Martin Hacker

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.

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.

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

Two‐photon (TP) excitation (820–1150 nm) and emission (280–700 nm) spectra for the fluorescent proteins (FPs) ECFP 3 , EGFP 3 and EYFP 3 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.

Programmable femtosecond laser pulses in the ultraviolet

Using a combination of a zero-dispersion compressor and spectrally compensated sum-frequency generation, we have produced amplitude-modulated femtosecond pulses in the UV at 200 nm.