Multimodal Nonlinear Microscopy for Therapy Monitoring of Cold Atmospheric Plasma Treatment

Abstract

Here we report on a non-linear spectroscopic method for visualization of cold atmospheric plasma (CAP)-induced changes in tissue for reaching a new quality level of CAP application in medicine via online monitoring of wound or cancer treatment. A combination of coherent anti-Stokes Raman scattering (CARS), two-photon fluorescence lifetime imaging (2P-FLIM) and second harmonic generation (SHG) microscopy has been used for non-invasive and label-free detection of CAP-induced changes on human skin and mucosa samples. By correlation with histochemical staining, the observed local increase in fluorescence could be assigned to melanin. CARS and SHG prove the integrity of the tissue structure, visualize tissue morphology and composition. The influence of plasma effects by variation of plasma parameters e.g., duration of treatment, gas composition and plasma source has been evaluated. Overall quantitative spectroscopic markers could be identified for a direct monitoring of CAP-treated tissue areas, which is very important for translating CAPs into clinical routine.

Coherent Anti-Stokes Raman Scattering Microscopy and Its Applications

“…Another recent trend has been focusing on label-free multimodal nonlinear microscopy based on CARS, twophoton excited fluorescence (TPEF), and SHG [91][92][93][94]. The coupling of two-photon fluorescence, second-harmonic generation, and coherent anti-Stokes Raman scattering has allowed for investigation of a broad range of biological phenomena concerning lipid metabolism, cancer development, cardiovascular disease, and skin biology.…”

FLIM data analysis based on Laguerre polynomial decomposition and machine-learning

“…The cell data were measured from a single fibroblast cell on the system described in Ref. 23 . Briefly, two synchronized picosecond (ps) pulse trains generated by a Ti: sapphire laser (831.7 nm) and an optical parametric oscillator (APE, Germany) (672.3 nm) are focused onto the cell by a objective (C-Apochromat, NA 1.1, Zeiss, Germany).…”