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Free keywords:
Animal; Biophysics; Cell Nucleus/ul [Ultrastructure]; Chromosomes/ul [Ultrastructure]; Drosophila melanogaster; Fluorescent Dyes; Indoles; Mice; Microscopy, Fluorescence/is [Instrumentation]; *Microscopy, Fluorescence/mt [Methods]; Photons; Support, Non-U.S. Gov't; 3T3 Cells; 2-photon fluorescence microscopy; Living cells; Laser; Protein; Dna; Spectroscopy; Liquids; Dapi
Abstract:
We have implemented continuous-wave two-photon excitation of near-UV absorbing fluorophores in a scanning near-field optical microscope (SNOM). The 647-nm emission of an Ar-Kr mixed gas laser was used to excite the UV-absorbing DNA dyes DAPI, the bisbenzimidazole Hoechst 33342, and ethidium bromide in a shared aperture SNOM with uncoated fiber tips. Polytene chromosomes of Drosophila melanogaster and the nuclei of 3T3 Balb/c cells labeled with these dyes were readily imaged. The fluorescence intensity showed the expected nonlinear (second order) dependence on the excitation power in the range of 8-180 mW. We measured the fluorescence intensity as a function of the tip-sample displacement in the direction normal to the sample surface in the single- and two-photon excitation modes (SPE, TPE). The fluorescence intensity decayed faster in TPE than in SPE.
