Two-photon near- and far-field fluorescence microscopy with continuous-wave excitation.
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.
Hell
S W
SW
High Resolution Optical Microscopy Group, Max Planck Institute for Biophysical Chemistry, D-37070 Göttingen, Germany.
Booth
M
M
Wilms
S
S
Schnetter
C M
CM
Kirsch
A K
AK
Arndt-Jovin
D J
DJ
Jovin
T M
TM