Nonlinear confocal imaging and photomanipulation with novel femtosecond laser sources

Nonlinear confocal microscopy provides a novel and promising tool for investigating biological processes in living specimen. The advantage of multi-photon excitation is its confinement to the focal volume. This effect enables a high spatial resolution for imaging, especially in axial direction. As wavelengths around 1 µm are a good compromise between low absorption and low scattering, a deep penetration into thick tissue samples is achieved. Due to their ultrabroadband tuning range, high stability and compactness, femtosecond fiber lasers are a promising alternative to free-space lasers based on e.g. Ti:sapphire [1].

Figure 1: Schematic view of the laser and imaging setup
Figure 2: Nucleus of a HeLa-229 cell after DNA damage induction at 1050 nm. Chromatin is displayed in red (Hoechst 33342), the recruited DNA repair protein XRCC1 in green (XRCC1-GFP). The size of the damage is restricted to a small volume inside the nucleus.

Multi-photon excitation can also be employed to manipulate living cells. One example is the induction of localized damage in DNA via three-photon absorption. The advantage here is that the damage is restricted to the focus of the objective lens. This allows us to watch the recruitment of DNA repair proteins to the lesioned sites [2]. A more detailed investigation of the different types of DNA damage which are induced by femtosecond laser irradiation showed that it is possible to specifically induce DNA strand breaks at an excitation wavelength of 1050 nm, whereas both UV photoproducts and strand breaks are generated pumping at 775 nm [3].

In order to visualize dynamic processes in living cells photoactivatable fluorescent proteins are becoming more and more popular. These proteins can be switched from a non-fluorescent state to a fluorescent state by irradiation at the activation wavelength. This property can be used to determine protein mobilities in live cells. By combining multi-photon DNA damage induction with two-photon photoactivation, the influence of DNA damage binding properties of nuclear proteins can be investigated.

References

[1] D. Träutlein, F. Adler, K. Moutzouris, A. Jeromin, A. Leitenstorfer und E. Ferrando-May
"Highly versatile confocal microscopy system based on a tunable femtosecond Er:fiber source"
J. Biophoton. 1, 53 (2008)

[2] U. Camenisch, D. Träutlein, F. C. Clement, J. Fei, A. Leitenstorfer, E. Ferrando-May und H. Naegeli
"Two-stage dynamic DNA quality check by xeroderma pigmentosum group C protein"
The EMBO Journal 28, 2387 (2009)

[3] D. Träutlein, M. Deibler, A. Leitenstorfer und E. Ferrando-May
"Specific local induction of DNA strand breaks by infrared multi-photon absorption"
Nucleic Acids Res. 38, e14 (2010)