Polarized light microscope. The polarization microscope is based on the behavior that certain components of the cell and tissues have , when viewed with polarized light .
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- 1 Polarized microscopy
- 1 Principle of operation
- 2 Measurement of birefringence
- 3 Differences from conventional microscopes
- 4 Specimen birefrigente
- 2 Sources
Two polarizers have been added to this microscope (one between the condenser and the sample and the other between the sample and the observer). The material used for this is a quartz crystal and a Nicol crystal, passing only the light that vibrates in a single plane (polarized light). This light produces clarity or darkness in the field of the microscope, depending on whether the two nicoles are parallel or crossed.
This type of microscope is used to better identify crystalline or fibrous substances (such as the cytoskeleton), amyloid substance, asbestos, collagen, urate crystals, keratin, silica, and others of exogenous origin.
If the material is isotropic, the propagation of the polarized light is done at the same speed , regardless of the direction of the plane of incidence of the polarized light. These substances or structures are characterized by having the same refractive index in all directions. On the other hand, in an anisotropic material, the propagation speed of polarized light is different depending on the direction considered. A material with these characteristics is also called birefringent because it has two different refractive indices that correspond to the respective different transmission speeds.
Measurement of birefringence
Polarized light allows to distinguish the internal birefringent particles
Birefringence can be expressed quantitatively by the difference that exists between the two refractive indices (N e -N o ) that are associated with the higher or lower speed ray.
In practice, it is measured with the delay polarization microscope expressed in fraction of wavelength, which experiences light in one plane with respect to the speed it presents in another plane perpendicular to the previous one. The delay depends on the thickness of the specimen (d):
Birefringence (β) = N e – N o = √ / d
The measurement of the delay is carried out with the use of various compensators that are introduced into the optical system.
The birefringence of biological materials is generally very small, being on the order of 0.01 to 0.001. The measurement of small delays requires very sensitive compensators.
Differences from conventional microscopes
The polarization microscope differs from conventional microscopes by the addition of two polarization elements: the polarizer and the analyzer, which can be made of a polaroid sheet or by Nicol prisms of calcite. The polarizer mounts under the condenser and sends plane polarized light to the object. A similar system, the analyzer, is placed above the objective lenses.
When the analyzer is rotated around 360 or the field of view appears bright and dark alternately every 180 o of rotation. The two positions of maximum light transmission are obtained, when the analyzer is placed parallel to the polarizer, at an angle of 90 or the extinction (non-transmission) of the polarized light is carried out.
Under these conditions if a birefrigerant specimen is placed, the plane of polarization will be deviated according to the delay introduced by the object. Control with polarized light consists of rotating the specimen on a special rotating stage and finding the points of maximum or minimum brightness.
Once the birefringence has been demonstrated and the delay measured, it is necessary to determine the axis and the sign of the birefringence. In most biological fibers, the birefringence is uniaxic and can be positive, if the refractive index along the fiber axis is greater than in the plane perpendicular to it and negative in the opposite case.
The determination of the sign is carried out by interposing a birefrigerant material with axes whose higher or lower speed are known.
With the refinement of currently available polarization microscopy methods, delays of the order of 0.1mμ (1A or ) can be measured with a resolution of 0.3μ.