S.Pakalnis, V.Sitas, H.Schneckenburger, R.Rotomskis
Picosecond absorption spectroscopy of biologically active pigments NADH, FMN and fluorescence marker Rhodamine-123
Materials and Methods 

Rh-123, NADH and FMN were obtained from Fluka (Neu-Ulm, Germany) and were used without further purification. The crude of the chemicals were dissolved in aqueous solution or phosphate buffer solution (PBS) at pH 7.0. There was no differences whether phosphate buffer or aqueous solution were used in our experiments.

Picosecond time resolved absorption spectrophotometer is based on the Nd3+ glass laser as a driving generator. A single pulse selected from a pulse train is amplified, then two beams are formed. The first beam, after its frequency has been triplicated, is used for sample excitation. A picosecond continuum source of D2O pumped by the second beam of the main irradiation was used for sample probing. As a result, a sensitivity of >10-3 optical density units can be obtained, the sample absorption being ~ 1 unit of optical density units. The time resolution of the spectrophotometer is up to 3 ps. The measurements were made in a 1 mm cuvette. The absorption of the sample at the excitation wavelength (third harmonic of neodymium glass laser ( l= 352 nm)) varied from 0.1 to 0.8 in optical density. The duration of pump and probe pulses was about 5 ps.

The measurements of the difference absorption spectra DA are based on the detection of absorption spectra of buffer solutions at different delay times between pump and probe pulses:

D A(l ,t)=A(l ,0) - A(l ,t),

where A(l ,0) is the absorption spectrum of the sample before excitation and A(l ,t) is the absorption spectrum at the selected delay time t between pump and probe pulses. Generally, the spectrum DA(l ,t) includes the effects of absorption bleaching due to a decrease of the ground state population, induced absorption from the excited state of biomolecules, as well as amplification of theprobing pulse by stimulated emission. 

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