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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.
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