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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. Tracking Gold Nanorods’ Interaction with Large 3D Pancreatic-Stromal Tumor Spheroids by Multimodal Imaging: Fluorescence, Photoacoustic, and Photothermal Microscopies

   https://doi.org/10.1038/s41598-020-59226-6  

   Darrigues, Emilie ; Nima, Zeid A. ; Nedosekin, Dmitry A. ; Watanabe, Fumiya ; Alghazali, Karrer M. ; Zharov, Vladimir P. ; Biris, Alexandru S. ( February 2020 , Scientific Reports)

   Pancreatic cancer is one of the most complex types of cancers to detect, diagnose, and treat. However, the field of nanomedicine has strong potential to address such challenges. When evaluating the diffusion and penetration of theranostic nanoparticles, the extracellular matrix (ECM) is of crucial importance because it acts as a barrier to the tumor microenvironment. In the present study, the penetration of functionalized, fluorescent gold nanorods into large (>500 μm) multicellular 3D tissue spheroids was studied using a multimodal imaging approach. The spheroids were generated by co-culturing pancreatic cancer cells and pancreatic stellate cells in multiple ratios to mimic variable tumor-stromal compositions and to investigate nanoparticle penetration. Fluorescence live imaging, photothermal, and photoacoustic analysis were utilized to examine nanoparticle behavior in the spheroids. Uniquely, the nanorods are intrinsically photoacoustic and photothermal, enabling multi-imaging detection even when fluorescence tracking is not possible or ideal.

    

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3. 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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4. Blinking-Based Multiplexing (BBM): Harnessing Molecular Photophysics for Single-Emitter Classification

   Wustholz, K. ; Hoy, G. ; DeSalvo, G ; Kogan, I. ; Haile, S. ; Smith, E. ( April 2023 , Bulletin of the American Physical Society)

   Single-molecule fluorescence approaches have revolutionized biological and materials microscopy. However, many questions can only be addressed by multicolor imaging of multiple targets, a capability that is limited by the small subset of available, well-performing, and spectrally-distinct fluorescent probes. We recently introduced an alternative single-molecule multiplexing approach termed blinking-based multiplexing (BBM), wherein individual molecules are classified on the basis of their intrinsic blinking dynamics. We demonstrate accurate (>93.5%) binary classification of spectrally-overlapped rhodamine and quantum dot emitters using BBM, even when substantial blinking heterogeneity is observed. Classification can be accomplished using change point detection (CPD) analysis of blinking dynamics or a deep learning (DL) algorithm, the latter of which provides up to 96.6% accuracy. Here, we use CPD and DL algorithms to probe the excitation power, environmental, and molecular dependence of BBM. In addition to providing new opportunities in single-molecule spectroscopy and imaging, BBM represents a new take on single-molecule research, where blinking dynamics can be harnessed for more than just traditional localization or nanoreporting. 

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5. Extended depth-of-field light-sheet microscopy improves imaging of large volumes at high numerical aperture

   https://doi.org/10.1063/5.0101426  

   Keomanee-Dizon, Kevin ; Jones, Matt ; Luu, Peter ; Fraser, Scott E. ; Truong, Thai V. ( October 2022 , Applied Physics Letters)

   Light-sheet microscopes must compromise among field of view, optical sectioning, resolution, and detection efficiency. High-numerical-aperture (NA) detection objective lenses provide higher resolution, but their narrow depth of field inefficiently captures the fluorescence signal generated throughout the thickness of the illumination light sheet when imaging large volumes. Here, we present ExD-SPIM (extended depth-of-field selective-plane illumination microscopy), an improved light-sheet microscopy strategy that solves this limitation by extending the depth of field (DOF) of high-NA detection objectives to match the thickness of the illumination light sheet. This extension of the DOF uses a phase mask to axially stretch the point-spread function of the objective lens while largely preserving lateral resolution. This matching of the detection DOF to the illumination-sheet thickness increases the total fluorescence collection, reduces the background, and improves the overall signal-to-noise ratio (SNR), as shown by numerical simulations, imaging of bead phantoms, and imaging living animals. In comparison to conventional light sheet imaging with low-NA detection that yields equivalent DOF, the results show that ExD-SPIM increases the SNR by more than threefold and dramatically reduces the rate of photobleaching. Compared to conventional high-NA detection, ExD-SPIM improves the signal sensitivity and volumetric coverage of whole-brain activity imaging, increasing the number of detected neurons by over a third. 

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