CC BY
18B-I-16
Advances in confocal Mueller matrix polarimetry
J. Ramella-Roman1, I. Saytashev2, S. Sudipta3
1Florida International University, Biomedical Engineering and Herbert Wertheim College of Medicine, Miami, USA
2Florida International University, Herbert Wertheim College of Medicine, Miami, USA 3Florida International University, Physics, Miami, USA
Introduction
Mueller Matrix Polarimetry is a linear modality that has been used extensively in the determination of orientation and retardation of fibrous tissues due to its sensitivity to birefringence. To this day, our understanding of the back reflected polarized signal is limited. We have developed a confocal microscopic system aimed at discriminating the provenance of the polarized signature into a multiscattering environment.
Methods
We have developed an instrument that combines two polarization imaging techniques, Muller Matrix reflectance microscopy and Muller Matrix confocal polarimetry, and integrated these modalities into a Nonlinear Microscope (NLM). This system allows for the collection of the total back-reflected Mueller Matrix image as well as depth dependent non-linear and confocal Mueller Matrix images.
The system is based on a pre-compensated femtosecond laser. The reflected light at fundamental wavelength is separated from epi-detected NLM by a short-pass dichroic mirror and directed to CMOS camera placed at the image forming conjugate plane. Dual Liquid Crystal Variable Retarders are used at both illumination and detection to construct the back reflected Mueller Matrix.
Results/Discussion
We acquired stacks consisting of 11 images with NLM images as well as depolarization, orientation, linear retardation, diattenuation, and total retardation maps. Polarization properties were assembled in 3D maps with RGB colormap. Typical data is shown in Fig. 1.
TPEF SHG Depolarization Orientation Linear retardation Diattenuation Total retardation
05 1 -90* 90* 0* 25» 0 10* 25
Fig. 1.
Strong two-photon excited fluorescence (TPEF) from epithelial cells of unstained corneas is due to NAD(P)H visualizing intracellular space, however cellular nuclei are represented as low-intensity spots. A change from epithelium to stroma is visualized by transformation of NL signals from TPEF to Second Harmonic Generation (SHG). We observed increase in depolarization and diattenuation, and decrease in linear retardation once the epithelial layer ended. Interestingly, there is a steady increase in retardation past 30 |im depth, which is associated with the presence of collagenous lamellae. Furthermore we observed a significant retardation at epithelial layer obtained by Lu-Chipman decomposition.
Conclusions
We have developed an imaging system that provides depth-resolved TPEF and SHG imaging to achieve 3D reconstruction of cellular and collagen distribution and confocal Mueller Matrix imaging to measure polarization properties and relate it to the total backscattered Mueller Matrix. Our results demonstrate the proof-of-concept depth resolved Mueller Matrix imaging validated by NL microscopy with a great potential in preclinical and clinical use.
