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.

Multiphoton confocal microscopy

An
OPO increases the excitation range of multiphoton 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.

Source: Carl Zeiss