We present the experimental and theoretical study of the two-photon excited polarized fluorescence of p-terphenyl dissolved in cyclohexane/paraffin. The fluorescence was produced within a two-color two-photon (2C2P) excitation scheme utilizing simultaneous absorption of two femtosecond laser pulses at 400 nm and at 800 nm with the total excitation energy of 4.649 eV. The fluorescence was detected by a time correlated single photon counting (TCSPC) system with two detectors. Using different combinations of the absorbed photon polarizations we extracted seven time-dependent molecular parameters from experiment that contain all information on the dynamics of the three-photon process under study. The analysis of the obtained molecular parameter values was based on the ab initio calculations of the vertical excitation energies and transition matrix elements in p-terphenyl and allowed for determination of the whole structure of the two-photon absorption tensor, fluorescence lifetime, and the rotational correlation time. The obtained results imply that the fluorescence in the conditions of our experiment was governed mostly by the d(z) component of the fluorescence transition dipole moment that is parallel to the molecular long axis Z. The tensor was found to be symmetric. The two-photon excitation in p-terphenyl occurs simultaneously via two channels, one of them resulting in the population of the totally symmetric excited state and the other in the population of the nontotally symmetric excited state. Moreover, the energetically allowed pure electron transitions are dipole forbidden and become allowed by vibronic coupling.

The use of two-color two-photon (2c2p) excitation easily extends the wavelength range of Ti:sapphire lasers to the UV, widening the scope of its applications especially in biological sciences. We report observation of 2c2p excitation fluorescence of p-terphenyl (PTP), 2-methyl-5-t-butyl-p-quaterphenyl (DMQ) and tryptophan upon excitation with 400 and 800 nm wavelengths using the second harmonic and fundamental wavelength of a mode-locked Ti:sapphire femtosecond laser. This excitation is energetically equivalent to a one-photon excitation wavelength at 266 nm. The fluorescence signal is observed only when both wavelengths are spatially and temporally overlapping. Adjustment of the relative delay of the two laser pulses renders a cross correlation curve which is in good agreement with the pulse width of our laser. The fluorescence signal is linearly dependent on the intensity of each of the two colors but quadratically on the total incident illumination power of both colors. In fluorescence microscopy, the use of a combination of intense IR and low-intensity blue light as a substitute for UV light for excitation can have numerous advantages. Additionally, the effect of differently polarized excitation photons relative to each other is demonstrated. This offers information about different transition symmetries and yields deeper insight into the two-photon excitation process.

We present the first realization of a Two-Color Two-Photon Laser-Scanning Microscope (2c2pLSM) and UV fluorescence images of cells acquired with this technique. Fluorescence is induced by two-color two-photon absorption using the fundamental and the second harmonic of a Ti:Sa femtosecond laser. Simultaneous absorption of an 800 nm photon and a 400 nm photon energetically corresponds to one-photon absorption at 266 nm. This technique for Laser-Scanning Microscopy extends the excitation wavelength range of a Ti:Sa powered fluorescence microscope to the UV. In addition to the known advantages of multi-photon microscopy like intrinsic 3D resolution, reduced photo damage and high penetration depth 2c2pLSM offers the possibility of using standard high numeric aperture objectives for UV fluorescence imaging. The effective excitation wavelength of 266 nm corresponds especially well to the excitation spectrum of tryptophan. Hence, it is an ideal tool for label free fluorescence studies and imaging of intrinsic protein fluorescence which originates mainly from tryptophan. Thus a very sensitive natural lifetime probe can be used for monitoring protein reactions or changes in conformation. First measurements of living MIN-6 cells reveal differences between the UV fluorescence lifetimes of the nucleus and cytoplasm. The significance of this method was further demonstrated by monitoring the binding of biotin to avidin.

A short overview of the principles and applications of the two-colour two-photon (2C2P) excitation of fluorescence by using femtosecond pulses is given. Fluorescence is generated by the simultaneous absorption of an 800 nm photon and a 400 nm photon of overlapping laser beams of a titanium:sapphire laser. Two examples of its application are presented: firstly, it is used to monitor the enzymatic cleavage of bovine serum albumin (BSA) by elastase. The fluorescent amino acid tryptophan present in BSA is excited corresponding to an effective one-photon wavelength of 266 nm. Secondly, it is shown how one can utilize the different polarizations of the excited beams for determining the symmetry of the excited states of molecules, exemplarily shown for p-terphenyl in cyclohexane. Further applications and experiments for 2C2P are suggested for using it in UV-fluorescence microscopy and for determining the properties of the electronic states of biomolecules by using differently polarized photons.

In the last decade, the two-photon fluorescence laser-scanning microscopy (TPLSM) has become an indispensable tool for the bioscientific and biomedical research. TPLSM techniques as well as their applications are currently experiencing a dramatic evolution and represent the focus of many biophysical research projects. In this work, we compare in detail two steady-state TPLSM techniques, i.e. single-beam scanning microscopy combined with point-detection (SB-PMT) and multi-beam scanning microscopy combined with synchronous detection (MB-CCD), as far as their technical characteristics relevant for the bioscientific research are concerned, i.e. optical performance and imaging speed. We demonstrate that the SB-PMT technique is more adequate for deep-tissue imaging (few 100 µm depth) than the MB-CCD technique, whereas only the MB-CCD technique enables high-speed imaging for characterizing the dynamics of fast biological phenomena. Novel applications of these techniques are additionally discussed. Moreover, we employ a time-resolved TPLSM technique, i.e. biexponential fluorescence lifetime imaging based on the cellular fluorescence of the nicotinamide pyridine dinucleotides NADH and NADPH, which allows us to probe for the first time the redox cellular metabolism of MIN6 cells (mutated insulin producing pancreatic β-cells) as well as to show the potential of this method for the specific and dynamic investigation of NADH-and NADPH-dependent cellular processes.