Non-Linear Microscopy


Non-linear optical microscopy, especially harmonic generation microscopy (HGM), are promising microscopy technologies for many biological applications.
HGM summarizes both Second Harmonic Generation (SHG) and Third Harmonic Generation (THG). These are processes where either two or three photons interact with the biological structures to be visualized and result in the direct emission of a photon with half or one-third of the wavelength. As the conditions for HGM are limited to the areas of high photon density, HGM offers a sub-micron optical resolution. Furthermore, as HGM does not involve energy dissipation into the sample, it exhibits low photodamage and low photobleaching, which is highly desirable when working with biological samples.

In a recent publication, Chung et al. (2019) demonstrated that HGM becomes a critical modality for optical virtual skin biopsy with SESS-based laser sources such as Cycle SOPRANO. HGM benefits from the femtosecond driving source in the 1.15–1.35 μm range. Most of the resulting SHG and THG light falls in the visible wavelength range and can be efficiently detected. In addition, longer excitation wavelengths correspond to less photon energy and reduce the risk of photodamage. In their publication, Cheng et al. (2019) used laser pulses between 1.15 and 1.35 μm with >10 nJ pulse energy and 100 fs pulse duration to excite SHG in the collagen fibers and THG in the epidermis of human skin tissue.

Figure 1 – HGH imaging of ex vivo human skin from the back part at different penetration depth excited by 1.25-μm femtosecond pulses. (a) SC (stratum corneum) and SG (stratum granulosum) in epidermis at 20-mm depth. (b) SS (stratum spinosum) and SB (stratum basale) in epidermis at 39-μm depth. (c) SS, SB, and DP (dermal papillae) between epidermis and dermis at 62-μm depth. (d) Collagen fibers in dermis at 113-μm deoth. Scale bar: 50 μm. (Chung et al., 2019)

In the scope of the presented, Chung et al. (2019) performed a systematic investigation of HGM imaging of ex vivo human skin. Results showed that excitation wavelengths in the range of 1.15-1.25 μm have low optical attenuation in tissue and allow greater imaging depth for HGM in human skin. The latter makes HGM with a fiber-based SESS source such as the SOPRANO laser a promising tool for noninvasive virtual skin biopsy in clinical applications. It will advance and simplify the study of histopathology, morphology, and skin diseases.

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