Reactive oxygen species (ROS) play an essential role as cellular messengers, functioning as redox regulators under normal physiological conditions. However, the excessive production of ROS in cells and organs due to disorders such as diabetes mellitus, inflammation, cardiovascular, cancer, and neurodegenerative disease leads to oxidative stress, which may be an early indication of progressive pathology . Currently, ROS -specific indicators which requires labelling are used for ROS quantification. Therefore, a label-free imaging technique is desirable for assessing the level of ROS. In this work, we introduced a novel imaging method to quantitatively identify ROS in cells and tissues named autofluorescence multispectral imaging (AFMI). This technique involved a custom-built spectral imaging system with 18 spectral channels with distinctive excitation and emission wavelength spanning specific excitation (365 nm-495 nm) and emission (420 nm-700 nm) wavelength ranges. Such a system can extract rich spectral information related to the ROS present in cells and tissue. We correlated the spectral information obtained from AFMI to the level of ROS acquired from CellROX imaging, which served the reference ROS value in this study. Further, our analyses were repeated for UV sensitive applications where an excitation spectrum less than 400nm was avoided. Quantitative analysis of the spectral images showed a strong multispectral signature correlating the spectral variable with the ROS level in the cells and tissue. Our results showed that ROS levels can be determined non-invasively using AFMI, which potentially can be translated to future clinical applications where ROS are known to correlate with progressive disease.
Our approach to multispectral fluorescence microscopy can non-invasively identify the biomolecular composition of cells and capture complex biological heterogeneity which is fundamentally important for biological research and medical diagnostics. We have applied this technology to demonstrate the embryo quality for chromosomal abnormalities (containing euploid and aneuploidy cells) and understanding the biochemical signatures of polycystic ovarian syndrome (PCOS) oocytes. We then explored oocyte quality following treatment with the NAD+ precursor NMN. These findings demonstrate the utility of our approach to the multispectral assessment of autofluorescence for the non-destructive, label-free assessment of clinically relevant problems.
Pain is currently assessed using subjective measurements, often not aligning with clinical symptoms. Therefore, objective pain level assessments, using minimally-invasive and molecular methods, are needed to assess disease activity and response to treatment in osteoarthritis and rheumatoid arthritis. We report sophisticated quantitative biochemical “signatures” from the label-free hyperspectral imaging (HSI) of cartilage tissue for the characterization of molecular composition, structure and functional status. Further study on sinuvium tissue provides evidence that HSI could be used as a novel technique to delineate disease state. Additionally, HSI could be used to objectively separate individuals based on pain severity providing molecular correlates of pain.
Type 1 diabetes occurs when insulin secreting beta cells in pancreatic islets are destroyed leading to elevated glucose and ill health. Islet transplantation is an effective therapy, but islets are often damaged by the isolation process resulting in numerous, repeated transplants to achieve insulin independence. We have applied hyperspectral microscopy to damaged islets and have shown through the assessment of native cell autofluorescence we can detect heterogenous forms of damage (elevated ROS, inflammatory signalling and warm ischemia) in mouse islets. This approach has great potential to be translated clinically to minimise the burden of suboptimal islet transplantations.
we developed mitochondrially targeted biodegradable polymerpoly(lactic-co-glycolic acid) nanocarriers incorporating a photosensitiser verteporfin, ultrasmall (2-5 nm) gold nanoparticles as radiation enhancers and triphenylphosphonium acting as the mitochondrial targeting moiety. Upon X-ray radiation our nanocarriers generated cytotoxic amounts of singlet oxygen within the mitochondria, triggering the loss of membrane potential and mitochondria-related apoptosis of cancer cells. Our X-PDT strategy effectively controlled tumour growth with only a fraction of radiotherapy dose (4 Gy) and improved the survival rate of a mouse model bearing colorectal cancer cells. It may offer a paradigm-shifting treatment alternative for patients who need neoadjuvant radiotherapy but wish to avoid long term detrimental effect on functional outcome by undergoing X-PDT using only a fraction of the conventional radiotherapy.
