Polarization Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 319

The situation becomes even more complex when considering that optical anisotropy is
really a 3D property. Using a conventional polarized light microscope, one measures only a
projection of the 3D property onto the plane that is perpendicular to the optical axis of the
microscope. We have recently devised two different approaches that attempt to analyze the
3D aspect of birefringence in a biological sample. The first method relies on oblique
illumination, essentially tilting the beam path through the specimen. By combining
measurements made with four different tilt angles, it is possible to reconstruct the 3D
birefringence properties [31,32] .
The other method involves a micro lens array that is placed in the image plane of the
objective lens. The camera is placed behind the micro lenses and captures a hybrid image
of the specimen ( Figure 15.10 ). Since the camera is located in a plane that is conjugate to
the back focal plane of the micro lenses, it records a large array of small conoscopic
images, each specific to a small sample area. While this imaging method reduces the lateral
resolution, it systematically records the specimen birefringence as a function of tilt angle of
the beam path [33] . Both methods are related to each other but require vastly different
implementation. The first method is implemented through a variable aperture mask in the
condenser aperture. It only slightly decreases the resolution of the microscope, but raw
images are difficult to interpret directly. The second method reduces the resolution by about
a factor 10, producing an array of conoscopic images that are readily interpreted based on
the classic approach of conoscopic imaging.
15.6 Conclusion
Polarized light microscopy allows one to nondestructively follow the dynamic organization
of living cells and tissues at the microscopic as well as submicroscopic levels. Imaging with
polarized light reveals information about the organization of the endogenous molecules that
built the complex and highly dynamic architecture of cells and tissues. While polarized
light microscopy is not sensitive to the chemical nature of the constituent molecules, it is
sensitive to the structural, anisotropic nature of macromolecular assemblies such as the
submicroscopic alignment of molecular bonds and filaments.
Polarization analysis can also be applied to fluorescence imaging, combining the
molecular specificity of fluorescence labeling with the structural specificity of
polarization. Polarized fluorescence has its origin in the dipole radiation patterns of most
fluorophores, including fluorescent proteins. Recently, we have extended the use of the
liquid crystal universal compensator to analyzing the assembly dynamics of higher-order
septin structures in yeast, fungi, and mammalian cells [34,35] . The study took advantage
of the polarized fluorescence emitted by every single green fluorescent protein (GFP)
fluorophore. GFP was fused to septin with a rigid linker that limits the GFP's ability to