Polarizing Microscopes – Principle, Parts, Uses

What is Polarizing Microscope?

A polarizing microscope is an optical microscope that allows one to see birefringent materials—materials that give off two rays of light moving at varying speeds and indices of refraction—and contains certain added components for enhanced viewing contrast and more precise structural detail. As for the components of the microscope procedure that relate to the polarizing microscope, there are two filters, the polarizer and the analyzer. The polarizer resides beneath the microscopic stage and filters the light that enters the device to ensure that it travels and vibrates along one plane.

The light emitted from the polarizer when it is activated is referred to as plane-polarized light. When plane-polarized light reaches an object that is anisotropic/birefringent, it splits into two rays known as the ordinary and extraordinary. They vibrate at different rates, meaning they become polarized perpendicularly to one another. The analyzer exists above the objective lens and allows the viewer to see the specimen as it sits perpendicular to the polarizer. Thus, like the polarizer, the analyzer can hold the two rays in a perpendicular orientation; however, it allows for overlap; thus, the analyzer allows for perpendicular crossing of the two rays.

When activated, the overlap of the two planes creates interference colors and textures which represent certain information about the internal arrangement and constitution of the sample. The uses of the polarizing microscope extend to geology, biology, and materials science. For instance, geologists examine rocks with a polarizing microscope to assess mineral content. Biologists examine biological tissue such as muscle or bone with a polarizing microscope to assess crystal structure. Materials scientists examine polymers, crystals, and other anisotropic substances with a polarizing microscope to assess structural integrity. The polarizing microscope came about in the early nineteenth century.

The first of such microscopes would run on the Nicol prism, a polarizing light creator, invented by William Nicol in 1829. In 1830, the first practical polarizing microscope was created by Giovanni Battista Amici. But the petrographic polarizing microscope didn’t come about until 1875, when Rudolf Fuess created it and Harry Rosenbusch spoke of it in that year’s review of mineralogy. Ultimately, however, as optics progressed, Nicol prisms were soon obsolete. Other polarizing materials were invented that rendered the polarizing microscope more functional and less complicated across disciplines.

Non-Polarized to Polarized Light Convertion

The way the polarized light microscope works is that it takes non-polarized light and converts it into polarized light via the absorption of vibrational motion of light traveling in one direction. Such mechanisms may occur naturally with certain minerals (e.g., tourmaline) or via human-made films/filters that create the same results. For instance, the filters within Polaroid cameras are made up of microscopic crystallites of iodoquinine sulfate aligned in a preferred direction and then trapped in a polymeric filter. The trapping is required so that once the crystals are aligned, they do not move and take on a different orientation. The device that converts natural/non-polarized light to plane-polarized light is called a polarizer.

Principles of Polarized Light Microscopes

Essentially, a polarised light microscope is comprised of a light source and an analyser, with the sample situated in between. Thus, as the polarised light source moves toward the sample, the resultant light is plane-polarised after dissipating through the sample. Next, the plane-polarised light strikes a substance that shows double refraction at perpendicular angles, splitting the light into two waves known as common and extraordinary rays. After this point, the rays continue their path through the sample via a series of steps. Ultimately, however, the analyser yields the rays through constructive interference and destructive interference. The ultimate goal of all these actions is to yield an image.

Parts of a Polarized Light Microscope

1. Polarizers

The polarised light microscope’s distinguishing features are the polarizing filters. In general, polarizing filters are composed of two parts: the polarizer and the analyzer. The polarizer is found beneath the specimen stage and is rotatable 360 degrees; it polarizes all light that hits the specimen. The analyzer is found above the objective and is sometimes rotatable as well; it takes the errant rays emitted from the specimen and turns them into one, unified image.

2. Specialized Stage

This is the specimen stage that rotates 360 degrees to ensure that the specimen is always in alignment with the objective plane.

3. Strain-Free Objectives

Any strain placed on the objective during installation can alter the optical properties of the lens, resulting in a reduction in performance.

Additionally, if the lens is attached too tightly on the frame, strain can be introduced. In addition, anti-reflection coatings and refractive characteristics must be precisely evaluated to guarantee polarisation and enhanced contrast.

4. Rotating Turret

The use of a rotating nosepiece. The majority of polarizing microscopes are set up with rotating stages and lenses. Thus, the rotating nosepiece allows the viewer to continue seeing the center of the specimen in the field of view even if the stage has been adjusted.

5. Compensator and Retardation Plates

Many polarizing microscopes have compensators and/or retardation plates. These sit between the crossed polarizers to enhance the optical path difference through a particular sample. This further enhances image contrast. Thus, the purpose of a polarizing microscope is to enhance image contrast to better view the different anisotropic subcellular structures.

