Starch granules as a probe for the polarization at the sample plane of a high resolution multiphoton microscope

Because of its polarization sensitivity, SHG microscopy can provide information about the orientation and degree of structural organization inside biological samples. To fully exploit the above potential, the state of the polarization at the sample plane needs to be known. In this work we present starch granules as a reliable probe for the polarization state of the excitation beam at the sample plane of a high resolution multiphoton microscope. Polarization dependent SHG series of images demonstrated the radial distribution of SHG active molecules inside starch granules. This allowed the granule to exhibit symmetrical SHG emission regions. The pattern rotates along with the rotation of a λ/2 waveplate and thus, can demonstrate the polarization at the sample plane. Maximum signal in the forward detected geometry appears when imaging starch granules exactly at the hemisphere plane. Symmetric SHG regions rotating with the incoming linear polarization were also recorded in the backward detected geometry. A portion of the backwards detected SHG signal, which corresponds to two rotating equator arcs, does not overlap with the forward SHG signal. Importantly, polarization measurements, performed either in the forward or the backwards directions, have demonstrated the suitability and flexibility of this technique for both detection schemes. As result, observation of the starch signal allowed us to know the polarization of our SHG microscope. Furthermore, by coding this information in an angular representation, we corrected the input values in a theoretical model that predicts the average orientation of SHG active molecules. This has allowed us to map the mean orientation of SHG active molecules in body walls muscle of Caenorhabditis elegans, with pixel resolution.