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1. 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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2. Spectral scanning and fluorescence lifetime imaging microscopy (FLIM) enable separation and characterization of C. elegans autofluorescence in the cuticle and gut

   https://doi.org/10.1242/bio.060613  

   Hulsey-Vincent, Heino J; Cameron, Elizabeth A; Dahlberg, Caroline L; Galati, Domenico F (December 2024, Biology Open)

   ABSTRACT Caenorhabditis elegans gut and cuticle produce a disruptive amount of autofluorescence during imaging. Although C. elegans autofluorescence has been characterized, it has not been characterized at high resolution using both spectral and fluorescence lifetime-based approaches. We performed high resolution spectral scans of whole, living animals to characterize autofluorescence of adult C. elegans. By scanning animals at 405 nm, 473 nm, 561 nm, and 647 nm excitations, we produced spectral profiles that confirm the brightest autofluorescence has a clear spectral overlap with the emission of green fluorescent protein (GFP). We then used fluorescence lifetime imaging microscopy (FLIM) to further characterize autofluorescence in the cuticle and the gut. Using FLIM, we were able to isolate and quantify dim GFP signal within the sensory cilia of a single pair of neurons that is often obscured by cuticle autofluorescence. In the gut, we found distinct spectral populations of autofluorescence that could be excited by 405 nm and 473 nm lasers. Further, we found lifetime differences between subregions of this autofluorescence when stimulated at 473 nm. Our results suggest that FLIM can be used to differentiate biochemically unique populations of gut autofluorescence without labeling. Further studies involving C. elegans may benefit from combining high resolution spectral and lifetime imaging to isolate fluorescent protein signal that is mixed with background autofluorescence and to perform useful characterization of subcellular structures in a label-free manner. 

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3. Generative adversarial network enables rapid and robust fluorescence lifetime image analysis in live cells

   https://doi.org/10.1038/s42003-021-02938-w  

   Chen, Yuan-I; Chang, Yin-Jui; Liao, Shih-Chu; Nguyen, Trung Duc; Yang, Jianchen; Kuo, Yu-An; Hong, Soonwoo; Liu, Yen-Liang; Rylander, III, H. Grady; Santacruz, Samantha R.; et al (January 2022, Communications Biology)

   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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4. 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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5. Noninvasive metabolic profiling of cumulus cells, oocytes, and embryos via fluorescence lifetime imaging microscopy: a mini-review

   https://doi.org/10.1093/humrep/dead063  

   Venturas, Marta; Yang, Xingbo; Sakkas, Denny; Needleman, Dan (April 2023, Human Reproduction)

   Abstract A major challenge in ART is to select high-quality oocytes and embryos. The metabolism of oocytes and embryos has long been linked to their viability, suggesting the potential utility of metabolic measurements to aid in selection. Here, we review recent work on noninvasive metabolic imaging of cumulus cells, oocytes, and embryos. We focus our discussion on fluorescence lifetime imaging microscopy (FLIM) of the autofluorescent coenzymes NAD(P)H and flavine adenine dinucleotide (FAD+), which play central roles in many metabolic pathways. FLIM measurements provide quantitative information on NAD(P)H and FAD+ concentrations and engagement with enzymes, leading to a robust means of characterizing the metabolic state of cells. We argue that FLIM is a promising approach to aid in oocyte and embryo selection. 

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