Two-photon near- and far-field fluorescence microscopy with continuous-wave excitation |
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Authors: | Hell S W Booth M Wilms S Schnetter C M Kirsch A K Arndt-Jovin D J Jovin T M |
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Affiliation: | High Resolution Optical Microscopy Group, Max Planck Institute for Biophysical Chemistry, D-37070 G?ttingen, Germany. |
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Abstract: | 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. |
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