6. Light Intensity Adjustment

Change the diopter according to your eye. The diopter wheel controls focus. If this is not set according to your vision, you will be out of focus even if the ocular is clear.

7. Interpupillary Distance Adjustment

With both hands on either side of the observation head, take the left and right sides and turn the two sides until one circle of light is visible to accommodate the interpupillary distance. The interpupillary distance will be indicated by notched gradations in reference to the eye distances of the operator, as symbolized by “•” located on the eyepiece holder. The interpupillary distance adjustment ranges from 48-75 mm.

8. Diopter Adjustment

View through the right eyepiece with the right eye only and focus on the specimen. Then, view through the left eyepiece with the left eye; if the image appears blurry, adjust accordingly with the dioptric adjustment ring.

9. Diopter adjustment

Adjustment range is ± 5 diopter. An adjustment graduated ring will register the amount, and this adjustment should be reflected in the dioptric correction of the operator.

10. Aperture diaphragm

The condenser diaphragm has a numerical aperture (N.A.) which can be adjusted to increase/decrease image contrast. Adjusting the N.A. value will change resolution, contrast, and depth of focus. The diaphragm lever slides left/right to decrease/increase the N.A. value. A good N.A. for a specimen with low contrast is about 70%-80% less than that of the objective. If needed, remove one eyepiece and look into the empty sleeve to properly adjust the diaphragm of the condenser for an appropriate image.

11. Rechargeable batteries

When the unit is plugged into the power supply, the battery charging LED for the battery illuminates. RED LED: battery charging. GREEN LED: battery charged. When the power supply is unplugged, the LED is off. When the unit is used with batteries, the LED is ALWAYS OFF.

12. Strain-Free Lenses

Strain-free lenses are a common feature with polarized light microscopy condensers. One has a slot for the polariser, the other has the polarizing element integrated within the condenser, underneath the aperture diaphragm. Much of the polarized light microscope ocular includes a cross wire reticle (or graticule) superimposed upon the centered field of view. The important accessories for the ocular are generally the photomicroscopy reticle, which superimposes a grid upon the field of view—as well as helps to focus and take photographs of the specimen—and includes a series of frames that delineate which portion of the viewfield will be photographed—digitally or in film.

13. Eyepieces

The eyepieces of a polarised light microscope have a cross-wire reticle (or graticule) to mark the centre of the field of view. Frequently, the cross wire reticle is replaced by a photomicrography reticle that aids in focusing the specimen and composing photographs with a set of frames enclosing the viewfield to be shot digitally or on film. A point pin that slips into the observation tube sleeve ensures the correct orientation of the eyepiece relative to the polarizer and analyzer.

14. Bertrand Lens

A. Bertrand lens is found in the intermediate tube or observation tubes and serves to project the interference pattern observed at the back focal plane of the objective into the focal plane of the image formed in the microscope. This provides the user access to the back focal plane of the objective within view, allowing for rotation of the aperture diaphragm to produce and view interference patterns as shown in Figure 2. That’s the anticipated characteristics of a uniaxial crystal under polarized light; however, FIG. 2(c) is what’s expected characteristic of a uniaxial crystal with a first-order retardation plate in the field of view.

Basic Properties of Polarized Light

Polarized light is light that, unlike usual general light that vibrates randomly in all directions, chooses a specific plane in which to vibrate. Some important notes about polarized light:

• Polarization planes- The orientation of the oscillation of light waves is called the plane of polarization. Light can be polarized horizontally, vertically, or at an intermediate angle.
• Polarization direction- The term “polarization direction” refers to the electric field relative to the incident light waves. It is essential to know two types of polarized light: “linear polarization,” which means that the electric field exists in one direction, and “circular polarization,” which means that the electric field rotates around the direction of propagation.
• Extinction- Light is said to be extinguished when the relative orientation of the sample’s plane of polarization is 90° to the analyzer’s plane of polarization (a specific polarizing filter). This is relevant to observe the birefringence of some samples in the polarizing microscope, which essentially means that the light is completely blocked by the analyzer.
• Brewster angle – The angle of incidence at which light striking a transparent material is fully polarized. This angle depends on the refractive index of the material and the wavelength of the light.
• Double refraction – The property of some transparent materials that are crystalline. They allow light to be split into two rays vibrating at right angles to each other. This phenomenon is called birefringence, and can be observed in the polarizing microscope.

