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Nonlinear optical phenomena cover a broad research area. The emphasis is mostly on the generation of higher harmonics to be used in laser designs or on the characterization capabilities of nonlinear optics. The latter ability of nonlinear optics is important when combined with a microscope to detect simultaneously multiphoton fluorescence and second-harmonic generation. Submicron size features can then be investigated separately and information on their structure can be revealed by second-harmonic generation. For example, the point group symmetry can be determined in situ and in vivo in complex media. Moreover, nonlinear optical microscopy has several additional advantages: the generation and detection of nonlinear signals is intrinsically confocal and degradation, if present, only occurs at a localized places in the structure. In biological structures, multiphoton fluorescence and second-harmonic generation do not necessarily occur in the same type of the structure. This can be exploited to visualize different structures in one sample by simultaneous detection of two-photon fluorescence and second-harmonic generation. Also, the incident beam can be tuned to fit in the biological window of biological structures, which gives second-harmonic generation microscopy a significant advantage over linear microscopy due to absorbance issues in the visible wavelength range. We exploit these advantages to characterize collagen-bearing biological structures. Collagen is the dominant structural protein in connective tissue in mammals. Being the most abundant protein in the mammal clade, it is essential for the very existence of it. Collagen is a protein with a very strict quaternary structure. The most simple Ramachandran model states that an amino-acid sequence of Glycine-prolin-hydroxyprolin leads to a right-handed helical structure. The inherent stability is such that a sole helix cannot exist for a prolonged period of time, it will therefore combine with 2 near identical helices, resulting in the formation of the superhelical structure tropocollagen. Subsequently tropocollagen will align in a linear direction forming the fibers composing collagen-tissue. Due to the superhelical nature of collagen, this structure is ideal to be probed by second-harmonic generation.
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