Non-destructive, label free identification of cell cycle phase in cancer cells by multispectral microscopy of autofluorescence

Abstract

BackgroundCell cycle analysis is important for cancer research. However, available methodologies have drawbacks including limited categorisation and reliance on fixation, staining or transformation. Multispectral analysis of endogenous cell autofluorescence has been shown to be sensitive to changes in cell status and could be applied to the discrimination of cell cycle without these steps.MethodsCells from the MIA-PaCa-2, PANC-1, and HeLa cell lines were plated on gridded dishes and imaged using a multispectral fluorescence microscope. They were then stained for proliferating cell nuclear antigen (PCNA) and DNA intensity as a reference standard for their cell cycle position (G1, S, G2, M). The multispectral data was split into training and testing datasets and models were generated to discriminate between G1, S, and G2 + M phase cells. A standard decision tree classification approach was taken, and a two-step system was generated for each line.ResultsAcross cancer cell lines accuracy ranged from 68.3% (MIA-PaCa-2) to 73.3% (HeLa) for distinguishing G1 from S and G2 + M, and 69.0% (MIA-PaCa-2) to 78.0% (PANC1) for distinguishing S from G2 + M. Unmixing the multispectral data showed that the autofluorophores NADH, FAD, and PPIX had significant differences between phases. Similarly, the redox ratio and the ratio of protein bound to free NADH were significantly affected.ConclusionsThese results demonstrate that multispectral microscopy could be used for the non-destructive, label free discrimination of cell cycle phase in cancer cells. They provide novel information on the mechanisms of cell-cycle progression and control, and have practical implications for oncology research.

Non-invasive, label-free optical analysis to detect aneuploidy within the inner cell mass of the preimplantation embryo

“…Therefore, it is tempting to postulate that aneuploidy may accelerate this process of change in metabolic pathway for mouse blastomeres and potentially chimeric blastocysts, leading to the observed altered metabolic profile. Collectively, our results align with the current understanding of how aneuploid cells display altered cellular metabolism compared to euploid cells (Williams, et al , 2008, Zhu, et al , 2018) and confirms the capability of label-free hyperspectral microscopy to discriminate cells based on cellular autofluorescence (Campbell, et al , 2019, Gosnell, et al , 2016b, Mahbub, et al , 2019).…”

“…As metabolites and co-enzymes of metabolic pathways are naturally fluorescent, including nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADPH) and flavins, there is potential to exploit this fluorescence as a non-invasive indicator of aneuploidy. Importantly, these endogenous fluorophores can be directly distinguished by their excitation and emission profiles without the need for exogenous fluorescence labels (Campbell, et al , 2019). Both NAD(P)H and flavins are increasingly used to reflect cellular metabolism in oocytes and early embryos (Aparicio, et al , 2013, Dumollard, 2004, Sugimura, et al , 2014, Sutton-McDowall, et al , 2016, Thompson, et al , 2016).…”

“…This technique excites endogenous fluorophores with a wide range of excitation and emission wavelengths as opposed to using single (or dual) excitation wavelength(s). Notably, hyperspectral imaging has demonstrated the capability to discriminate between different types of cancer cells (Campbell, et al , 2019, Gosnell, et al , 2016b), bovine embryos with good and poor developmental potential (Santos Monteiro, et al , 2020, Sutton-McDowall, et al , 2017), as well as quantifying the metabolic content of oocytes from young and aged mice (Bertoldo, et al , 2020).…”

Non-invasive assessment of exfoliated kidney cells extracted from urine using multispectral autofluorescence features

“…This data yields the spectral profile of autofluorescent features for each cell, including parameters such as average channel intensities, channel intensity ratio 12 , pixel standard deviations and skewness 13 . This gives each cell a unique signature, arising directly from its intracellular biochemistry and organisation, which has been used in the literature for the sensitive discrimination of specific cellular characteristics, which have included cell cycle stage 14 , the presence of inflammatory disease 15 , levels of reactive oxygen species 16 , neoplasia 10 , 17 and age 18 . We have previously demonstrated increased oxidative stress markers in the proximal tubules of animals with early diabetic or obesity related kidney disease 19 , 20 .…”

“…Oxidative stress is a key factor affecting kidney function 21 , 22 . Multispectral assessment of cell autofluorescence has been specifically shown to be sensitive to metabolic changes and oxidative stress 14 , 16 . As such, it could be expected that the application of multispectral autofluorescence microscopy to exfoliated renal tubule cells will enable the sensitive, non-invasive characterisation of kidney dysfunction, providing a new window into physiology of this deep organ.…”