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Polarized Light Microscopy

The polarized light microscope is designed to observe and photograph specimens that are visible primarily due to their optically anisotropic character. In order to accomplish this task, the microscope must be equipped with both a polarizer, positioned in the light path somewhere before the specimen, and an analyzer (a second polarizer), placed in the optical pathway between the objective rear aperture and the observation tubes or camera port. Image contrast arises from the interaction of plane-polarized light with a birefringent (or doubly-refracting) specimen to produce two individual wave components that are each polarized in mutually perpendicular planes. The velocities of these components are different and vary with the propagation direction through the specimen. After exiting the specimen, the light components become out of phase, but are recombined with constructive and destructive interference when they pass through the analyzer. Polarized light is a contrast-enhancing technique that improves the quality of the image obtained with birefringent materials when compared to other techniques such as darkfield and brightfield illumination, differential interference contrast, phase contrast, Hoffman modulation contrast, and fluorescence.

Introduction - Although much neglected and undervalued as an investigative tool, polarized light microscopy provides all the benefits of brightfield microscopy and yet offers a wealth of information, which is simply not available with any other optical microscopy technique. The technique exploits optical properties of anisotropy to reveal detailed information about the structure and composition of materials, which are invaluable for identification and diagnostic purposes.

Polarization of Light - Natural sunlight and most forms of artificial illumination transmit light waves whose electric field vectors vibrate in all perpendicular planes with respect to the direction of propagation. When the electric field vectors are restricted to a single plane by filtration then the light is said to be polarized with respect to the direction of propagation and all waves vibrate in the same plane.

Optical Birefringence - Anisotropic crystals have crystallographically distinct axes and interact with light in a manner that is dependent upon the orientation of the crystalline lattice with respect to the incident light. When light enters a non-equivalent axis in a anisotropic crystal, it is refracted into two rays each polarized with the vibration directions oriented at right angles to one another, and traveling at different velocities. This phenomenon is termed double- or bi-refraction (or birefringence) and is seen to a greater or lesser degree in all anisotropic crystals.

Microscope Configuration - Although similar to the common brightfield microscope, the polarized light microscope contains additional components that are unique to instruments of this class. These include a polarizer and analyzer, strain-free objectives and condenser, a circular graduated stage capable of 360-degree rotation, Bertrand lens, and an opening in the microscope body or intermediate tube for compensators, such as a full-wave retardation plate, quartz wedge, or quarter-wavelength plate. Removal of the polarizer and analyzer (while other components remain in place) from the light path renders the instrument equal to a typical brightfield microscope with respect to the optical characteristics.

Polarized Microscope Alignment - In polarized light microscopy, proper alignment of the various optical and mechanical components is a critical step that must be conducted prior to undertaking quantitative analysis between crossed polarizers alone, or in combination with retardation plates and compensators. Several essential components must be correctly positioned with respect to both the microscope optical axis and to other mechanical and optical components. The series of alignment steps outlined in this section are intended for general use with polarized light microscopes and should be applicable to both student and research-level instruments.

Compensators and Retardation Plates - Optical anisotropy is studied in the polarized light microscope with accessory plates that are divided into two primary categories: retardation plates that have a fixed optical path difference and compensators, which have variable optical path lengths. Addition of a retardation plate or compensator to the polarized light microscope produces a highly accurate analytical instrument that can be employed to determine the relative retardation (often symbolized by the Greek letter G) or optical path difference between the orthogonal wavefronts (termed ordinary and extraordinary) that are introduced into the optical system by specimen birefringence. The terms relative retardation, used extensively in polarized light microscopy, and optical path difference (D or OPD), are both formally defined as the relative phase shift between the orthogonal wavefronts, expressed in nanometers.

Michel-Levy Birefringence Chart - The birefringence of a anisotropic material can be estimated when observed and/or photographed in a polarized light microscope. A relationship between interference color and retardation can be graphically illustrated in the classical Michel-Levy interference color chart, presented in this section. The graph plots retardation on the abscissa and specimen thickness on the ordinate. Birefringence is determined by a family of lines that emanate radially from the origin, each with a different measured value of birefringence corresponding to thickness and interference color.

