OPS>TECHNOLOGY>BIREFRINGENCE

Birefringence

What is imaged ?

Birefringence and Diattenuation are material properties that can occur when there is molecular order, that is, when the average molecular orientation is non-random, as in crystals or in aligned polymeric materials. Diattenuation can only occur when the material absorbs or otherwise reduces the transmittance of the light passing through the material. Birefringence occurs in absorbing and transparent materials and expresses the difference in refractive index for light polarized parallel and perpendicular to the axis of molecular alignment.

The molecular alignment creates a slow and a fast axis in the material. Light that is polarized parallel to the fast axis travels faster through the material than light that is polarized parallel to the slow axis. This leads to a differential phase shift between the polarization components of the transmitted light. The differential phase shift is called retardance. The Birefringence OpenPolScope measures and presents images of the retardance and orientation of fast and slow axis in the specimen.

Birefringece Setup
Birefringece Setup (Inverted Scope)

Hardware

The OpenPolScope augments the traditional polarizing microscope with specific hardware and software components.

Required hardware components:

General: Wide-field microscope stand with monochromatic light source, such as a halogen lamp with bandpass filter, for best results use strain-free objective and condenser optics;
CCD or equivalent camera supported by Micro-Manager software;
Computer with Windows 7, ImageJ, and Micro-Manager installed.

OpenPolScope specific: LC universal compensator; analyzer for circularly polarized light.

The LC universal compensator is made of a linear polarizer and two variable retarder plates implemented as liquid crystal devices. Unpolarized light enters on the side of the linear polarizer and exits as polarized light whose polarization can be set to any state, including circular polarization and linear polarization of any orientation. The LC settings are computer controlled through an electronic controller box.

The liquid crystal devices for the universal compensator can be custom ordered from several manufacturers.

Unlike the traditional polarizing microscope, which is typically equipped with linear polarizers, the OpenPolScope requires a circular polarization analyzer for measuring linear birefringence in the specimen. A circular polarizer is typically made from a linear polarizer and a quarter wave plate that are bonded together. Circular polarizers are manufactured to a specific wavelength, but are also available as achromatic circular polarizers.

The OpenPolScope Group at the MBL can assist in acquiring and optimizing the installation of hardware and software components. See Services

OpenPolScope Software

The OpenPolScope software synchronizes the LC settings and image acquisitions, calculates the retardance and slow axis orientation for each resolved image point, and presents the results as images.

Birefringence is one of three imaging modes of the OpenPolScope software which is built as Micro-Manager and ImageJ plugins.

Pol-Acquisition is a Micro-Manager plugin and is used for acquiring images. 

Pol-Analyzer is an ImageJ plugin and is used for viewing and reprocessing data. The Pol-Analyzer plugin requires Micro-Manager to be installed for reading image data acquired using Pol-Acquisition.

Retardance is typically measured as a distance in nm, signifying the relative distance between the two wavefronts that are associated with the two polarization components that pass through a birefringent material. The OpenPolScope measures the retardance for every resolved specimen point and presents the results of the computation as the retardance image.

In the retardance image, dark areas correspond to sample regions that have no or little birefringence. They are optically isotropic. Bright areas, on the other hand, correspond to sample regions that are birefringent. Their brightness directly corresponds to the retardance, irrespective of the orientation of the slow and fast axes.

In the orientation image, each pixel value gives the orientation of the slow axis as an angle between 0° and 180° with respect to the horizontal axis in the image. The red lines indicate the slow axis orientation on regular grid points. The orientation lines can be overlaid on any of the images.

In the retardance image with orientation colors, hue represents the slow axis orientation and brightness represents retardance.

Orientation image
orientation image with orientation lines
Retardance image
retardance
Retardance/Orientation colormap image
retardance with orientation colors

 

Example

Birefringence CraneFly Spermatocyte This retardance image was generated using a Nikon Microphot SA equipped with a liquid crystal universal compensator, a 60x/1.4 plan apochromat oil immersion objective and an achromat aplanat oil immersion condenser.  The cell in the center is a primary spermatocyte from the crane fly, Nephrotoma suturalis.  With the OpenPolScope, birefringence is revealed with striking contrast, regardless of specimen orientation, and thus, the spindle fibers, composed of bundled microtubules, are clearly imaged as bright structures on the non-birefringent (or weakly birefringent) background cytoplasm.  At the periphery of the spindle is a mantle of elongated mitochondria, as well as numerous highly birefringent vesicular structures. The pole-to-pole distance is approximately 25µm. Credit: James R. LaFountain, University at Buffalo.

 

© OpenPolScope | All Rights Reserved | Contact
Last Page Update on July 24 2015 17:39