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LASER DIAGNOSTICS AND SPECTROSCOPY
Fluorescence lifetime imaging of porous silicon nanoparticles in heart
muscle cells
M.B. Gongalsky1, D. Akimov2, E. Tolstik3, V. Sivakov2, L.A. Osminkina1
1- Lomonosov Moscow State University, Faculty of Physics, Leninskie Gory 1, 119991 Moscow, Russia 2- Leibniz Institute of Photonic Technology, Albert-Einstein-Strafie 9, 07745 Jena, Germany 3- Leibniz-Institut fur Analytische Wissenschaften - ISAS - e.V., Bunsen-Kirchhoff-Strafie 11, 44139 Dortmund, Germany Email: mgongalsky@gmail.com
Porous silicon nanoparticles (pSiNPs) are promising biocompatible and biodegradable agents for various diagnostics and therapy including anticancer treatments [1]. In aqueous media pSiNPs demonstrate a variety of remarkable fluorescent properties attributed to recombination in Si or SiOx phases [2]. Simple models are based on quantum confinement effect of charge carriers in Si/SiO2 core/shell structures. That provides relatively long fluorescence decay times in the range of 1-100 ms due to indirect band gap of Si. However, under strong excitation very fast recombination (1-10 ps) may occur in the same core/shell structures [3]. Differences of the decay time allow to discriminate objects in fluorescence lifetime imaging technique (FLIM). This work is aimed to demonstrate capabilities of advanced time-resolved bioimaging of pSiNPs in biological systems. Previously it was shown that time-gated approach for imaging of pSiNPs can increase signal-to-noise ratio about 1000 times [4]. But it has never been tested in non-linear regime.
In present study we used mesoporous silicon nanoparticles, obtained by consequent electrochemical etching of Si wafers (orientation - (100), specific resistivity 1-10 mQ*cm) in HF solution and grinding in a planetary ball mill (balls and glasses - ZnO2). As a result 200-nm pSiNPs with porosity about 50-70%, pores and nanocrystals sizes about 10 nm are formed. However, during the formation, storage in aqueous solutions and incubation in cell cultures pSiNPs were additionally oxidized and core/shell structure with reduced 3-6 nm Si core were formed. PSiNPs were incubated with cardiac myoblast cells (H9c2) for 24 hours. The bioimaging of pSiNPs inside H9c2 cells is important, because they can potentially induce cardiotoxicity by themselves or by a drug loaded into their pores.
Images of pSiNPs in H9c2 were obtained by an inverted microscope at wavelength of 746 nm, which approximately corresponds to maximum of PL emission spectrum of pSiNPs. Fluorescence was excited by two photons with wavelength of 833 nm generated by a femtosecond Ti:sapphire laser. PSiNPs were distinguished on FLIM images by their ultrafast and slow fluorescence times. Ultrafast decay times was less than 100 ps, which was much faster than typical decay times of biological compounds (1-5 ns). Slow decay times were longer than 100 ns and appeared as visually constant component. Therefore, they were attributed to microsecond-scale recombination of excitons confined in Si/SiO2 quantum dots.
Thus, we demonstrated for the first time, that FLIM can be used for imaging of porous silicon nanoparticles inside human cells. They can be distinguished with very high signal-to-noise ratio by their specific lifetimes, which are much shorter or much longer than typical autofluorescence lifetime. The proposed approach is promising for monitoring of silicon nanoparticles as containers for drug delivery or sono/photosensitizers in anticancer treatments.
This work was supported by the Russian Science Foundation (Grant № 22-75-10107).
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