Tuesday, February 4, 2014

Microcope Sample Characterization by Light

Light behavior is revealed in different forms including reflection, refraction, dispersion, diffraction, interference, absorption, polarization and excitation. Each of these forms are described in detail below and provide a basis for understanding the various specimen characterization methods by light action with the microscope.


Reflection of light from a smooth surface (such as a mirror), takes place along a definite direction determined by the direction of the incident ray, and is called regular or specular reflection. Reflection from a rough or matte surface occurs in many directions and the incident beam is said to be "diffused" or "scattered". Non-luminous objects become visible by diffused reflection.

light reflection illustration
Light reflection from a smooth surface.

light reflection from a rough surface
Light reflection from a rough surface.


A ray of light of a single wavelength  undergoes an abrupt change of direction upon passing obliquely from one medium to another. The effects of refraction are responsible for a variety of familiar phenomena, such as the apparent bending of an object that is partially submerged in water and the mirages observed on a dry, sandy desert. The refraction of visible light is also an important characteristic of microscope lenses that enables them to focus a beam of light onto a single point.

light refraction
Light refraction


The spreading out of light into its component colors is known as dispersion. For example, when a narrow slit is illuminated by white light which is then passed through a prism, refraction takes place and the constituent colors are spread out into an array called a spectrum.

ligh dispersion image
Light Dispersion


Light bends slightly around edges when it is obstructed. The spreading of a beam of light into the region behind an obstacle is known as diffraction. When we view a specimen, whether directly or with a microscope, the image seen is composed of a myriad of overlapping points of light emanating from the plane of the specimen. Therefore, the appearance and integrity of the image from a single point of light holds a significant amount of importance with regards to formation of the overall image. Because the image-forming light rays are diffracted, a single point of light is never really seen as a point in the microscope, but rather as a diffraction pattern containing a central disk or spot of light having a finite diameter and encircled by a fading series of rings. As a result, the image of a specimen is never an exact representation of the specimen, and a lower limit is imposed on the smallest detail in the specimen that can be resolved.

light diffraction illustration
Light Diffraction


The colors produced by the interference of light waves at the front and back surfaces of very thin films are observed by reflection. Some natural substances have the property of double refraction, such as quartz, calcite and mica. Man-made devices such as diffraction gratings and others are often employed to produce interference effects. The phenomena of interference, like most of the other properties of light, are used in microscopy both as a tool to investigate object materials, or the phenomena produced naturally by the object material are interpreted as a characteristic.


The amount of light that is reflected from a transparent substance depends on the angle of incidence and the refractive index of the substance. At perpendicular incidence of a light beam only a part is reflected, the rest is being absorbed and transmitted. The amount of light absorbed depends on its thickness and the nature of the specimen. Absorption means the conversion of electromagnetic energy to heat energy.

absorption image illustration


Polarized light is used as a tool or is interpreted as an object characteristic. Light vibrations restricted to a single plane are said to be plane-polarized. Polarized light microscopy is a useful method to generate contrast in birefringent specimens. The beautiful variety of colors displayed by specimens under crossed polarizers is a result of the interference between light waves passing through the specimen.


Certain substances when exposed to or excited by light continue to emit light a fraction of a second after removal of the exciting source. This property of a substance is known as fluorescence. Microscopic samples are excited with a fluorescence microscope. Fluorescence is a very powerful analytical tool in light microscopy.

Source: McLaughlin, Robert. Special Methods in Light Microscopy. London: Microscope Publications Ltd., 1977. Print.