Topics in Light Microscopy

Anomalous Birefringence

Anomalous birefringence is simply the result of a material's birefringence changing in a significant way as a function of the wavelenght of light. It produces anomalous interference colors in the material being examined between crossed polarizing filters.

Crocidolite, Higher Birefringence in Red Light (longer Wavelengths)

Very thin fibers appear red between crossed polarizing filters. Thicker fibers appear blue because of the strong blue color of the mineral.

Silicon Carbide, Higher Birefringence in Blue Light (Shorter Wavelenghts)

Blue wavelengths cycle more rapidly than red wavelengths. Yellow interference color begins for thinner particles and first order red appears purple because blue is increasing well before red significantly decreases. This effect changes the color sequence through the whole range of microscopic silicon carbide particles.

Silicon Carbide Abrasive Under the Microscope

Apparent Birefringence

Polarized light can be depolarized by light scatter or by specular reflection at an interface that is not aligned with the orientation of the polarized beam. Birefringence can be induced by stress in materials that are otherwise isotropic. Ordered distributions of regular shaped isotropic particles or fibers with a different refractive indix than an isotropic matrix can induce an apparent birefringence. Click on the photographs below for more information.

Edge Effect Birefringence

Polarized light is depolarized at the interface between a conductive particle and a non-conductive mounting medium. This light halo effect with transmitted crossed polarized light indicates an opaque particle is a wear metal particle or at least is conductive. Graphite is sufficiently conductive to produce this effect. Pencil debris can be distinguished from combustion residue by this effect.

Graphite

Magnetite Spheres

Stress Birefringence

When a material is placed under stress the distribution of the electrons in the material is changed. The amount of change is different for each material and is a characteristic of the material. The photoelastic constant of the matrial is a measure of the electron displacement (strain) as a function of the load (stress) applied as long as the deformation is elastic, springs back when the load is removed. If the YOung's Modulus of the matrial is exceeded then some of the deformation becomes permenant. In some materials the applied load can be "frozen" in place, as in the case of high stress glass sheet. Polarized light can make the displacement visible. Both plastic deformation and elastic deformation result in an anisotropic distribution of electrons in the material that becomes visible as interference colors when the object is viewed between crossed linear or crossed circular polarizing filters. Click on the photographs below for more information.

Birefringence from Plastic Deformation in Human Skin Cells

Birefringence from Plastic Deformation in Dog Skin Cells

Stress Birefringence in Safety Glass

Resolution vs Visibility Through the Microscope

The resolution of objects viewed through the light microscope is a calculated value based on configuration of the microscope. Visibility is a function of contrast. To take advantage of resolution there must be sufficient contrast to see the subject but objects far below the resolution limit of a specific microscope configuration can be made visible with sufficient contrast. This is the same phenomena that allows us to see the stars in the night sky, all of which are far below the resolution limit of our eyes. A few examples through the microscope are provided below showing methods of improving the resolution and/or visibility of objects viewed through the "standard" light microscope. Click on the photographs below for more information.

Resolution and Ocular Magnification

The resolution of the microscope is determined by the objective and the condenser. The ocular must provide sufficient magnification for the observer to see what is being resolved. The images below provide a simple example.

$Darkfield Diffraction Pattern at Back Focal Plane$

Darkfield Image of 1.9 micrometer Diffration Grating

Brightfield vs Darkfield Illumination

$Diffraction Pattern at Back Focal Plane$

Brightfield Image of 1.9 micrometer Diffration Grating

$Darkfield Diffraction Pattern at Back Focal Plane$

Darkfield Image of 1.1 to 0.32 micrometer rulings

Brightfield vs Oblique Illumination

$Diffraction Pattern at Back Focal Plane$

Oblique Image of 1.9 micrometer Diffration Grating

$Diffraction Pattern at Back Focal Plane$

Phase Contrast vs Visibility Through the Microscope

Phase contrast microscopy makes "phase" objects more visible. Every object is a "phase" object strictly speaking but some objects are primarily phase objects, being nearly invisible using standard brightfield illumination. Phase constrast does not always improve visibility. Small objects with significantly different refractive indices than the medium in which they are mounted will be visible using darkfield illumination even when they are invisible using phase contrast. The "Phase Contrast Test Slide" is a good example, as shown below.

40X 0.65 NA Phase Contrast Image of Phase Contrast Test Slide

10X 0.25 NA Darkfield Image of Phase Contrast Test Slide

Polarized Light vs Visibility Through the Microscope

Objects with more than one refractive index can be made self-luminous by using crossed polarized light. This can create sufficient contrast to see the object.

Chrysotile Field 1 in 1.550 RI Liquid Viewed with Brightfield Illumination

Chrysotile Field 1 in 1.550 RI Liquid Viewed with Polarized Light

Asbestos Fibers and Visibility Through the Light Microscope

Much has been made of the need for transmission electron microscopy (TEM) "because the light microscope can't see asbestos fibers thinner than 0.25 micrometers". This quote is in error on many counts, but is a common misconception. First, it is not the microscope that "sees", but rather the human eye establishes the detection limit. If the light source is not "on" the resolution limit for the microscope has not changed but the eye won't see anything. Second, the ability to "see" (detect) an object is not dependent on resolution. This point has been made for the general case above but will be made again here in the specific case of asbestos fibers. Third, after over thirty years of diligent searching there has been no evidence that asbestos fibers shorter than five micrometers cause any health effect more detrimental than nuisance dust. Fresh crystalline silica is more detrimental in this respect. That challenges the "need" for an instrument that can "see" particles that are not apparently detrimental as a replacement for light microscopy. We will continue monitoring with the TEM because it is required in many governement and industrial regulations, but it is not a "superior" method in the case of hazardous asbestos detection or identification at sites of asbestos removal projects.

Cleared Filter Containing Amosite, Phase Contrast 40X Objective

10X 0.25 NA Darkfield Image of Cleared Filter Containing Amosite

Cleared Filter Containing Chrysotile, Phase Contrast 40X Objective

10X 0.25 NA Darkfield Image of Cleared Filter Containing Chrysotile

40X 0.65 NA Phase Contrast Image of HSE Groups 5, 6, and 7