CONTINUOUS-WAVE 2-PHOTON SCANNING NEAR-FIELD OPTICAL MICROSCOPY

We have implemented continuous-wave two-photon excitation of near-UV a bsorbing fluorophores in a scanning near-field optical microscope (SNO M). 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, a nd ethidium bromide in a shared aperture SNOM with uncoated fiber tips . Polytene chromosomes of Drosophila melanogaster and the nuclei of 3T 3 Balb/c cells labeled with these dyes were readily imaged. The fluore scence intensity showed the expected nonlinear (second order) dependen ce on the excitation power in the range of 8-180 mW. We measured the f luorescence intensity as a function of the tip-sample displacement in the direction normal to the sample surface in the single- and two-phot on excitation modes (SPE, TPE). The fluorescence intensity decayed fas ter in TPE than in SPE.