Jacques Babinet (1794-1872) - Jacques Babinet was a French physicist, mathematician, and astronomer born in Lusignan, who is most famous for his contributions to optics. Among Babinet's accomplishments are the 1827 standardization of the Ångström unit for measuring light using the red cadmium line's wavelength, and the principle (bearing his name) that similar diffraction patterns are produced by two complementary screens.

Max Berek (1886-1949) - Max Berek was a German physicist and mathematician, associated with the firm of E. Leitz, who designed a wide spectrum of optical instruments, in particular for polarized light microscopy and several innovative camera lenses. Professor Berek is credited as the inventor of the Leica camera lens system at their Wetzlar factory.

Sir David Brewster (1781-1868) - Sir David Brewster was a Scottish physicist who invented the kaleidoscope, made major improvements to the stereoscope, and discovered the polarization phenomenon of light reflected at specific angles. In his studies on polarized light, Brewster discovered that when light strikes a reflective surface at a certain angle (now known as Brewster's Angle), the light reflected from that surface is plane-polarized. He elucidated a simple relationship between the incident angle of the light beam and the refractive index of the reflecting material.

Shinya Inoué (1921-Present) - Shinya Inoué is a microscopist, cell biologist, and educator who has been described as the grandfather of modern light microscopy. The pioneering microscopist heavily influenced the study of cell dynamics during the 1980s through his developments in video-enhanced contrast microscopy (VEC), which is a modification of the traditional form of differential interference contrast (DIC) microscopy. Inoué developed the method in parallel with Robert and Nina Allen and described his work at the same meeting of the American Society for Cell Biology as his fellow scientists. His seminal work, Video Microscopy, was published in 1986, and a second revised and updated edition, co-authored with Kenneth Spring, followed in 1997. The book is a cornerstone of microscopical knowledge and is highly regarded throughout the scientific community.

Walter C. McCrone (1916-2002) - Walter McCrone was an optical microscopist from Chicago who founded the world-famous McCrone Research Institute and made a significant number of contributions to microscopy as an investigational tool. McCrone's acclaimed work with the Shroud of Turin received worldwide attention in 1978 when he concluded that the Turin Shroud is a medieval painting. This observation was vindicated by radioactive carbon-14 dating techniques in 1988. In 2000, McCrone received the American Chemical Society National Award in Analytical Chemistry for his work on the Turin Shroud and for this enduring patience for the defense of his work for nearly 20 years.

Henri Hureau de Sénarmont (1808-1862) - Sénarmont was a professor of mineralogy and director of studies at the École des Mines in Paris, especially distinguished for his research on polarization and his studies on the artificial formation of minerals. He also contributed to the Geological Survey of France by preparing geological maps and essays. Perhaps the most significant contribution made by de Sénarmont to optics was the polarized light retardation compensator bearing his name, which is still widely utilized today.

Interactive Java Tutorials

Acoustical Model of Anisotropy - The anisotropic character of materials relates to those properties that have different values when measurements are made in different directions within the same material. This interactive tutorial explores how sound waves exhibit anisotropic character as a function of grain structure when traveling through a wooden block, which serves as an excellent model for the behavior of light passing through anisotropic crystals.

Double Refraction (Birefringence) - Calcite is a form of calcium carbonate, commonly referred to as Iceland spar, which has a rhombohedral crystalline shape. Light passing through a crystal of calcite is refracted into two rays, which are separated by a wide margin due to the strong birefringence of the crystal. This interactive tutorial simulates viewing of a ball-point pen and a line of text through a crystal of Iceland spar, producing a double image from the refracted light rays.

Brewster's Angle - A common source of polarized light is that reflected from a dielectric medium such as a window pane, sheet of paper, or a highway on a sunny day. This tutorial demonstrates the polarization effect on light reflected at a specific angle (the Brewster angle) from a transparent medium. Adjustable parameters include the incident beam wavelength, refractive index of the dielectric medium, and the rotation angle from which the tutorial is viewed by the visitor.

