After building and aligning the polarization-sensitive two-photon microscope system, according to the displayed schematic representation, test the optics alignment quality with an isotropic reference sample like fluorescein embodied in an amorphous polymer. To do so, switch on the excitation laser and measure the 360 degree polar graphs of the reference sample for a zero half-wave plate. Adjust the half-wave plate angle to correspond to the X and Y axes of the sample plane, and check if both photodiodes collect similar intensities of two photon-excited emission components.
To analyze the sample, mount the glass slide with this spherulite specimen on the Piezoelectric Stage, ensuring the face of the slide containing the cover slip faces the high numerical aperture immersion objective. Then secure the glass slide with tape. Next, by adjusting the X, Y, and Z positions using the microscope knobs, focus the objective on the spherulite in the solution.
Focus specifically on the central area of the entire structure, as spherulite diameters are usually in the range of tens of microns. Then center this spherulite in the field of view in the microscope plane. Determine the size of the XY scan by adjusting the number of Piezo Stage steps in X and Y directions to cover the entire structure's area.
Adjust the Piezo Stage parameters such as scanning speed, step size, and range to cover the area of the whole spherulite. Open the shutter, turn on the photodiodes, and collect the X and Y emission components of two-photon excited fluorescence intensity for every single step of the selected scanning area for the excitation beam polarized corresponding to the X and then the Y axes. Then scan this spherulite sample, resulting in two-photon excited fluorescence.
To perform full polarization analysis from the specific spot on the sample, turn off the excitation beam. Take specific coordinates of Piezo Stage corresponding to the chosen location on this spherulite where the information about the orientation of molecules is required with the submicron resolution. Adjust the Piezoelectric Stage to center the field of view at indicated X and Y coordinates.
After turning on the excitation beam, turn on the 180 degree rotation of the half-wave plate to perform a full 360 degree polarization analysis of the X and Y emission components of the two-photon excited fluorescence emission. The image of the insulin spherulite obtained after performing a 2PFM raster scan was consistent with those reported for a spherulite. The position of the half-wave plate corresponding to the excitation of the sample along the X and Y axes was also verified.
The polar graphs changed while performing a full polarization analysis from different selected spots. Outside the spherulite, the PLO graph looked like a collection of artifacts and random signal noise spikes. Properly measured polygraphs from highly ordered locations along the X and Y axes had different shapes and geometries depending on the local orientation and organization of fluorophores.
