A fluorescent ESIPT-based benzimidazole platform for the ratiometric two-photon imaging of ONOO–in vitro and ex vivo

In this work, we have developed an ESIPT benzimidazole-based platform for the two-photon cell imaging of ONOO– and a potential ONOO–-activated theranostic scaffold.


Measurement of Two-Photon Cross Section
The two-photon cross section (δ) was measured using Ti-sapphire femtosecond laser (Mai Tai HP).

Two-Photon Microscopy Imaging
RAW264.7 macrophages were cultured in DMEM (WelGene) for two days. Before imaging, medium was changed to serum free medium and the chosen ESIPT probe (5 μM) was added. Images were acquired using multiphoton microscopes (Leica TCS SP8 MP) with Ti-sapphire femtosecond laser. Excitation wavelength was 740 nm and 2.49 W output power, which corresponded to 1.20  10 6 W cm -2 average power in the focal plane.

Cell viability.
CCK-8 kit (AbCareBio CL) assay was conducted to assess the cytotoxicity of each probe. RAW264.7 macrophages were cultured in 96-well plate for one day, and then difference concentrations of MO-E2 (0-50 μM) were added. After incubation for 2 h, absorbances were measured at 450 nm.

Photostability
Photostability of MO-E2 in RAW264.7 macrophages was conducted by monitoring the changes in fluorescence intensities, which was induced by two photon excitation. The fluorescence intensities were obtained 1800 signals with 2 sec intervals for 1 h. The fluorescence intensities were collected at 380-600 nm upon excitation at 740 nm.

Preparation and Staining of Fresh Rat Hippocampal Slices
A 14-day-old SD rats was used to perform tissue imaging. Rat hippocampal slices were cut into 0.4 mm thickness and transferred to glass-bottomed dishes (NEST). Slices were stained with MO-E2 (50 μM) in DPBS (WelGene) and incubated for 1.5 h. Slices were washed with DPBS and observed using multiphoton microscopes.      9. Cell studies. a) λmax of the two-photon emission spectra in nm. b) Φ is the fluorescence quantum yield and δmax is the twophoton action cross sections in GM (1 GM = 10 -50 cm 4 s photon -1 ).