Quantitative image analysis and cell imaging of peroxynitrite based on an upconversion luminescence total internal reflection single particle imaging platform

With the rapid development of science and technology, single particle imaging technology has been greatly improved, and many more sensitive optical microscopes have been rapidly developed. Herein, we propose a single particle imaging method for quantitative image analysis and cell imaging of peroxynitrite based on upconversion luminescence total internal reflection imaging platform. Cyanine-dye-assembled composite upconversion nanoparticles (UCNPs) were prepared and used as luminescent probe. For the quantitative image analysis method, the numbers of photons are obtained by calculating the average brightness of 550 frames of sequence images based on the Brownian motion of the luminescent nanoparticles. Compared to the fluorescence spectrometer method, the quantitative image analysis method for sensing peroxynitrite has many advantages such as less sample consumption (≤10 μL), fast detection time (~0.1 s) and low detection limit (0.067 μM). Due to the unique advantages of UCNPs luminescent materials, such as low biotoxicity, near-infrared emission and high penetration depth, the luminescent materials are also applied to image and semiquantitatively detect of exogenous peroxynitrite in living cells based on the single particle imaging platform. This work not only greatly improves the efficiency in vitro study, but also provides a simple, sensitive and convenient semiquantitative detection method for single particle imaging of living cells.