Thursday, 22 February 2018

About Polarization

Utilizing the polarization method in light microscopy reveals a specific kind of information about the sample. Applying this contrast method, we less often talk about structure size and shape, resolution power or even a specific staining. In case of transparent samples, polarization contrast in first instance has one subject in mind: the potential BIREFRINGENCE of the sample and its implications on a structural insight.

BIREFRINGENCE is a widespread phenomenon, found in nature as well as in man-made materials. Starch grains, fibers, crystals and minerals, plastic sheets and die cast components, the structural basis is always the same: embedded within an amorphous matrix, structures with an intrinsic geometric pattern cause an impact on the speed of light and its oscillating direction when sent through the sample.

Chemical cristals

Starch grains

Mineral Section - Peridotita

Light as an electro-magnetic wave oscillates in 90° relation to its propagation direction.

Speed of light only is a constant in vacuum: c0= 299.792.458 m/sec. This constant is related to the refractive index (n) of vacuum, set per definition as n=1. If light passes a medium with a refractive index n>1, the speed of light is reduced.

C0= speed of light in vacuum / C= speed of light in medium
E.g. for water (n=1,33), the calculation is as follows: C=C0/1,33 = 225.407.85 m/sec. The refractive index is a value for the "optical viscosity" of a material. The more resistance the material gives to light, the higher the value. A short list:

Refractive Index (n)
Immersion Oil
Lead Sulfide

Note that the refractive index for immersion oil and glass is very similar. This allows to install the "homogeneous immersion" method in light microscopy: no boundary surface between oil and glass (cover slip, front lens), so no refraction, means maximum resolution power with immersion objectives.

In ISOTROPIC substances the refractive index (n) → speed of light is equal for all propagation directions. BIREFRINGENT samples only allow two oscillation directions for the light, with two different refractive indices: a fast (= ordinary; n1) and a slow (= extraordinary; n2) direction. The extraordinary ray is shifted laterally, that is why we get a double image.

Isotropic   |   Anisotropic (Birefringent)

The difference (n2-n1) is a material constant, directly influencing the retardation (∆) (unit: nm). The longer BIREFRINGENCE works on the light, the larger the retardation. That is why the sample thickness directly effects the total value of ∆. Transferring this value into visual information even gives a chance to deduct the material of crystals and minerals.

To detect BIREFRINGENCE, this is our microscope setup: A "Polarizer" is placed between light source and sample. As natural light (in this case we regard Halogen or LED light sources as "natural") oscillates in an endless number of directions, this special filter device allows only one oscillation direction to get through: linear polarized light.

After passing the sample, the "Analyzer", an identical piece of hardware, is placed in the optical path. The analyzer is positioned at 90° position in relation to the polarizer. For fine tuning, rotate one item (mostly the polarizer is more easily accessible) until the image background is perfectly dark. Once orientated at 90° to each other, any polarized light not being influenced by the sample will be blocked by the analyzer.

A BIREFRINGENT sample, only allowing two oscillation directions, will provoke a change of the oscillation direction of the linear polarized light, the light ray will be "split" into an "ordinary (o)" and "extraordinary (e)" component, thus provoking a retardation ∆. At least some ratio of light will pass the analyzer (take this is a vector calculation, not as a percentage calculation).

The analyzer forces the ordinary and extraordinary component to interfere. The result, an interference "color" is directly related to the retardation value caused by the sample. Most retardation values are <300nm, resulting in a black/white image. Only higher retardation values will cause colors.

In case the fast/slow propagation directions within the sample are exactly orientated as the polarizer/analyzer passage direction, there will be no BIREFRINGENCE effect visible. That is why a rotation of the sample/stage is of high interest to eliminate this possible coincidence.

Such a FILTER POL setup can be performed with a large number of transmitted light microscopes. The polarizer mostly can be placed on top of the light exit at the microscope foot. Placing the analyzer is more difficult. In simple models, the observation tube is taken off, inserting the analyzer near the rear side of revolving nosepiece.

A dedicated Pol objective is labelled with a red inscription and a "P" following the NA value. The glass elements within this objective are mounted "strain-free", without any tension which could lighten up the image background. A specialized POL condenser may be a perfect addition, but it is not mandatory.

Pol Objective with standard labelling

For semi-quantitative measurements of retardation in order to evaluate the chemical basis of the sample, a rotating stage as well as "compensators" are essential, additional birefringent samples added to the ray path to shift the retardation value into a colorful region higher than 300nm. In this case a dedicated POL microscope with rotating stage and compensator slot is necessary.

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