Photo-Induced Cell Damage Analysis for Single- and Multifocus Coherent Anti-Stokes Raman Scattering Microscopy

Abstract

In this study, we investigated photo-induced damage to living cells during single-and multifocus excitations for coherent antiStokes Raman scattering (CARS) imaging. A near-infrared pulsed laser (709 nm) was used to induce cell damage. We compared the photo-induced cell damage in the single-and the multifocus excitation schemes with the condition to obtain the same CARS signal in the same frame rate. For the evaluation of cell viability, we employed 4′,6-diamidino-2-phenylindole (DAPI) fluorophores that predominantly stained the damaged cells. One-and two-photon fluorescence of DAPI fluorophores were, respectively, excited by an ultraviolet light source and the same near-infrared light source and were monitored to evaluate the cell viability during near-infrared pulsed laser irradiation. We found lower uptake of DAPI fluorophores into HeLa cells during the multifocus excitation compared with the single-focus excitation scheme in both the one-and the two-photon fluorescence examinations. This indicates a reduction of photo-induced cell damage in the multifocus excitation. Our findings suggested that the multifocus excitation scheme is expected to be suitable for CARS microscopy in terms of minimal invasiveness.

Live-imaging of Bioengineered Cartilage Tissue using Multimodal Non-linear Molecular Imaging

“…However, with CARS microscopy, a number of questions remain as to whether: (i) live-imaging using CARS microscopy is fully non-invasive; (ii) cell development remains unaltered; and (iii) the cells remain viable and robust for further use in clinical applications following live-imaging with CARS microscopy. Studies have reported on thresholds of photo-induced cell damage by CARS microscopy, commonly by visualising direct cell morphological changes 13 , and detecting formation of apoptotic membrane protrusions 14 , or by analysing and comparing nuclear staining between damaged and non-damaged cells after laser exposure 15 . The induced damage and changes are obvious at the levels of damage thresholds.…”

Label-Free Biomedical Imaging Using High-Speed Lock-In Pixel Sensor for Stimulated Raman Scattering

“…This figure shows that the SRS image of the lipid droplet is successfully obtained at the resonance Raman shift. For future works, SRS lock-in CMOS pixel array will be used in conjunction with multi-focus irradiation where less photo-damage is achieved [ 18 , 19 ]. Imaging time can be drastically improved and imaging at video rate using small CMOS image sensor could be achieved.…”

“…In single beam irradiation method, higher laser power is needed in order to achieve high-speed imaging. Considering the photo-damage to the biological sample is mainly induced by multiphoton events, which is proportional to the peak power of the incident laser, higher laser power increases the potential of greater photo-damage to the biological samples [ 18 , 19 , 28 ]. For single-focus and multi-focus systems obtaining signals at the same frame rate, multi-focus imaging requires longer exposure time but lesser peak laser power on each point.…”

“…For single-focus and multi-focus systems obtaining signals at the same frame rate, multi-focus imaging requires longer exposure time but lesser peak laser power on each point. Such a technique reduces the induced photo-damage to the biological samples [ 18 , 19 ]. Conventional SRS imaging requires a separate high-frequency lock-in amplifier for each channel to demodulate the small SRS signal, which hinders parallel detection, as such system leads to higher hardware complexity and unrealistic cost.…”

“…For single-focus and multi-focus systems obtaining signals at the same frame rate, multi-focus imaging requires longer exposure time but lesser peak laser power on each point. Such a technique reduces the induced photo-damage to the biological samples, which is highly demanded, especially for living cell imaging [ 18 , 19 ]. The parallel detection of SRS signals needs a customized pixel that has a function of demodulation of the modulated laser to extract weak SRS signal in huge background, and high modulation/demodulation frequency to reduce the influence of the substantially-large laser 1/f noise.…”