Callibration procedure and characterization of a commercial multiphoton microscope to measure Polarization Second Harmonic Generation microscopy

Polarization Second Harmonic Generation enables to quantify some of the underlying molecular details of key biological structures within muscle, axons and collagen at suboptical scales. However, it is only available in dedicated labs. In this work we detail a procedure to adapt and calibrate a commercial multiphoton microscope (Leica TCS-SP5) to acquire PSHG images reliably. For this, two different procedures were developed: (1) using inexpensive starch granules as polarization probe to estimate incident laser polarization within a region, and (2) using a fluorophore in solution to estimate the incident intensity arriving to each pixel of the image. This works might help to broaden the users of PSHG acquisition in microscopy as well as to provide the elements to correct for experimental errors.