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. 2011 Mar 4;2(4):805-16.
doi: 10.1364/BOE.2.000816.

Mechanisms of high-order photobleaching and its relationship to intracellular ablation

S Kalies, K Kuetemeyer, A Heisterkamp

Mechanisms of high-order photobleaching and its relationship to intracellular ablation

S Kalies et al. Biomed Opt Express. .

Abstract

In two-photon laser-scanning microscopy using femtosecond laser pulses, the dependence of the photobleaching rate on excitation power may have a quadratic, cubic or even biquadratic order. To date, there are still many open questions concerning this so-called high-order photobleaching. We studied the photobleaching kinetics of an intrinsic (enhanced Green Fluorescent Protein (eGFP)) and an extrinsic (Hoechst 33342) fluorophore in a cellular environment in two-photon microscopy. Furthermore, we examined the correlation between bleaching and the formation of reactive oxygen species. We observed bleaching-orders of three and four for eGFP and two and three for Hoechst increasing step-wise at a certain wavelength. An increase of reactive oxygen species correlating with the bleaching over time was recognized. Comparing our results to the mechanisms involved in intracellular ablation with respect to the amount of interacting photons and involved energetic states, we found that a low-density plasma is formed in both cases with a smooth transition in between. Photobleaching, however, is mediated by sequential-absorption and multiphoton-ionization, while ablation is dominated by the latter and cascade-ionization processes.

Keywords: (170.1020) Ablation of tissue; (170.3880) Medical and biological imaging; (180.4315) Nonlinear microscopy; (190.4180) Multiphoton processes.

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Figures

Fig. 1.
Fig. 1.
Schematic set-up for multiphoton imaging and logging of photobleaching kinetics.
Fig. 2.
Fig. 2.
Wavelength-dependence of the multiphoton-order for Hoechst 33342 and eGFP. Each data point represents the mean ± standard deviation of at least five experiments. The multiphoton-order was about two over the whole wavelength range for eGFP, while it increased from two up to three for Hoechst.
Fig. 3.
Fig. 3.
Wavelength-dependence of the photobleaching-order for eGFP at different laser-parameters. The dotted line illustrates its behaviour. Each data point represents the mean ± standard deviation of at least five experiments. While the photobleaching-order was independent of repetition rate and NA, there was a step increase of one at about 840 nm.
Fig. 4.
Fig. 4.
Wavelength-dependence of the photobleaching-order for Hoechst at different repetition rates. The dotted line illustrates its behaviour. Each data point represents the mean ± standard deviation of at least five experiments. The bleaching-order varied slightly around two from 720 up to 900 nm and increased to three at 950 nm.
Fig. 5.
Fig. 5.
Correlation of ROS formation and high-order photobleaching. (a) Multiphoton images from different points of time during photobleaching. ROS concentration increased during the drop in Hoechst fluorescence. Scale bar: 8 µm. (b) Time-dependence of ROS formation in comparison to Hoechst photobleaching for three half-life periods. Each data point represents the mean ± standard deviation of at least five cells. The half-life period of photobleaching and the half-saturation value of ROS formation were approximately at the same time (yellow box).
Fig. 6.
Fig. 6.
Influence of eGFP photobleaching on the degradation of Hoechst molecules in its environment. (a) Multiphoton images of Hoechst and eGFP before and after photobleaching of eGFP. Scale bar: 16 µm. (b) Loss of relative Hoechst fluorescence intensity referred to an absolute bleaching of eGFP for different half-life periods and both repetition rates. Each data point represents the mean ± standard error of at least eight experiments. The loss of Hoechst fluorescence intensity referred to an absolute bleaching of eGFP is within the range of 50–80%.
Fig. 7.
Fig. 7.
Schematic of high-order photobleaching and ablation of Hoechst molecules. Violet arrows correspond to wavelengths from 720 up to 920 nm, yellow arrows to 920 nm and above. In photobleaching two or three photons evoke the excitation of Hoechst and another two photons are sequentially absorbed in saturated one photon transitions, while in ablation multiphoton-ionization occurs by the quasi-simultaneous absorption of four or five photons.
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