Birefringent Crystals in Polarized Light - In order to examine how birefringent anisotropic crystals interact with polarized light in an optical microscope, the properties of an individual, isolated crystal can be considered. The specimen material in this tutorial is a hypothetical tetragonal birefringent crystal having an optical axis oriented in a direction that is parallel to the long axis of the crystal. Light entering the crystal from the polarizer will be traveling perpendicular to the optical axis of the crystal, regardless of the crystal orientation with respect to the polarizer and analyzer transmission axes. The virtual microscope viewport presents the crystal as it would appear in the eyepieces of a microscope under crossed-polarized illumination as it is rotated around the microscope optical axis.

Birefringence in Calcite Crystals - As light travels through an anisotropic material, the electromagnetic waves become split into two principal vibrations, which are oriented mutually perpendicular to each other and perpendicular to the direction that the waves propagate. The wave whose electric vector vibrates along the major axis of the index ellipse is termed the slow wave, because the refractive index for this wave is greater than the refractive index for the other wave. The wave vibrating perpendicular to the slow wave is termed the fast wave. This tutorial explores double refraction or birefringence in calcite (calcium carbonate), a colorless, transparent, rhombohedral crystalline salt that is the most common such material found naturally.

Polarization of Light - When light travels through a linear polarizing material, a selected vibration plane is passed by the polarizer, while electric field vectors vibrating in all other orientations are blocked. Linearly polarized light transmitted through a polarizer can be either passed or absorbed by a second polarizer, depending upon the electric vector transmission azimuth orientation of the second polarizing material. This tutorial explores the effect of rotating two polarizers on an incident beam of white light.

Polarization of Light (3-D Version) - When non-polarized white light encounters a linear polarizer that is oriented with the transmission azimuth positioned vertically to the incident beam, only those waves having vertical electric field vectors will pass through. Polarized light exiting the first polarizer can be subsequently blocked by a second polarizer if the transmission axis is oriented horizontally with respect to the electric field vector of the polarized light waves. The concept of using two polarizers oriented at right angles with respect to each other is commonly termed crossed polarization and is fundamental to the concept of polarized light microscopy. This tutorial explores the effects of two polarizers having adjustable transmission axes on an incident beam of white light, and enables the visitor to translate the optical train in three dimensions.

Electromagnetic Wave Propagation - Electromagnetic waves can be generated by a variety of methods, such as a discharging spark or by an oscillating molecular dipole. Visible light is a commonly studied form of electromagnetic radiation, and exhibits oscillating electric and magnetic fields whose amplitudes and directions are represented by vectors that undulate in phase as sinusoidal waves in two mutually perpendicular (orthogonal) planes. This tutorial explores propagation of a virtual electromagnetic wave and considers the orientation of the magnetic and electric field vectors.

Polarized Light Waveforms - The ordinary and extraordinary light waves generated when a beam of light traverses a birefringent crystal have plane-polarized electric vectors that are mutually perpendicular to each other. In addition, due to differences in electronic interaction that each component experiences during its journey through the crystal, there is usually a phase shift that occurs between the two waves. This interactive tutorial explores the generation of linear, elliptical, and circularly polarized light by a pair of orthogonal light waves (as a function of the relative phase shift between the waves) when the electric field vectors are added together.

Nicol Prisms - Several versions of prism-based polarizing devices were once widely available, and these were usually named after their designers. The most common polarizing prism (illustrated in the tutorial window) was named after William Nicol, who first cleaved and cemented together two crystals of Iceland spar with Canada balsam in 1829. Nicol prisms were first used to measure the polarization angle of birefringent compounds, leading to new developments in the understanding of interaction between polarized light and crystalline substances. This interactive tutorial explores the generation of orthogonal or mutually perpendicular (ordinary and extraordinary) waves as the result of light transmission through a Nicol prism.

The Fresnel or Refractive Index Ellipsoid - The Fresnel, or refractive index, ellipsoid describes the dielectric properties measured in all directions through a material. Measurements through the radius yields the refractive index (n) or the square root of the dielectric constant for waves whose electric displacement vectors lie in the direction of the ellipsoid radius. This tutorial explores variations in the shape and dimensions of the ellipsoid as a function of refractive index.

