Phase-contrast microscopy

Phase-contrast microscopy visualizes differences in the refractive indexes of different parts of a specimen relative to unaltered light. It is an optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visible when shown as brightness variations. Phase contrast is a method used in microscopy and developed in the early 20th century by Frits Zernike. Phase is only useful on specimens that are colorless and transparent and usually difficult to distinguish from their surroundings. We call these specimens "phase objects". Examples of phase objects include cell parts in protozoans, bacteria, sperm tails and other types of unstained cells. Principle Highly refractive structures bend light to a much greater angle than do structures of low refractive index. The same properties that cause the light to bend also delay the passage of light by a quarter of a wavelength or so. A phase ring in condenser allows a cylinder of light through in phase. Light that is unaltered hits the phase ring in the lens and is excluded. Light that is slightly altered by passing through a different refractive index is allowed through. Light passing through cellular structures is retarded because they have a higher refractive index than the surrounding medium.

Use
Phase contrast is preferable to bright field microscopy when high magnifications (400x, 1000x) are needed and the specimen is colorless or the details so fine that color does not show up well. Cilia and flagella, for example, are nearly invisible in bright field but show up in sharp contrast in phase contrast. Today, two main types of phase contrast are positive and negative.Positive phase contrast reveals medium to dark gray images on a lighter grey background; these images often have a bright halo along the edge of the sample.Negative phase contrast is the opposite. The specimen appears lighter with a dark background; they also have a dark halo outlining the image. Advantages The advantages of the phase contrast microscope are its capacity to observe living cells in a natural state, can provide far more information than specimens that need to be killed, fixed or stain to view under a microscope, High-contrast, high-resolution images, Ideal for studying and interpreting thin specimens, Ability to combine with other means of observation, such as fluorescence, Modern phase contrast microscopes, with CCD or CMOS computer devices, can capture photo and/or video images Disadvantages Disadvantages and limitations of phase contrast are Annuli or rings limit the aperture to some extent, which decreases resolution.This method of observation is not ideal for thick organisms or particles because thick specimens can appear distorted. Images may appear grey or green, if white or green lights are used, respectively, resulting in poor photomicrography. Shade-off and Halo effect will also occur.Shade-off occurs with larger particles, results in a steady reduction of contrast moving from the center of the object toward its edges. Halo effect, where images are often surrounded by bright areas, which obscure details along the perimeter of the specimen