Abstract
Based on its polarization dependency, second harmonic generation (PSHG) microscopy has been proven capable to structurally characterize molecular architectures in different biological samples. By exploiting this polarization dependency of the SHG signal in every pixel of the image, average quantitative structural information can be retrieved in the form of PSHG image histograms. In the present study we experimentally show how the PSHG image histograms can be affected by the organization of the SHG active molecules. Our experimental scenario grounds on two inherent properties of starch granules. Firstly, we take advantage of the radial organization of amylopectin molecules (the SHG source in starch) to attribute shifts of the image histograms to the existence of tilted off the plane molecules. Secondly, we use the property of starch to organize upon hydration to demonstrate that the degree of structural order at the molecular level affects the width of the PSHG image histograms. The shorter the width is the more organized the molecules in the sample are, resulting in a reliable method to measure order. The implication of this finding is crucial to the interpretation of PSHG images used for example in tissue diagnostics.
Highlights
Up to day, developments towards the maximum acquisition of the available information in second harmonic generation (SHG) imaging are based on the dependency of the produced SHG signal on the excitation linear polarization (PSHG) [1,2,3,4,5,6,7,8,9]
The resulting modulation in the detected SHG signal is usually fitted into a theoretical model that is taken from the nonlinear optics in crystals
I.e., increasing the organization, we show that the PSHG image histogram becomes narrower
Summary
Developments towards the maximum acquisition of the available information in SHG imaging are based on the dependency of the produced SHG signal on the excitation linear polarization (PSHG) [1,2,3,4,5,6,7,8,9]. The resulting modulation in the detected SHG signal is usually fitted into a theoretical model that is taken from the nonlinear optics in crystals. This model assumes cylindrical symmetry and by analyzing the SHG modulation, the ratio of two non-vanishing, independent elements of the χ(2) susceptibility tensor can be determined [2,3,4,5,6,7,8,9]. By joining the tensorial and the hyperpolarizability approaches, the SHG effective angle θe between the β axis and the long axis of the cylindrical symmetry can be estimated [2,3]
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