Abstract
Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20–24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm−2 at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm−2, emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.
Highlights
Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently
To use the limited photon budget efficiently in live-cell experiments and reduce photobleaching and phototoxicity, low irradiation intensities confined to micron-thin planes[1], e.g., light-sheet and Bessel beam plane illumination microscopy, have been used in combination with super-resolution structured illumination microscopy[2,3,4]
We used U2OS, COS-7 and HeLa cells seeded in petri dishes with an imprinted 500 μ m relocation grid, irradiated them under localization microscopy conditions (0–3 kW cm−2) and observed them afterwards for 20–24 hours under standard culture conditions using an automated cell observation system
Summary
We used U2OS, COS-7 and HeLa cells seeded in petri dishes with an imprinted 500 μ m relocation grid, irradiated them under localization microscopy conditions (0–3 kW cm−2) and observed them afterwards for 20–24 hours under standard culture conditions using an automated cell observation system. Photodamage analysis was based on the fact whether the irradiated cells appear healthy and show cell division during the 20–24 hour after irradiation or not. In each experiment we irradiated 20–50 cells and counted the number of ‘dead’ (apoptotic + f rozen) cells (Table 1). We performed irradiation experiments with U2OS cells at different irradiation intensities (0–3 kW cm−2) at 514 nm for 240 s (Fig. 2 and Supplementary Fig. 2). ‘frozen’ 254.1 313.9 331.6 253.6 n.d. 211.6 n.d
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