Fluorescence lifetime imaging with a megapixel SPAD camera and neural network lifetime estimation

Žičkus, Vytautas; Wu, Ming-Lo; Morimoto, Keiko; Kapitany, Valentin; Fatima, Areeba; Turpin, Alex; Insall, Robert H.; Whitelaw, Jamie; Machesky, Laura M.; Bruschini, Claudio; Faccio, Daniele; Charbon, Edoardo

doi:10.1038/s41598-020-77737-0

Cited by 56 publications

(46 citation statements)

References 67 publications

(76 reference statements)

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“…com/inatamara/NyxSense) and could be used by the community to test, further improve, or simply use the methodology we proposed. Spectrally resolved FLIM is readily available commercially, and several bespoke implementations aimed to make available cost-effective and user-friendly solutions have been also published (e.g., [6,42,43,51,56,58,61,63,76], promising increased availability of such sophisticated assays in the near future. We showed that the combination of spectrally resolved time phasors (sTP → ) with the spectral phasors (SP or TSP) permitted efficient demixing of three FRET pairs, presenting a low level of direct acceptor excitation using only six control signatures.…”

Section: Discussionmentioning

confidence: 99%

Single-Cell Biochemical Multiplexing by Multidimensional Phasor Demixing and Spectral Fluorescence Lifetime Imaging Microscopy

et al. 2021

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Revealing mechanisms underpinning cell function requires understanding the relationship between different biochemical reactions in living cells. However, our capabilities to monitor more than two biochemical reactions in living cells are limited. Therefore, the development of methods for real-time biochemical multiplexing is of fundamental importance. Here, we show that data acquired with multicolor (mcFLIM) or spectrally resolved (sFLIM) fluorescence lifetime imaging can be conveniently described with multidimensional phasor transforms. We demonstrate a computational framework capable of demixing three Forster resonance energy transfer (FRET) probes and quantifying multiplexed biochemical activities in single living cells. We provide a comparison between mcFLIM and sFLIM suggesting that sFLIM might be advantageous for the future development of heavily multiplexed assays. However, mcFLIM—more readily available with commercial systems—can be applied for the concomitant monitoring of three enzymes in living cells without significant losses.

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Section: Discussionmentioning

confidence: 99%

Single-Cell Biochemical Multiplexing by Multidimensional Phasor Demixing and Spectral Fluorescence Lifetime Imaging Microscopy

et al. 2021

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“…Following on this work, Zickus et al. 96 used an MLP trained with simulated data [ Figs. 3(a) and 3(b) ], in combination with image stitching, to retrieve a 3.6-megapixel (

) wide field FLIM image reconstruction using a time-resolved single-photon avalanche diode (SPAD) array.…”

Section: Deep Learning For Macroscopic Fluorescence Lifetime Imagingmentioning

confidence: 99%

Deep learning in macroscopic diffuse optical imaging

et al. 2022

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. Significance : Biomedical optics system design, image formation, and image analysis have primarily been guided by classical physical modeling and signal processing methodologies. Recently, however, deep learning (DL) has become a major paradigm in computational modeling and has demonstrated utility in numerous scientific domains and various forms of data analysis. Aim : We aim to comprehensively review the use of DL applied to macroscopic diffuse optical imaging (DOI). Approach : First, we provide a layman introduction to DL. Then, the review summarizes current DL work in some of the most active areas of this field, including optical properties retrieval, fluorescence lifetime imaging, and diffuse optical tomography. Results : The advantages of using DL for DOI versus conventional inverse solvers cited in the literature reviewed herein are numerous. These include, among others, a decrease in analysis time (often by many orders of magnitude), increased quantitative reconstruction quality, robustness to noise, and the unique capability to learn complex end-to-end relationships. Conclusions : The heavily validated capability of DL’s use across a wide range of complex inverse solving methodologies has enormous potential to bring novel DOI modalities, otherwise deemed impractical for clinical translation, to the patient’s bedside.

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“…Additionally, multispectral FLIM was implemented by Ghezzi et al 9 using an 18 × 1 linear SPAD array. Moreover, Zickus et al 10 aimed for real-time FLIM using a time-gated SPAD array capable of achieving widefield FLIM of 500 × 1024 pixels with lifetime estimation performed by a custom artificial neural network (ANN) that was trained on simulated lifetime decay curves. Given the advantages of the SPAD array (Table 1), this technology is likely to increase in popularity for FLIM.…”

Section: Wide-field Flimmentioning

confidence: 99%

Recent innovations in fluorescence lifetime imaging microscopy for biology and medicine

et al. 2021

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Significance: Fluorescence lifetime imaging microscopy (FLIM) measures the decay rate of fluorophores, thus providing insights into molecular interactions. FLIM is a powerful molecular imaging technique that is widely used in biology and medicine.Aim: This perspective highlights some of the major advances in FLIM instrumentation, analysis, and biological and clinical applications that we have found impactful over the last year.Approach: Innovations in FLIM instrumentation resulted in faster acquisition speeds, rapid imaging over large fields of view, and integration with complementary modalities such as singlemolecule microscopy or light-sheet microscopy. There were significant developments in FLIM analysis with machine learning approaches to enhance processing speeds, fit-free techniques to analyze images without a priori knowledge, and open-source analysis resources. The advantages and limitations of these recent instrumentation and analysis techniques are summarized. Finally, applications of FLIM in the last year include label-free imaging in biology, ophthalmology, and intraoperative imaging, FLIM of new fluorescent probes, and lifetime-based Förster resonance energy transfer measurements.Conclusions: A large number of high-quality publications over the last year signifies the growing interest in FLIM and ensures continued technological improvements and expanding applications in biomedical research.

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Fluorescence lifetime imaging with a megapixel SPAD camera and neural network lifetime estimation

Cited by 56 publications

References 67 publications

Single-Cell Biochemical Multiplexing by Multidimensional Phasor Demixing and Spectral Fluorescence Lifetime Imaging Microscopy

Single-Cell Biochemical Multiplexing by Multidimensional Phasor Demixing and Spectral Fluorescence Lifetime Imaging Microscopy

Deep learning in macroscopic diffuse optical imaging

Recent innovations in fluorescence lifetime imaging microscopy for biology and medicine

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