||Two-photon microscopy has an outstanding optical sectioning capability due to its nonlinear excitation process. While observing tendon tissues with its second harmonic generation microscopy, because of the non-centrosymmetric structure, tendons can be observed directly. In addition, the excited process has no energy level transitions, meeting the conservation of momentum, therefore the sample will not be damaged by the light source, making it well suited for long time observations.|
Unlike fluorescents, second harmonic signals are under the conditions of no energy level transitions, thus it does not apply to the fluorescent signal-based super-resolution microscopies, however, it could be combined with structured illumination microscopy. The original structured illumination microscopy was based on a wide field setup, yet two-photon microscopy systems require extremely high excitation intensities to produce the two-photon excitation on its samples, consequently, scanning systems were chosen to overcome such issue. This paper will combine the scanning second harmonic generation microscope and structured illumination microscopy to improve the resolution.
Second harmonic signals are coherent signals, its imaging theory is different to the incoherent signal’s. In this paper, it is proven that the reconstruction image has a higher frequency information based on the Second Harmonic Generation Scanning Structured Illumination Microscopy theory. In simulation, under four different periods: 1047 nm, 873 nm, 748.6 nm and 655 nm, the resolution is enhanced by 1.28, 1.35, 1.44, and 1.52 times respectively; in experiments, by using chicken wings tendon as samples, resolution were improved by 1.29 and 1.38 times; and in the Y direction, 1.3 and 1.4 times the resolution improvement were achieved.
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