Birefringence Variations with Crystal Orientation - When a beam of light is incident on a birefringent crystal, the waves are split upon entry into orthogonal polarized components (termed ordinary and extraordinary) that travel through the molecular lattice along different pathways, depending on their orientation with respect to the crystalline optical axis. If the incident beam is oblique to the optical axis, the waves diverge during their journey through the crystal. In contrast, the orthogonal wave components follow a co-linear pathway when the incident light beam enters the crystal either parallel or perpendicular to the optical axis. This interactive tutorial explores variations in birefringence that result from orientational variations between the crystal optical axis and the incident light beam.

Compensation Accessory Plates and Wedges - A careful examination of anisotropy as a function of specimen orientation permits identification of the refractive index difference and orientation of the extraordinary and ordinary light rays produced by birefringent materials. This tutorial explores how compensators may be employed to help determine orientation parameters of anisotropic materials.

de Sénarmont Compensators - A de Sénarmont compensator is composed of a linear polarizer combined with a quarter-wavelength retardation plate, and is capable of producing either linear, elliptical, or circularly polarized light, depending upon the orientation of the polarizer vibration axis with respect to the fast and slow axes of the retardation plate. This interactive tutorial explores the relationship between wavefronts emanating from the compensator as the polarizer is rotated through its useful range.

Interactive Michel-Levy Birefringence Chart - Quantitative analysis of the interference colors observed in birefringent samples is usually accomplished by consulting a Michel-Levy chart similar to the one illustrated in the tutorial window below. As is evident from this graph, the polarization colors visualized in the microscope and recorded onto film or captured digitally can be correlated with the actual retardation value, thickness, and birefringence of the specimen. The chart is relatively easy to use with birefringent samples if two of the three required variables are known. This interactive tutorial enables visitors to determine the interference color associated with all three values by clicking on selected regions of the interactive chart. A large version of the tutorial is also available.

Polarized Light Virtual Microscopy Java Tutorials

Polarized Light Virtual Microscopes - When a birefringent material is placed between crossed polarizers in an optical microscope, light incident upon the material is split into two component beams whose amplitude and intensity vary depending upon the orientation angle between the polarizer and permitted vibration directions of the material. Use this link to explore our tutorials on polarized light microscopy.

Polarized Light Digital Image Gallery

Digital Image Gallery - As a contrast-enhancing optical technique, polarized light microscopy is unsurpassed in the magnificent array of colors and beautiful textures generated through interference between orthogonal wavefronts at the analyzer. Useful for observation of mineral thin sections, hairs, fibers, particles, bones, chemical crystals, polymers, and a wide variety of other specimens, polarized light can be employed for both quantitative as well as qualitative investigations. Visit this gallery to observe how polarized light can be of advantage in the observation of specimens that would otherwise exhibit poor contrast and be difficult to distinguish from the background.

Selected Literature and Web Resources

Polarized Light Literature References - A number of high-quality books and review articles on polarized light microscopy have been published by leading researchers in the field. This section contains periodical location information about these articles, as well as providing a listing of selected original research reports and books describing the classical techniques of optical crystallography and polarized light microscopy.

Polarized Light Microscopy Web Resources - Although much neglected and undervalued as an investigative tool, polarized light microscopy provides all the benefits of brightfield microscopy and yet offers a wealth of information, which is simply not available with any other optical microscopy technique. This section is a compendium of web resources focused on all aspects of polarized light microscopy, optical crystallography, and related techniques.

Contributing Authors

Mortimer Abramowitz, Peter Dimitruk and William K. Fester - Scientific Equipment Group, Olympus America Inc., Two Corporate Center Drive, Melville, New York, 11747.

Savile Bradbury - 61 Hill Top Road, Oxford OX4 1PD, United Kingdom.

Philip C. Robinson - Department of Ceramic Technology, Staffordshire Polytechnic, College Road, Stoke-on-Trent, ST4 2DE, United Kingdom.

Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.

Brian O. Flynn, John C. Long, Matthew J. Parry-Hill, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.


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