In the last ten years multi-photon-microscopy has become increasingly popular to enhance contrast and resolution for modern microscopic imaging due to the utilization of non-linear optical effects. Amongst many methods of multi-photon-microscopy, two-photon fluorescence microscopy (2-PFM) is the most popular with several hundred systems installed world wide every year.
Since the two-photon absorption process needs a high density of photons (intensity up to 10 MW/cm²), pulsed lasers with a pulse duration in the sub-picosecond time scale should be used. This is because the virtual absorption of a photon of non-resonant energy lasts only for a ver short period (10-15 - 10-18s). During this time a second photon must be absorbed to reach an excited state.
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2-PFM image of Cresyl Violet coloured paper fibre. The image demonstrates the applicability of a „femtoTRAINTM Yb“ system for excitation of cresil violet dye. The measuring line corresponds to 50 μm. | SHG (Second Harmonic Generation) signal obtained for urea crystals illuminated by a „femtoTRAINTM Yb“ laser. Here SHG as an endogenous contrast source originating from urea crystals was used. The measuring line corresponds to 50 μm. |
