Maintaining polarization in polarimetric multiphoton microscopy

Abstract

Polarimetric measurements in multiphoton microscopy can reveal information about the local molecular order of a sample. However, the presence of a dichroic through which the excitation beam propagates will generally scramble its polarization. We propose a simple scheme whereby a second properly-oriented compensation dichroic is used to negate any alteration regardless of the wavelength and the initial polarization. We demonstrate how this robust and rapid approach simplifies polarimetric measurements in second-harmonic generation, two-photon excited fluorescence and coherent anti-Stokes Raman scattering. Illustration of the polarization maintaining strategy with the compensating dichroic oriented such that its s- and p-axes are interchanged with these of the primary dichroic.

Intravital assessment of myelin molecular order with polarimetric multiphoton microscopy

“…CARS imaging was performed in a polarization-resolved manner (P-CARS). This approach takes advantage of the high level of symmetry in myelin sheaths 15 20 21 . Indeed, CH 2 molecules are not randomly distributed within the diffraction limited excitation volume.…”

“…Each image is recorded with a different orientation of the collinear polarization of the two excitation beams. As the linear polarizations are rotated 21 , the CARS signal intensity is modulated and this modulation informs on the molecular dipole distribution 23 24 25 . Figure 1e shows CARS images of a Mauthner axon taken with the orthogonal orientations of the polarization from Fig.…”

“…It is already well known that nonlinear susceptibility tensor elements or equivalent parameters obtained from polarization dependence experiments relate to the degree of molecular alignment 23 39 . For CARS, this aspect has been demonstrated in crystal 23 40 and applied to biological samples 21 24 25 41 . As defined here, an increasing MM during development informs on the myelin sheath lamellae becoming more organized, CH 2 molecular dipoles becoming more aligned.…”

“…Additional experimental evidence of this fact can be found in Bélanger et al . 21 . To further support this observation, we performed EM on 3 and 5 dpf zebrafish and validated the increase in myelin lamellae organization.…”

A large‐field polarisation‐resolved laser scanning microscope: applications to CARS imaging

“…In all of the aforesaid scanning imaging techniques it is of crucial importance that the polarisation distortions induced by the optical elements, in particular dichroic mirrors (DMs), are made as small as possible, for example, by compensating them with a combination of λ/2 and λ/4 waveplates (Bélanger et al, 2009) or by adding a matched dichroic to the optical path (Bélanger et al, 2015). Distortions introduced by dichroics typically vary strongly when the light incidence angle is changed by the galvanometric mirrors during the raster scanning and it does not seem feasible to compensate those distortions simply by adjusting the retarders in real time during the image acquisition.…”