Observation Microorganism

Introduction

Phase contrast microscopy, first described in 1934 by Dutch physicist Frits Zernike, is a contrast-enhancing optical technique that can be utilized to produce high-contrast images of transparent specimens, such as living cells (usually in culture),

Microorganisms, thin tissue slices, lithographic patterns, fibers, latex dispersions, glass fragments, and subcellular particles (including nuclei and other organelles).

Unstained specimens that do not absorb light are called phase objects because they slightly alter the phase of the light diffracted by the specimen, usually by retarding such light approximately 1/4 wavelength as compared to the undeviated direct light passing through or around the specimen unaffected.

Unfortunately, our eyes as well as camera film, are unable to detect these phase differences. To reiterate, the human eye is sensitive only to the colors of the visible spectrum (variations in light frequency) or to differing levels of light intensity (variations in wave amplitude).

Principle

Phase contrast microscopy translates small changes in the phase into changes in amplitude (brightness), which are then seen as differences in image contrast.

Unstained specimens that do not absorb light are known as phase objects. 

This is because they slightly change the phase of light that is diffracted by them; the light is usually phase-shifted by about ¼ wavelength compared to the background light.

Phase contrast enables high contrast images to be produced by further increasing the difference of the light phase. It is this characteristic that enables background light to be separated from specimen diffracted light. 

The difference of the light phase is increased by slowing down (or advancing) the background light by a ¼ wavelength, with a phase plate just before the image plane. 

When the light is focused on the image plane, the diffracted and background light cause destructive (or constructive) interference which decreases (or increases) the brightness of the areas that contain the sample, in comparison to the background light.

Light from a tungsten-halogen lamp goes through the condenser annulus in the substage condenser before it reaches the specimen. This allows the specimen to be illuminated by parallel light that has been defocused. Some of the light that passes through the specimen will not be diffracted.

These light waves form a bright image on the rear aperture of the objective. The light waves that are diffracted by the specimen pass the diffracted plane and focus on the image plane only. 

This allows the background light and the diffracted light to be separated. The phase plate then changes the background light’s speed by ¼ wavelength.

When the light is focused on the image plane, the diffracted and background light will cause destructive or constructive interference, which changes the brightness of the areas that contain the sample in comparison to the background light. 

Working

1. Install the phase rings in the condenser.
2. Remove the eyepieces and replace these with the phase contrast centring telescope.
3. Put the phase contrast telescope into focus, so that the phase plate and phase ring are in focus.
4. Put the lowest magnification phase objective and corresponding phase annulus in place.
5. Look at the phase plate and phase ring through the phase telescope.
6. Using the adjustment screws on the condenser, centre the phase plate and phase ring so the segmented circle of light sits on the black ring.
7. Once all objectives have been aligned and centred, remove the phase contrast-centring telescope and replace this with the eyepieces.

Application

Phase-contrast microscopy is especially useful for studying microbial motility, determining the shape of living cells, and detecting bacterial structures such as endospores and inclusions.

These are clearly visible because they have refractive indices markedly different from that of water. Phase-contrast microscopes also are widely used to study eukaryotic cells .