Ruprecht-Karls-Universitšt Heidelberg

Overview Electrophysiology Nonlinear Microscopy Calcium Imaging Modelling Image Processing Single Molecules

Nonlinear Microscopy

Second Harmonic Generation

SHG signals from myofibrils
Figure 1 SHG signals from myofibrils change under rotation of the excitation laser polarization
Figure 2 Two-photon excited fluorescence of Bodipy-Fl labeled actin in skeletal muscle

Second harmonic generation (or frequency doubling) is an effect that can be observed in certain nonlinear optical media, which emit radiation at a doubled frequency 2w when excited by an external electric field of the frequency w. The effect is well known for a number of crystals like quartz, but was also discovered for some biological structures like collagen or skeletal muscle myosin-II.

We are interested in the physical properties of second harmonic generation in these biological structures and applications for tissue imaging. The SHG signal intensity, for example, depends on the fiber orientation and the polarization of the incident light. Theoretical predictions for this dependency can be derived from standard equations considering crystal symmetries and then be tested with our experimental setup.

Multiphoton Fluorescence

Two or more photons of an infrared laser can be absorbed simultaneously by a fluorescent molecule to promote it to an excited state. The emission wavelength is the same as for standard single photon excitation.

As the effect of multi-photon excitation needs high energy densities, it occures mainly in the small center of the laser focus. Fluorescence is limited to this spot, which provides multi-photon microscopes with inherent high resolution. The main advantage over confocal microscopes is the higher resolution in deep tissue layers.