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9 October 2003 New two-photon excitation chromophores for cellular imaging
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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.
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Laura D'Alfonso, Giuseppe Chirico, Maddalena Collini, Giancarlo Baldini, Alberto Diaspro, Paola Ramoino, Alessandro Abbotto, Luca Beverina, and Giorgio A. Pagani "New two-photon excitation chromophores for cellular imaging", Proc. SPIE 5139, Confocal, Multiphoton, and Nonlinear Microscopic Imaging, (9 October 2003);

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