Harvesting the two-photon advantage...
...with TILL's two-photon microscopes
Crayfish stretch receptor neuron, FITC-immunostained for tubulin, 4 optical z-sections with 10Ám spacing, scale bar is 25Ám
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.Greater penetration depth
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.Less tissue damage
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).
Another benefit of two-photon excitation is that many different dyes can be excited simultaneously without changing the laser wavelength. Thus, TPE avoids the illumination-wavelength-dependent focus error that can lead to artefacts in confocal microscopy with multiple dyes.Harvesting the two-photon advantage
TILL's two-photon microscopes, the inverted Two-Photon-iMIC and the upright Intravital2P are equipped to entirely harvest the TPE advantages: The novel VoiceCoil z-Drive accounts for greater imaging depth with its huge travel range of 7mm and speed (10ms settling time after 2Ám step). The Yanus laser scanner, also used in Stefan Hells lab for STED microscopy, excels in large scan fields as well as fast frame rates and complex scan modes e.g. for calcium imaging. GaAsP detector modules with >40% quantum efficiency in combination with collecting lens systems ensure that no fluorescent light is lost. Last but not least, our modular Intravital2P XY stage is prepared for a large variety of samples, be it slides, dishes, or mice.