Multiphoton confocal microscopes from Carl Zeiss
now permit the simultaneous use of two NLO lasers or one laser with an
optical parametric oscillator (OPO). Both components are fully
integrated and expand the functionality of the multiphoton systems.
In dual laser systems different laser wavelengths simultaneously excite
several fluorescent dyes or proteins. Without time loss, users can image
specimens with one wavelength and manipulate them in the multiphoton
mode with another. The automatic free beam adjustment gives the system a
high degree of stability and reproducibility and ensures exact overlay
of the two excitation beams. Such dual laser systems are used, above
all, in intravital microscopy, e.g. for examining functional
correlations in the brain of a mouse.
An OPO increases the excitation range of multi- photon microscopy to up
to 1300 nanometers and therefore covers in particular the absorption
peak of red fluorescent proteins such as mCherry, mPlum and tdTomato.
This efficient, long-wave excitation enables excellent specimen
protection. The potentially very high light intensities of the OPO
lasers interact with specific structures in the tissue, leading to a
doubling and tripling of the oscillation frequency. This non-linear
effect of frequency doubling (SHG) occurs, for example. in striated
skeletal muscle and collagen. Frequency tripling is especially visible
on regions where structures with inconsistent optical density converge.
These include lipid-water boundaries - for example, between membrane and
cytoplasm.
