Harvesting the two-photon advantage...

When it comes to fluorescence imaging of living or freshly excised tissue, two-photon excitation (TPE) microscopy has two major advantages over confocal microscopy: The combination of increased penetration depth and reduced tissue damage.

Penetration depth is dependent on the scattering properties of the sample, but usually you can expect to reach at least twice the depth that a confocal microscope reaches in the same sample. As in confocal microscopy, the images created by two-photon microscopes are thin optical sections, providing better image contrast within thick tissues, and by recording several images in different depth one can obtain 3D-representations of the tissue. The image on the left is an example for two-photon microscope images.

TPE evokes fluorescence only in a very small volume within the focus of the objective. Images are recorded by scanning the focal spot over the sample plane. Because TPE does not evoke fluorescence outside of the focal plane, and because the infra-red light that is used is less harmful for biological tissues, there is much less tissue damage. Thus, two-photon microscopy is not only suited for fast high resolution imaging in fresh, thick tissue blocks, but it is also suited for work in living tissues and animals, for example long term observations (e.g. cell growth, embryonic development) and opto-physiological studies (e.g. calcium imaging).