Continuous wave two-photon scanning near-field optical microscopy.
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.
Kirsch
A K
AK
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Subramaniam
V
V
Striker
G
G
Schnetter
C
C
Arndt-Jovin
D J
DJ
Jovin
T M
TM
D016475:3T3 Cells; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002467:Cell Nucleus / Q000648:ultrastructure; D002875:Chromosomes / Q000648:ultrastructure; D004331:Drosophila melanogaster; D005456:Fluorescent Dyes; D007211:Indoles; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D017785:Photons
D005456:Fluorescent Dyes; D007211:Indoles; C007293:DAPI