NLOM technologyhas the potential to provide critical information for the fast and reliable in-situ analysis of various CH objects without endangering the integrity of the samples. NLOM techniques are based on the integration of tightly focused near-infrared (NIR) femtosecond laser beams, allowing for the extraction of information from deeper layers of the sample due to the reduced optical scattering of such wavelengths.
Moreover, due to the non-linear nature of the process, the NLOM provides high axial and lateral resolution images, intrinsic 3D optical sectioning, and prolonged period of irradiation while minimizing photodamage effects. These significant advantages for CH diagnostics are related to the fact that, in contrast to single-photon techniques, only a small volume of the medium (~ 1μm3) interacts with the laser light, diminishing thermal deposition or other photodamage effects on the object under investigation.
The non-linear techniques commonly employed to image the samples are: Multi-photon excitation fluorescence (MFEF), Second and Third harmonic Generation (SHG, THG). MPEF is a non-linear absorption effect, while optical higher harmonic generation procedures including SHG and THG are coherent, non-linear scattering phenomena. SHG and THG are intrinsic properties of the material providing minimal sample disturbance due to their energy conservation characteristics.
This technology, in the form of three different imaging modalities, provides simultaneously the in-depth compositional characterization of CH objects (via MPEF), the high-resolution morphological delineation (via THG), as well as the sub-micron distribution of non-centrosymmetric molecules (via SHG), including collagen and starch.
