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.
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