Quantitative non-invasive cell characterisation and discrimination based on multispectral autofluorescence features

Gosnell, Martin E.; Anwer, Ayad G.; Mahbub, Saabah B.; Perinchery, Sandeep Menon; Inglis, David W.; Adhikary, Partho P.; Jazayeri, Jalal; Cahill, Michael A.; Saad, Sonia; Pollock, Carol; Sutton‐McDowall, Melanie L.; Thompson, Jeremy G.; Goldys, Ewa M.

doi:10.1038/srep23453

Cited by 76 publications

(80 citation statements)

References 52 publications

(71 reference statements)

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“…In this work we utilized a fluorescence microscope adapted for multispectral imaging by adding a number of custom-made fluorescent channels (Gosnell et al, 2016a(Gosnell et al, , 2016bHabibalahi et al, 2019;Rehman et al, 2017) to characterize native fluorescence of oocytes. Images were taken in the following channels listed in the table below.…”

Section: Hyperspectral Imagingmentioning

confidence: 99%

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NAD+ Repletion Rescues Female Fertility during Reproductive Aging

et al. 2020

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Graphical Abstract Highlights d Declining NAD(P)H is associated with oocyte dysfunction during reproductive aging d Oocyte quality and fertility can be restored by NMN treatment in aged mice d Supplementation of embryo media with NMN improves developmental milestones d SIRT2 overexpression mimics benefits of NMN but is unlikely to mediate its effects SUMMARYReproductive aging in female mammals is an irreversible process associated with declining oocyte quality, which is the rate-limiting factor to fertility.Here, we show that this loss of oocyte quality with age accompanies declining levels of the prominent metabolic cofactor nicotinamide adenine dinucleotide (NAD + ). Treatment with the NAD + metabolic precursor nicotinamide mononucleotide (NMN) rejuvenates oocyte quality in aged animals, leading to restoration in fertility, and this can be recapitulated by transgenic overexpression of the NAD + -dependent deacylase SIRT2, though deletion of this enzyme does not impair oocyte quality. These benefits of NMN extend to the developing embryo, where supplementation reverses the adverse effect of maternal age on developmental milestones. These findings suggest that late-life restoration of NAD + levels represents an opportunity to rescue female reproductive function in mammals.

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Section: Hyperspectral Imagingmentioning

confidence: 99%

“…To prepare spectral images for quantitative analysis and spectral unmixing, first, a multistep image preparation procedure was carried out (Gosnell et al, 2016a(Gosnell et al, , 2016bMahbub et al, 2017;Vidal and Amigo, 2012). This procedure removes sources of errors, reduces noise and standardizes the spectral images.…”

Section: Hyperspectral Imagingmentioning

confidence: 99%

NAD+ Repletion Rescues Female Fertility during Reproductive Aging

et al. 2020

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“…It has been demonstrated that free and protein-bound NADH also display slightly different emission spectra, with the protein-bound NADH peaking at 440 nm and the free NADH at 470 nm. This difference can be used, for example, to differentiate between cell groups (69,70). Although the discriminating capacity between NADH populations is much smaller than with fluorescence lifetimes, it can increase the confidence of discrimination as an additional parameter (71).…”

Section: Nadh Autofluorescence Spectroscopymentioning

confidence: 99%

NADH Autofluorescence—A Marker on its Way to Boost Bioenergetic Research

et al. 2018

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More than 60 years ago, the idea was introduced that NADH autofluorescence could be used as a marker of cellular redox state and indirectly also of cellular energy metabolism. Fluorescence lifetime imaging microscopy of NADH autofluorescence offers a marker-free readout of the mitochondrial function of cells in their natural microenvironment and allows different pools of NADH to be distinguished within a cell. Despite its many advantages in terms of spatial resolution and in vivo applicability, this technique still requires improvement in order to be fully useful in bioenergetics research. In the present review, we give a summary of technical and biological challenges that have so far limited the spread of this powerful technology. To help overcome these challenges, we provide a comprehensible overview of biological applications of NADH imaging, along with a detailed summary of valid imaging approaches that may be used to tackle many biological questions. This review is meant to provide all scientists interested in bioenergetics with support on how to embed successfully NADH imaging in their research.

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“…The (AFMI) system used in this study is based on an adapted IX83 Olympus microscope as previously reported [17,24,25]. It provides narrow excitation wavelength ranges (±5nm) from high power LEDs and several filter cubes to produce defined spectral regions, which span the excitation (340 nm-510 nm) and emission (420 nm-650 nm) wavelength ranges.…”

Section: Autofluorescence Multispectral Imaging (Afmi)mentioning

confidence: 99%

Non-invasive real-time imaging of reactive oxygen species (ROS) using multispectral auto-fluorescence imaging technique: a novel tool for redox biology

Habibalahi¹,

Moghari

Campbell

et al. 2020

Preprint

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AbstractDetecting reactive oxygen species (ROS) that play a critical role as redox modulators and signalling molecules in biological systems currently requires invasive methods such as ROS - specific indicators for imaging and quantification. We developed a non-invasive, real-time, label-free imaging technique for assessing the level of ROS in live cells and thawed cryopreserved tissues that is compatible with in-vivo imaging. The technique is based on autofluorescence multispectral imaging (AFMI) carried out in an adapted fluorescence microscope with an expanded number of spectral channels spanning specific excitation (365 nm-495 nm) and emission (420 nm-700 nm) wavelength ranges. We established a strong quantitative correlation between the spectral information obtained from AFMI and the level of ROS obtained from CellROX staining. The results were obtained in several cell types (HeLa, PANC1 and mesenchymal stem cells) and in live kidney tissue. Additioanly, two spectral regimes were considered: with and without UV excitation (wavelengths > 400 nm); the latter being suitable for UV-sensitive systems such as the eye. Data were analyzed by linear regression combined with an optimization method of swarm intelligence. This allowed the calibration of AFMI signals to the level of ROS with excellent correlation (R= 0.84, p=0.00) in the entire spectral range and very good correlation (R= 0.78, p=0.00) in the limited, UV-free spectral range. We also developed a strong classifier which allowed us to distinguish moderate and high levels of ROS in these two regimes (AUC= 0.91 in the entire spectral range and AUC = 0.78 for UV-free imaging). These results indicate that ROS in cells and tissues can be imaged non-invasively, which opens the way to future clinical applications in conditions where reactive oxygen species are known to contribute to progressive disease such as in ophthalmology, diabetes, kidney disease, cancer and neurodegenerative diseases.

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Quantitative non-invasive cell characterisation and discrimination based on multispectral autofluorescence features

Cited by 76 publications

References 52 publications

NAD+ Repletion Rescues Female Fertility during Reproductive Aging

NAD+ Repletion Rescues Female Fertility during Reproductive Aging

NADH Autofluorescence—A Marker on its Way to Boost Bioenergetic Research

Non-invasive real-time imaging of reactive oxygen species (ROS) using multispectral auto-fluorescence imaging technique: a novel tool for redox biology

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