Operating Procedure of Polarized Light Microscope

1. Turn on the light source and stabilize it.
2. Place the specimen slide on the stage and secure it.
3. Focus on the specimen using a 10x objective lens.
4. Close the field diaphragm to its smallest setting.
5. Insert the polarizer below the condenser.
6. Place the analyzer above the objective lens.
7. Ensure the polarizer and analyzer are crossed perpendicularly.
8. Rotate the stage to observe changes in the specimen.
9. Adjust focus and illumination for a clear image.
10. Insert compensators or retardation plates if needed.
11. Capture images or record observations for analysis.
12. Follow the manual for model-specific instructions.

How does a Polarized Light Microscope Works?

Compared to relative dark-field and bright-field illumination, fluorescence, differential interference contrast, phase contrast, and Hoffman modulation contrast, polarized light is another form of contrast enhancement; however, it only complements the appearance of birefringent materials. Therefore, although polarized light microscopes can be temperamental, they are stable in some parameters for the quantitative and qualitative assessment of various anisotropic materials.

Although there is an abundance of literature on qualitative polarizing microscopy—suggesting that qualitative assessments are more common—it seems that quantitative studies are less common but more difficult to perform, mainly by geologists, mineralogists, and chemists. However, in the last decade, various anisotropic subcellular structures have been evaluated for birefringence by biologists. The polarized light microscope works to observe and capture images of materials based on their anisotropic optical characteristics. However, the polarized light microscope requires a polarizer in the light path in front of the sample and an analyzer (second polarizer; see Figure 1) between the rear aperture of the objective and the eyepiece/camera port.

The resulting contrast is created from plane-polarized light directed onto a birefringent (or doubly refracting) material so that the two components of the wave are polarized in perpendicular planes. These velocities are defined as the ordinary and extraordinary wavefronts (Figure 1) and are produced when light passes through the variable and visible material of interest. However, when the two components of the wave exit the sample, they are no longer aligned; yet, when they reach the analyzer, they are reoriented by constructive or destructive interference.

Thus, Figure 1 illustrates these phenomena occurring for the wavefront field of a theoretical birefringent material. Finally, the figure shows the physical and optical components required for a modern polarized light microscope. Similar to brightfield, the ability to view under polarized light allows for absorption color and sharp boundaries where minerals break up due to different refractive indices. But it also allows the observer to determine whether the material is isotropic or anisotropic.

In addition, the use of contrast enhancement factors plays a role in the visual properties and implies anisotropy, which provides essential identification and diagnostic information about composition and structure. An isotropic material (many gases, liquids, unstressed glasses, and cubic crystals) is one that is the same in all directions because it is tested in all directions. Isotropic materials have a constant refractive index and provide no preferred direction of vibration of light during transmission. Therefore, the reverse is true for anisotropic materials, which constitute 90% of all solid materials.

With anisotropic materials, the optical properties depend on the incidence of the direction of light relative to the crystallographic axes. These materials have different refractive indices depending on the direction of light propagation through the material and the orientation of the vibration planes. With anisotropic materials, they are like beam splitters; they take a ray of light and make two perpendicular rays. Therefore, a polarizing microscope reveals the attributes of the anisotropic material based on the interference of rays that can be joined along the same optical access line.

Although polarized light microscopy is largely relegated to geological study (rocks and their thin sections), it can be applied to a number of materials: natural minerals and their artificial counterparts, cements, ceramics, mineral fibrous materials, composites, synthetic polymers, starch, natural lignocellulosic materials, urea, and many other natural biological polymers and multicomponent arrangements.

Therefore, this form of microscopy extends to many other fields outside of geology: materials science, chemistry, biology, metallurgy, medicine, and all of these fields can obtain qualitative and quantitative results with relative ease. Understanding the basics of polarized microscopy is a more complicated undertaking than other types of microscopy, but the tangible advantages over brightfield make it worth learning this experiment.

Applications of Polarized Light Microscope

• Use for Identifying and analyzing minerals in geological samples.
• Used to Examining muscle fibers, collagen, and tissue structures in biology.
• Used to Assessing polymers, ceramics, and composites in materials science.
• Detecting crystals in biological samples for medical diagnosis.
• Used for Identifying fibers and materials in forensic investigations.

Advantages

Here is the some Advantages of Polarized Light Microscope;

• It has Enhances image contrast for better structure visibility.
• This Provides detailed analysis of internal structures and stress patterns.
• It is Suitable for quantitative and qualitative studies.
• It Allows non-destructive examination of specimens.
• It is Useful across geology, biology, and materials science.

Limitations

Here is the some Limitations of Polarized Light Microscope

• It Only works with birefringent materials.
• The Sample preparation process can be complex.
• It has Limited magnification compared to electron microscopes.
• It Requires skilled operation and experience.
• Potential for misinterpretation if not carefully managed.

Polarized Light Microscope Images