Photobleaching and phototoxicity of mitochondria in live cell fluorescent super-resolution microscopy

Abstract

Photobleaching and phototoxicity can induce detrimental effects on cell viability and compromise the integrity of collected data, particularly in studies utilizing super-resolution microscopes. Given the involvement of multiple factors, it is currently challenging to propose a single set of standards for assessing the potential of phototoxicity. The objective of this paper is to present empirical data on the effects of photobleaching and phototoxicity on mitochondria during super-resolution imaging of mitochondrial structure and function using Airyscan and the fluorescent structure dyes Mitotracker green (MTG), 10-N-nonyl acridine orange (NAO), and voltage dye Tetramethylrhodamine, Ethyl Ester (TMRE). We discern two related phenomena. First, phototoxicity causes a transformation of mitochondria from tubular to spherical shape, accompanied by a reduction in the number of cristae. Second, phototoxicity impacts the mitochondrial membrane potential. Through these parameters, we discovered that upon illumination, NAO is much more phototoxic to mitochondria compared to MTG or TMRE and that these parameters can be used to evaluate the relative phototoxicity of various mitochondrial dye-illumination combinations during mitochondrial imaging.

Keywords: Fluorescent dye; Mitochondria; Photobleaching; Phototoxicity; Super-resolution.

Fig. 1.

Assessing photobleaching in time-lapse imaging with NAO and MTG. (A) Image of mitochondria stained with NAO and MTG before and after exposure to 488 nm illumination for 300 s. (B) NAO and MTG fluorescence intensities versus frames. N ≥ 3 independent experiments. (Intensity: 0.5 %; Pixel time: 3.54 μs; Time frame: 1 s).

Fig. 2.

Phototoxicity causes mitochondria ultrastructure to be destroyed. (Green) Time-lapse images of mitochondria stained with 100 nM NAO in HeLa cells using Zeiss Airyscan. (Red) Image with STED after sufficient time to cause mitochondria to become spherical shows similar morphology. N ≥ 3 independent experiments. (HeLa cell).

Fig. 3.

Mitochondria segmentation and structure analysis. The binarization mask is used for single mitochondria analysis. The statistical analysis shows the change of circularity of mitochondria in cells through the time-lapse experiment. The mitochondria in the HeLa cell were stained with 100 nM NAO (Intensity: 0.5 %; Pixel time: 3.54 μs; Time frame: 1 s).

Fig. 4.

Assessing NAO phototoxicity in loss of mitochondria membrane potential. (A) Time-lapse images of mitochondria in HeLa cells stained with 10 nM TMRE and 100 nM NAO, and exposure to 488 nm and 561 nm illuminations (Intensity: 0.5 %; Pixel time: 1.43 μs; Time frame: 1 s). (B) TMRE fluorescence intensity of mitochondria exposure to 561 nm only illuminations versus time.

Fig. 5.

Mitochondria stained with 10 nM NAO and 10 nM TMRE also experienced a rapid loss of fluorescence, loss of membrane potential, and morphological deformation when excited with 488/561 nm lasers. When excited with a 561 nm laser only, we did not observe the fast drop in TMRE intensity.

Fig. 6.

5 μM, 100 nM, and 10 nM NAO excited with 488 nm laser and measure emissions in 500–550 nm, 550–600 nm, 600–650 nm, 650–700 nm emission ranges.

Fig. 7.

Assessing cytotoxic effect through prolonged incubation time of NAO dyes in HEK293 cells. The HEK293 cells were stained with 100 nM NAO and 10 nM TMRE for 24 h before imaging. The mitochondria retained their tubular shapes and membrane potential. N = 3 independent experiments.

Fig. 8.

Phototoxicity was restricted in the illuminated region of the cell. Live cell mitochondria stained with NAO only showed structural swelling and lost mitochondrial membrane potential in the region exposed to the 488 nm illumination. N ≥ 3 independent experiments.