New Two-Photon Excitation Chromophores For Cellular Imaging. Laura D'alfonso ^ , Giuseppe Chirico *^ , Maddalena Collini ^ , Giancarlo Baldini ^ , Alberto Diaspro £ , Paola Ramoino $ , Alessandro Abbotto * , Luca Beverina * , Giorgio Pagani * ^ INFM and Dipartimento di Fisica, Università di Milano Bicocca £ INFM and Dipartimento di Fisica, Università di Genova $ Dipartimento per lo Studio del territorio de delle sue risorse, Università di Genova * Dipartimento di Scienza dei Materiali, Università di Milano Bicocca ABSTRACT The one photon and two photon excitation spectral properties (absorption, emission spectra, singlet lifetime) of a very efficient two photon absorber, dimethyl-pepep, have been measured in solution. The one photon excitation peak lye near 525 nm and the emission falls at 600 nm, where autofluorescence of cells is weak. The value of the singlet- triplet conversion rate, obtained by two-photon excitation fluorescence correlation spectroscopy, has a quadratic dependence on the excitation power and is comparable to that shown by the dye rhodamine. Preliminary results on stained cells from yeast Saccaromices cerevisiae and Paramecium primaurelia show that the dye preferentially stains DNA in the cell. A direct comparison with a DNA stainer, Dapi, is also performed. Some measurements of the dye functionalized to react with lysine and n-terminal residues of protein are presented. Moreover, this dye can be employed in order to follow in detail some cellular processes such as nuclei division. In vitro fluorescence titration of dimethyl-pepep with calf thymus DNA allowed to estimate the values of the dye-DNA association constant versus ionic strength, and an affinity close to that of ethidium bromide is found. Keywords: Two-photon excitation microscopy, Fluorescence lifetime, Fluorescence Imaging. 1. INTRODUCTION Cellular imaging is a long standing technique for the investigation of cell structure and functions. In particular, fluorescence microscopy has been widely adopted since it offers to investigate the details of a variety of physiological behaviors in the cell. Fluorescence microscopy, when coupled to confocal detection, offers enhanced resolution and three dimensional (3D) sectioning capabilities with respect to other optical microscopy techniques 1 . The selectivity for specific physiological behaviors within the cell is obtained by using fluorophores sensitive to the environment changes, such as Ca++, Mg++, K+ concentrations, voltage drops across membranes or the membrane polarity. More recently two-photon excitation microscopy has broken through the biomedical research, with a variety of cellular and tissue applications. The main advantage of this technique particularly for cellular applications is the intrinsic high spatial resolution, reduced Raman scattering and fluorescence background from the cell, and the reduced out of focus bleaching 2 . However the bleaching of the dyes in the scanning plane may be enhanced when compared to the conventional single photon confocal microscopy case. A wide effort is therefore being undertaken in order to develop efficient two-photon absorbers with high quantum efficiency and two-photon cross-section and with low bleaching for two-photon excitation 3-5 . We give here a detailed characterization employing both one and two photon excitation of an efficient two-photon absorber, dimethyl-pepep. This dye has a fluorescence brightness (the absorption cross-section times the quantum yield) as high as 1/10 of that of fluorescein 6 . Fluorescence correlation spectroscopy (FCS) has also been used in conjunction with photon counting histogram (PCH) technique, in order to determine the molecular brightness and the existence of a triplet state, whose conversion rate to the excited singlet state has been quantified. * Piazza della Scienza 3, 20126, Milano Italy; Tel.39-02-64482872; Fax:-39-02-64482894; Email: giuseppe.chirico@unimib.it.