Automated, unbiased methods of non-invasive cell monitoring able to deal with complex biological heterogeneity are fundamentally important for biomedical research. Label-free imaging provides information about endogenous autofluorescent metabolites, enzymes and cofactors. Our multispectral fluorescence imaging technique allows precise quantification of native fluorophores in cells and tissues. This study uses label-free multispectral analysis to extract different fluorophores and redox ratio from single cells (oocytes, cultured cancer cells) as well as blastocyst embryos. Additionally, we characterise the molecular composition, structure and functional status of ex vivo healthy bovine and osteoarthritic human knee articular cartilage to assess 2 types of experimental treatments.
Multispectral assessment of cell autofluorescence gives a direct window into the molecular processes occurring within those cells. This can be used to non-invasively characterise and classify various cellular properties without requiring fixation, dyes or transformation. Human mesenchymal stem/ stromal cells (MSCs) have great potential to contribute to regenerative medicine, especially with regards to autologous transplantation. However, this capacity is often limited by inherent properties of cell lines, which prevent their being sufficiently expanded after derivation for effective clinical application. The investigation of these properties requires numerous, time and labour-intensive assays. In this study we have used correlative microscopy based on multispectral images of cell autofluorescence then correlated to functional assays in order to construct multispectral signatures of numerous inherent cell characteristics. These included cell cycle status (indicating the proportion of cells undergoing cell division at a given time), cell ‘age’ (number of passages undergone, indicating capacity for further expansion), and β- galactosidase (a marker of senescence, indicating cells which can no longer divide). This study has established a single protocol, in place of multi-functional assays, to characterize the growth and differentiation capacity of hMSC lines using a non-invasive approach.
Despite its wide-spread use, the success rate of assisted reproductive technologies including in vitro fertilization is less than 20%. Most human embryos are mosaic for chromosome abnormalities: containing cells that are euploid (normal) and aneuploid (incorrect number of chromosomes). Currently, a cell biopsy is used in IVF clinics to diagnose aneuploidy in the embryo but this does not provide a diagnosis of how many cells are aneuploid in the entire embryo. Hence, the development of a non-invasive tool to determine the proportion of aneuploid cells would likely improve IVF success. Aneuploidy in human embryos leads to altered metabolism. The co-factors utilized in cellular metabolism are autofluorescent and can be used to predict the metabolic state of cells. Here we used hyperspectral imaging to noninvasively assess intracellular fluorophores and thus metabolism. In this study, we utilized a powerful model of embryo aneuploidy where we generate mouse embryos with differing ratios of euploid:aneuploid cells. We also used primary human fibroblast cells with known aneuploidies to make comparison with euploid cells. Hyperspectral imaging of 1:3 chimeric embryos showed a distinct spectral profile compare to the control/normal embryos. The abundance of FAD in the inner cell mass (cells that form the fetus) of euploid and aneuploid blastocysts was strikingly different. For human cell lines, we were able to clearly distinguish between euploid and aneuploid with different karyotypes. These data show hyperspectral imaging is able to distinguish cells based on their ploidy status making it a promising tool in assessing embryo mosaicism.
Automated and unbiased methods of non-invasive cell monitoring able to deal with complex biological heterogeneity are fundamentally important for biological research and medical diagnostics. Label-free cell imaging provides information about endogenous autofluorescent metabolites, enzymes and cofactors in cells. However, extracting high content information from autofluorescence imaging has been hitherto impossible. Here, we developed a multispectral fluorescence imaging technique which allows precise quantification of the native fluorophores in cells and tissues. With that approach we are now able to non-invasively image the aspects of biomolecular composition of cells and tissues; where many of these fluorophores (NADH, flavins, cytochrome C) are relevant to metabolism. We will discuss label-free detection of reactive oxygen species (ROS) and the cell cycle. Cell cycle and metabolism have a tight, bidirectional relationship, with the ability of the cell to commit to growth depending on the availability of metabolites, and the molecular mechanisms of the cell-cycle being linked to the regulation of metabolic networks. Cells entering the cell cycle increase glycolysis as they go from G1-phase into S-phase, this results in accumulation of the NADH relative to FAD which is also fluorescent.
Moreover, metabolic dysregulation is common across the spectrum of diseases, this next-generation methodology is able to detect major health conditions including neurodegeneration and cancer. This work also reports on approaches for early diagnosis of motor neurone disease (MND) and localisation of cancer margins for ocular surface squamous neoplasia. Our optimal discrimination approach (extracted features for treatment monitoring in MND and melanoma) enables statistical hypothesis testing and intuitive visualisations where previously undetectable differences become clearly apparent.
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