Spatial resolution enhancement in photon-starved STED imaging using deep learning-based fluorescence lifetime analysis

Chen, Yuan‐I; Chang, Yin-Jui; Sun, Yuansheng; Liao, Shih-Chu Jeff; Santacruz, Samantha R.; Yeh, Hsin‐Chih

doi:10.1039/d3nr00305a

Cited by 7 publications

(3 citation statements)

References 49 publications

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“…The Fourier Transform was applied to the acquired fluorescence decay histogram to analyze the FLIM data in the digital frequency domain [ 48 – 50 ]. The fluorescence decay histogram of each pixel was converted into a specific phasor and then plotted as a single point in the phasor plot under the corresponding modulation frequency condition ( ω k ).…”

Section: Methodsmentioning

confidence: 99%

Assessing the impact of extracellular matrix fiber orientation on breast cancer cellular metabolism

Pickett,

Chen,

Kamra

et al. 2024

Cancer Cell Int

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The extracellular matrix (ECM) is a dynamic and complex microenvironment that modulates cell behavior and cell fate. Changes in ECM composition and architecture have been correlated with development, differentiation, and disease progression in various pathologies, including breast cancer [1]. Studies have shown that aligned fibers drive a pro-metastatic microenvironment, promoting the transformation of mammary epithelial cells into invasive ductal carcinoma via the epithelial-to-mesenchymal transition (EMT) [2]. The impact of ECM orientation on breast cancer metabolism, however, is largely unknown. Here, we employ two non-invasive imaging techniques, fluorescence-lifetime imaging microscopy (FLIM) and intensity-based multiphoton microscopy, to assess the metabolic states of cancer cells cultured on ECM-mimicking nanofibers in a random and aligned orientation. By tracking the changes in the intrinsic fluorescence of nicotinamide adenine dinucleotide and flavin adenine dinucleotide, as well as expression levels of metastatic markers, we reveal how ECM fiber orientation alters cancer metabolism and EMT progression. Our study indicates that aligned cellular microenvironments play a key role in promoting metastatic phenotypes of breast cancer as evidenced by a more glycolytic metabolic signature on nanofiber scaffolds of aligned orientation compared to scaffolds of random orientation. This finding is particularly relevant for subsets of breast cancer marked by high levels of collagen remodeling (e.g. pregnancy associated breast cancer), and may serve as a platform for predicting clinical outcomes within these subsets [3–6].

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Section: Methodsmentioning

confidence: 99%

Assessing the impact of extracellular matrix fiber orientation on breast cancer cellular metabolism

Pickett,

Chen,

Kamra

et al. 2024

Cancer Cell Int

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“…In particular, the SPLIT method can separate the long-lived and short-lived fluorescence signal generated from the inner and outer parts of the STED microscopy probed region, respectively. 25,26,39,40 The SPLIT method represents the two decays as a linear combination of phasors. Thus the unmixing problem of the different components is solved by the SPLIT method by inverting a simple linear system (see Note 3 and Figs.…”

Section: Flism For Nanoscopy Imagingmentioning

confidence: 99%

Compact and effective photon-resolved image scanning microscope

Tortarolo,

Zunino,

Piazza

et al. 2024

Adv. Photon.

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.Fluorescence confocal laser-scanning microscopy (LSM) is one of the most popular tools for life science research. This popularity is expected to grow thanks to single-photon array detectors tailored for LSM. These detectors offer unique single-photon spatiotemporal information, opening new perspectives for gentle and quantitative superresolution imaging. However, a flawless recording of this information poses significant challenges for the microscope data acquisition (DAQ) system. We present a DAQ module based on the digital frequency domain principle, able to record essential spatial and temporal features of photons. We use this module to extend the capabilities of established imaging techniques based on single-photon avalanche diode (SPAD) array detectors, such as fluorescence lifetime image scanning microscopy. Furthermore, we use the module to introduce a robust multispecies approach encoding the fluorophore excitation spectra in the time domain. Finally, we combine time-resolved stimulated emission depletion microscopy with image scanning microscopy, boosting spatial resolution. Our results demonstrate how a conventional fluorescence laser scanning microscope can transform into a simple, information-rich, superresolved imaging system with the simple addition of a SPAD array detector with a tailored data acquisition system. We expected a blooming of advanced single-photon imaging techniques, which effectively harness all the sample information encoded in each photon.

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“…Given the photon-arrival time histogram of the probed region, a computational alternative can solve the hardware time-gated detection limitations, providing the same resolution improvement but without a strong SNR reduction. In particular, the SPLIT method can separate the long-lived and short-lived fluorescence signal generated from the inner and outer parts of the STED microscopy probed region, respectively [25,26,37,38]. The SPLIT method represents the two decays as a linear combination of phasors.…”

Section: Flism For Nanoscopy Imagingmentioning

confidence: 99%

A Compact and Effective Photon-Resolved Image Scanning Microscope

Tortarolo

Zunino

Piazza

et al. 2023

Preprint

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Fluorescence confocal laser-scanning microscopy (LSM) is one of the most popular tools for life science research. This popularity is expected to grow thanks to single-photon array detectors tailored for LSM. These detectors offer unique single-photon spatiotemporal information, opening new perspectives for gentle and quantitative super-resolution imaging. However, a flawless recording of this information poses significant challenges for the microscope data-acquisition system. Here, we present a data-acquisition module based on the digital frequency domain principle, able to record photons' essential spatial and temporal features. We use this module to extend the capabilities of established imaging techniques based on single-photon avalanche diode (SPAD) array detectors, such as fluorescence lifetime image scanning microscopy. Furthermore, we use the module to introduce a robust multi-species approach encoding the fluorophore's excitation spectra in the time domain. Lastly, we combine time-resolved stimulated emission depletion microscopy with image scanning microscopy, boosting spatial resolution. Our results demonstrate how a conventional fluorescence laser scanning microscope can transform into a simple, information-rich, super-resolved imaging system with the simple addition of a SPAD array detector with a tailored data acquisition system. We expected a blooming of advanced single-photon imaging techniques which effectively harness all the sample information encoded in each photon.

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Spatial resolution enhancement in photon-starved STED imaging using deep learning-based fluorescence lifetime analysis

Abstract: 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.

Cited by 7 publications

References 49 publications

Assessing the impact of extracellular matrix fiber orientation on breast cancer cellular metabolism

Assessing the impact of extracellular matrix fiber orientation on breast cancer cellular metabolism

Compact and effective photon-resolved image scanning microscope

A Compact and Effective Photon-Resolved Image Scanning Microscope

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