We report on scanning far- and near-field two-photon microscopy of cell nuclei stained with DAPI and bisbenzimidazole Hoechst 33342 (BBI-342) with the 647-nm laser line of a cw ArKr mixed-gas laser. Two-photon-excited fluorescence images are obtained for 50–200 mW of average power at the sample. A nearly quadratic dependence of fluorescence intensity on laser power confirmed the two-photon effect. The nonlinearity was further supported by evidence of three-dimensional sectioning in a scanning far-field microscope. We find that the cw two-photon irradiation sufficient for imaging within typically 5 s does not significantly impair cell cycling of BBI-342-labeled live cells. Finally, high-resolution imaging in scanning near-field microscopy with good contrast is demonstrated.
© 1998 Optical Society of America
(140.3460) Lasers and laser optics : Lasers
(170.0180) Medical optics and biotechnology : Microscopy
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(170.5280) Medical optics and biotechnology : Photon migration
Stefan W. Hell, Martin Booth, Stefan Wilms, Christoph M. Schnetter, Achim K. Kirsch, Donna J. Arndt-Jovin, and Thomas M. Jovin, "Two-photon near- and far-field fluorescence microscopy with continuous-wave excitation," Opt. Lett. 23, 1238-1240 (1998)