Label-Free Multiphoton Microscopy is a very powerful optical microscopy that
can be applied to study samples with no need for exogenous fluorescent probes,
keeping the main benefits of a Multiphoton approach, like longer penetration
depths and intrinsic optical sectioning, while opening the possibility of
serial examinations with different kinds of techniques. Among the many
variations of Label-Free MPM, Higher Harmonic Generation (HHG) is one of the
most intriguing due to its generally low photo-toxicity, which enables the
examination of specimens particularly susceptible to photo-damages. HHG and
common Two-Photon Microscopy (TPM) are well-established techniques, routinely
used in several research fields. However, they require a significant amount of
fine-tuning in order to be fully exploited and, usually, the optimized
conditions greatly differ, making them quite difficult to perform in parallel
without any compromise on the extractable information. Here we present our
custom-built Multiphoton microscope capable of performing simultaneously TPM
and HHG without any kind of compromise on the results thanks to two, separate,
individually optimized laser sources with full chromatic aberration
compensation. We also apply our setup to the examination of a plethora of ex
vivo samples in order to prove the significant advantages of our approach
