Flow cytometry visualization and real-time processing with a CMOS SPAD array and high-speed hardware implementation algorithm

Mai, Hanning; Poland, Simon P.; Rocca, F.; Treacy, Conor; Aluko, Justin; Nedbal, Jakub; Erdogan, A.T.; Gyöngy, István; Walker, Richard; Ameer-Beg, Simon; Henderson, R.

doi:10.1117/12.2544759

Cited by 3 publications

(3 citation statements)

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“…The data was read out and analysed after the measurement had finished. We note that it is in principle possible to obtain real-time FLIM image displays from average photon arrival times [ 32 , 50 – 57 ], but this method was not employed here.…”

Section: Resultsmentioning

confidence: 99%

Visualising varnish removal for conservation of paintings by fluorescence lifetime imaging (FLIM)

Wilda,

Burnstock,

Suhling

et al. 2023

Herit Sci

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The removal of varnish from the surface is a key step in painting conservation. Varnish removal is traditionally monitored by examining the painting surface under ultraviolet illumination. We show here that by imaging the fluorescence lifetime instead, much better contrast, sensitivity, and specificity can be achieved. For this purpose, we developed a lightweight (4.8 kg) portable instrument for macroscopic fluorescence lifetime imaging (FLIM). It is based on a time-correlated single-photon avalanche diode (SPAD) camera to acquire the FLIM images and a pulsed 440 nm diode laser to excite the varnish fluorescence. A historical model painting was examined to demonstrate the capabilities of the system. We found that the FLIM images provided information on the distribution of the varnish on the painting surface with greater sensitivity, specificity, and contrast compared to the traditional ultraviolet illumination photography. The distribution of the varnish and other painting materials was assessed using FLIM during and after varnish removal with different solvent application methods. Monitoring of the varnish removal process between successive solvent applications by a swab revealed an evolving image contrast as a function of the cleaning progress. FLIM of dammar and mastic resin varnishes identified characteristic changes to their fluorescence lifetimes depending on their ageing conditions. Thus, FLIM has a potential to become a powerful and versatile tool to visualise varnish removal from paintings. Graphical Abstract

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Section: Resultsmentioning

confidence: 99%

Visualising varnish removal for conservation of paintings by fluorescence lifetime imaging (FLIM)

Wilda,

Burnstock,

Suhling

et al. 2023

Herit Sci

Self Cite

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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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“…A new nonparametric empirical Bayesian framework [ 16 ] was adopted for lifetime analysis based on a statistical model, where the expectation–maximization algorithm was employed to solve the optimization problem. A hardware-friendly fitting-free center of mass (CMM) [ 17 , 18 , 19 ] algorithm was proposed to deliver fast analysis and has been applied to a flow-cytometry system [ 20 , 21 ]. Integral equation methods (IEM) [ 22 ] were also implemented in FPGA devices to provide real-time analysis.…”

Section: Introductionmentioning

confidence: 99%

Fast Analysis of Time-Domain Fluorescence Lifetime Imaging via Extreme Learning Machine

Zang

Xiao

Wang

et al. 2022

Sensors

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We present a fast and accurate analytical method for fluorescence lifetime imaging microscopy (FLIM), using the extreme learning machine (ELM). We used extensive metrics to evaluate ELM and existing algorithms. First, we compared these algorithms using synthetic datasets. The results indicate that ELM can obtain higher fidelity, even in low-photon conditions. Afterwards, we used ELM to retrieve lifetime components from human prostate cancer cells loaded with gold nanosensors, showing that ELM also outperforms the iterative fitting and non-fitting algorithms. By comparing ELM with a computational efficient neural network, ELM achieves comparable accuracy with less training and inference time. As there is no back-propagation process for ELM during the training phase, the training speed is much higher than existing neural network approaches. The proposed strategy is promising for edge computing with online training.

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Flow cytometry visualization and real-time processing with a CMOS SPAD array and high-speed hardware implementation algorithm

Cited by 3 publications

References 0 publications

Visualising varnish removal for conservation of paintings by fluorescence lifetime imaging (FLIM)

Visualising varnish removal for conservation of paintings by fluorescence lifetime imaging (FLIM)

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

Fast Analysis of Time-Domain Fluorescence Lifetime Imaging via Extreme Learning Machine

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