Multiphoton Femtosecond Microscopy for 3D Imaging
of Intracellular Fluorophores
König, K. and Halbhuber, K.-J.
Institute of Anatomy II, University Jena, Teichgraben 7, D-07743 Jena, Germany
Multiphoton microscopy, in particular two-photon microscopy, is a novel type of 3D microscopy based on the simultaneous absorption of two photons. Near infrared photons can be used for excitation of visible intracellular and intramolecular fluorescence with high spatial resolution in a sub-femtoliter volume. To perform high scan rate two-photon 3D fluorescence imaging, the application of ultrashort laser pulses of moderate peak power but low average power (pulsed microbeams) is required. In near infrared femtosecond microscopes, non-destructive imaging of two-photon excited fluorophores in various human and culture cells was demonstrated for < 2 mW average powers, < 200 W peak powers, and 400 GW/cm2 intensities (700-800 nm, ~150 fs, ~100 MHz). In contrast to conventional one-photon microscopy, there are no photobleaching and destructive effects in out-of-focus regions because the fluorophore excitation is limited to the sub-femtoliter focus volume of the high numerical aperture objective. In addition to inherent 3D spatial resolution in two-photon scanning microscopes, high temporal resolution in the picosecond range can be used to perform fluorescence lifetime imaging (" 
mapping, 4D-microscopy). Time-resolved, two-photon autofluorescence images of the coenzyme NADH in spermatozoa and Chinese hamster ovar cells are presented. In particular, the influence of UVA photostress on the intracellular redox state was studied. In addition, three dimensional 780 nm excited fluorescence images of Fura2-labeled macrophages during phagocytosis of green emitting microspheres were obtained.
| LOCATION |
DATE |
TIME |
| Lecture Hall II |
Sunday, April 5 |
03:00 pm |