the examination of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle. Although previous intensity-based TPEF studies in non-human primates have successfully imaged several classes of retinal cells and elucidated aspects of both rod and cone photoreceptor function, fluorescence lifetime imaging (FLIM) of the retinal cells under light-dark visual cycle has yet to be fully exploited. Here we demonstrate a FLIM assay of photoreceptors and retinal pigment epithelium (RPE) that reveals key insights into retinal physiology and adaptation. We found that photoreceptor fluorescence lifetimes increase and decrease in sync with light and dark exposure, respectively. This is likely due to changes in all-trans-retinol and all-trans-retinal levels in the outer segments, mediated by phototransduction and visual cycle activity. During light exposure, RPE fluorescence lifetime was observed to increase steadily over time, as a result of all-trans-retinol accumulation during the visual cycle and decreasing metabolism caused by the lack of normal perfusion of the sample. Our system can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes under different conditions of light and dark exposure.

     
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  2. Mapping molecular deformation and forces in protein biomaterials is critical to understanding mechanochemistry.

     
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    Free, publicly-accessible full text available December 7, 2024
  3. Free, publicly-accessible full text available February 13, 2025
  4. Since the early 1990s, single-molecule detection in solution at room temperature has enabled direct observation of single biomolecules at work in real time and under physiological conditions, providing insights into complex biological systems that the traditional ensemble methods cannot offer. In particular, recent advances in single-molecule tracking techniques allow researchers to follow individual biomolecules in their native environments for a timescale of seconds to minutes, revealing not only the distinct pathways these biomolecules take for downstream signaling but also their roles in supporting life. In this review, we discuss various single-molecule tracking and imaging techniques developed to date, with an emphasis on advanced three-dimensional (3D) tracking systems that not only achieve ultrahigh spatiotemporal resolution but also provide sufficient working depths suitable for tracking single molecules in 3D tissue models. We then summarize the observables that can be extracted from the trajectory data. Methods to perform single-molecule clustering analysis and future directions are also discussed.

     
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  5. In this work, a deep learning-based method, STED-flimGANE, is introduced to achieve enhanced STED imaging resolution under a low STED-beam power and photon-starved conditions.

     
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  6. Periasamy, Ammasi ; So, Peter T. ; König, Karsten (Ed.)
  7. Abstract

    Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to quantify molecular compositions and study molecular states in complex cellular environment as the lifetime readings are not biased by fluorophore concentration or excitation power. However, the current methods to generate FLIM images are either computationally intensive or unreliable when the number of photons acquired at each pixel is low. Here we introduce a new deep learning-based method termedflimGANE(fluorescencelifetimeimaging based onGenerativeAdversarialNetworkEstimation) that can rapidly generate accurate and high-quality FLIM images even in the photon-starved conditions. We demonstrated our model is up to 2,800 times faster than the gold standard time-domain maximum likelihood estimation (TD_MLE) and thatflimGANEprovides a more accurate analysis of low-photon-count histograms in barcode identification, cellular structure visualization, Förster resonance energy transfer characterization, and metabolic state analysis in live cells. With its advantages in speed and reliability,flimGANEis particularly useful in fundamental biological research and clinical applications, where high-speed analysis is critical.

     
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  8. Achilefu, Samuel ; Raghavachari, Ramesh (Ed.)
    Invented in 2010, NanoCluster Beacons (NCBs) (1) are an emerging class of turn-on probes that show unprecedented capabilities in single-nucleotide polymorphism (2) and DNA methylation (3) detection. As the activation colors of NCBs can be tuned by a near-by, guanine-rich activator strand, NCBs are versatile, multicolor probes suitable for multiplexed detection at low cost. Whereas a variety of NCB designs have been explored and reported, further diversification and optimization of NCBs require a full scan of the ligand composition space. However, the current methods rely on microarray and multi-well plate selection, which only screen tens to hundreds of activator sequences (4, 5). Here we take advantage of the next-generation-sequencing (NGS) platform for high-throughput, large-scale selection of activator strands. We first generated a ~104 activator sequence library on the Illumina MiSeq chip. Hybridizing this activator sequence library with a common nucleation sequence (which carried a nonfluorescent silver cluster) resulted in hundreds of MiSeq chip images with millions of bright spots (i.e. light-up polonies) of various intensities and colors. With a method termed Chip-Hybridized Associated Mapping Platform (CHAMP) (6), we were able to map these bright spots to the original DNA sequencing map, thus recovering the activator sequence behind each bright spot. After assigning an “activation score” to each “light-up polony”, we used a computational algorithm to select the best activator strands and validate these strands using the traditional in-solution preparation and fluorometer measurement method. By exploring a vast ligand composition space and observing the corresponding activation behaviors of silver clusters, we aim to elucidate the design rules of NCBs. 
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