In this study, we have characterized the high-order photobleaching of the two fluorophores eGFP and Hoechst 33342. For the first time, we examined high-order photobleaching in a large wavelength range and made a comparison to another laser-mediated process in microscopy: intracellular ablation.
The measurements of the multiphoton-order yielded values of approximately two for eGFP, while it increased from two to three with the wavelength for Hoechst (see ). Our findings for Hoechst are in agreement with Gryczynski et al. [24
]. For eGFP, our results correspond to the large two-photon action cross-section [25
] over the whole measured wavelength range.
The photobleaching-order measurements were independent of the repetition rate and NA for both fluorophores (see ). Chen et al. found similar data, concerning the NA, for a mutant of the fluorophore GFP [11
]. Therefore, diffusion processes in the milli- and microsecond regime, as in the reaction with molecular oxygen, should be negligible in high-order photobleaching, as reported by Dittrich et al. [14
]. Our results underline their thesis, that triplet population does not play a key role in high-order photobleaching.
We observed that for normalized fluorescence intensity, the half-life period depended on the wavelength. It increased with the two-photon action cross-section of both fluorophores (data not shown). If photobleaching occurs from the first excited singlet state, the half-life period should be independent of the wavelength [10
]. Therefore, we suggest that bleaching of eGFP and Hoechst occurs from a highly excited state or the ground state.
Our results indicate that eGFP bleaching is dominated by multiphoton-ionization of the fluorophore from ground state. The ionization threshold of the eGFP chromophore is in the range of 4.6 to 6.2 eV [26
]. This could be reached by a quasi-simultaneous absorption of three or four photons (compare with photobleaching-orders in ), but not with a two-photon excitation in the first singlet (2,6 eV [27
]) followed by further sequential-absorptions in higher states. This is underlined by the fact that eGFP is likely missing any relevant states far above the first singlet [27
]. Therefore, high-order photobleaching of eGFP is independent from two-photon excitation. The step increase in the photobleaching-order at a wavelength of approximately 840 nm can be explained by the multiphoton-ionization of a tryptophan residue 13 to 15 Å
] next to the eGFP chromophore. Its ionization energy is 4.5 eV [29
], corresponding to the measured transition wavelength of 840 nm divided by the number of interacting photons. This leads to the production of a hydroxyl radical or a free electron [30
] and therefore to bleaching by chemical deconstruction. Hence, bleaching is mediated by multiphoton-ionization of the chromophore itself and the tryptophan residue. As multiphoton-ionization becomes less effective with increasing wavelength, the concurrent rise of the half-life period can be well explained.
A study by Bourgeois et al. [31
] suggests that the intracellular ablation threshold also has a cubic dependence on excitation power for GFP at a wavelength of 720 nm. For this reason we conclude, that high-order photobleaching as well as ablation of eGFP involves three or four photons and occurs via multiphoton-ionization from ground state. Furthermore, both processes provide free electrons.
We presume that our findings for the photobleaching-order of Hoechst (see ) are closely related to a two-photon excitation in the first singlet followed by several sequential-absorption events in higher states. In contrast to eGFP, Hoechst has electronic states above the first singlet [32
] in which another one-photon absorption is likely because of the very large photon density [8
]. Bleaching is then evoked by one or more one-photon absorptions after excitation into the first singlet. These transitions are not counted in photobleaching-order, as they are saturated [8
]. This is why our findings for the photobleaching-order of Hoechst are similar to the ones for the multiphoton-order (compare and ). By reaching the ionic state (5.5 eV [33
]) or a highly excited state, the Hoechst fluorophore forms an electron-cation pair, which can split up in a polar environment [13
]. Kuetemeyer et al. reported ablation threshold dependencies on the fourth and fifth order of laser power at 720 and 950 nm for Hoechst-stained biomolecules, respectively [22
]. As ablation is mediated by multiphoton-ionization, we can conclude that the same amount of photons is involved (see ) and free electrons are provided in both processes.
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 (more ...)
If we take into account the formation of free electrons, we have to point out that high-order photobleaching likely produces a low-density plasma [16
]. This is quite similar to ablation. By observing a linear dependence of the half-life period on multiphoton image-size (data not shown), we found that high-order photobleaching is also an accumulation of single-pulse events [22
]. Therefore, we are convinced that there is a smooth transition between high-order photobleaching and ablation.
The augmentation of reactive oxygen species correlating with bleaching over time (see ), indicates the formation of a low-density plasma. Reactive oxygen species are produced by the interaction of biomolecules, water or oxygen with free electrons [16
]. Thus, the cell gets confronted with oxidative stress, but intracellular radical scavengers like ascorbic acid or glutathione [21
] are able to neutralize this, as we found no major effects on cell viability. However, a significant effect of the low-density plasma became obvious in the non-direct bleaching of Hoechst in the eGFP expressing RAT1 cells (see ). It likely results from the interaction of free electrons or reactive oxygen species with dye molecules.
According to the literature, a mammalian cell nucleus with a volume of about 100 fl [34
] contains about 6 · 108
Hoechst molecules at the used dye concentration [35
]. Therefore, the average distance of two Hoechst molecules was 2rH
≈ 7 nm. A concentration of at least 0.2 µ
M eGFP molecules is necessary to make eGFP fluorescence stronger than autofluorescence [36
]. As eGFP fluorescence was relatively strong in our experiments, we assume that the eGFP concentration was about 1 µ
M, corresponding to 60,000 molecules in the cell nucleus. Also taking the ratio of relative fluorescence intensity loss Rloss
≈ 0.7 (see ) into account, we were able to estimate the interaction range of electrons or ROS with fluorophores, equal to the low-density plasma radius:
where C is the ratio between Hoechst and GFP molecules in the nucleus. The low-density plasma radius was calculated to be in the range of 30 to 50 nm corresponding well to the diffusion length of singlet oxygen being less than 50 nm [36
] in cells. As the low-density plasma influences fluorophore-molecules, it is possible that biomolecules were attacked, too. This issue needs further investigations.
To conclude, we have shown that high-order photobleaching is quite similar to ablation. Both processes are equal regarding the amount of involved photons and the accumulation of single-pulse events. High-order photobleaching, however, is mediated by sequential-absorption and multiphoton-ionization, while ablation is dominated by the latter and additionally occurring cascade-ionization processes. For this reason, the involved energetic states are not always the same. We propose that there are smooth transitions between laser-mediated processes in the low-density plasma regime. This comprises the transitions from sequential-absorption to multiphoton-ionization and finally cascade-ionization, both depending on the fluorophores and biomolecules as the major source for free electrons.