Two-photon excitation and emission spectra of the green fluorescent protein variants ECFP, EGFP and EYFP.

Summary Two-photon (TP) excitation (820-1150 nm) and emission (280-700 nm) spectra for the fluorescent proteins (FPs) ECFP3, EGFP3 and EYFP3 produced in human tumour cells were recorded. TP excitation spectra of pure and highly enriched samples were found to be more differentiated in comparison with their one-photon (OP) spectra. They exhibited more pronounced main and local maxima, which coincided among different purity grades within small limits. TP and OP emission spectra of pure and enriched samples were identical. However, in crude samples, excitation was slightly blue-shifted and emission red-shifted. The data indicate that both OP and TP excitation routes led to the same excited states of these molecules. The emission intensity is dependent on the pH of the environment for both types of excitation; the emission intensity maximum can be recorded in the alkaline range. Reconstitution of emission intensity after pH quenching was incomplete, albeit that the respective spectral profiles were identical to those prequenching. When emission data were averaged over the whole range of excitation, the resulting emission profile and maximum coincided with the data generated by optimal excitation. Therefore, out-of-maximum excitation, common practice in TP excitation microscopy, can be used for routine application.

Femtosecond-pulse sequence compression by Gires-Tournois interferometers.

We demonstrate the compression of femtosecond-pulse sequences by phase-modulating resonators, such as Gires-Tournois interferometers. The experiments are based on the precompensation of the complex phase response of the resonator by a high-resolution liquid-crystal pulse shaper. This method can be utilized for lowering peak intensities at critical points in optical setups, as well as for encryption or decryption of ultra-short pulses.

Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging.

Two-photon absorption and emission spectra for fluorophores relevant in cell imaging were measured using a 45 fs Ti:sapphire laser, a continuously tuneable optical parametric amplifier for the excitation range 580-1150 nm and an optical multichannel analyser. The measurements included DNA stains, fluorescent dyes coupled to antibodies as well as organelle trackers, e.g. Alexa and Bodipy dyes, Cy2, Cy3, DAPI, Hoechst 33342, propidium iodide, FITC and rhodamine. In accordance with the two-photon excitation theory, the majority of the investigated fluorochromes did not reveal significant discrepancies between the two-photon and the one-photon emission spectra. However, a blue-shift of the absorption maxima ranging from a few nanometres up to considerably differing courses of the spectrum was found for most fluorochromes. The potential of non-linear laser scanning fluorescence microscopy is demonstrated here by visualizing multiple intracellular structures in living cells. Combined with 3D reconstruction techniques, this approach gives a deeper insight into the spatial relationships of subcellular organelles.

Optimization of Kalpha bursts for photon energies between 1.7 and 7 keV produced by femtosecond-laser-produced plasmas of different scale length.

The conversion efficiency of a 90 fs high-power laser pulse focused onto a solid target into x-ray Kalpha line emission was measured. By using three different elements as target material (Si, Ti, and Co), interesting candidates for fast x-ray diffraction applications were selected. The Kalpha output was measured with toroidally bent crystal monochromators combined with a GaAsP Schottky diode. Optimization was performed for different laser intensities as well as for different density scale lengths of a preformed plasma. These different scale lengths were realized by prepulses of different intensities and delay times with respect to the main pulse. Whereas the Kalpha yield varied by a factor of 1.8 for different laser intensities, the variation of the density scale length could provide a gain factor up to 4.6 for the Kalpha output.

Iterative Fourier transform algorithm for phase-only pulse shaping.

We demonstrate the adaptation of an iterative Fourier transform algorithm for the calculation of theoretical spectral phase functions required for pulse shaping applications. The algorithm is used to determine the phase functions necessary for the generation of different temporal intensity profiles. The performance of the algorithm is compared to two exemplary standard approaches. i.e. a Genetic Algorithm and a combination of a Simplex Downhill and a Simulated Annealing algorithm. It is shown that the iterative Fourier transform algorithm converges much faster than both alternative methods.