PHASE CONTRAST MICROSCOPY

Introduction:

Most cells are too small to be seen by the naked eyes, the study of cells has depended heavily on the use of microscopes. Microscopy is the technique which involves the instruments microscopes that make the small things visible to the naked eyes.

 Antony van Leeuwenhoek, in the 1670s, was able to observe a variety of different types of cells by using a microscope. The light microscope remains a basic tool of cell biologists, with technical improvements allowing the visualization of ever-increasing details of cell structure.

However, the light microscope is not sufficiently powerful to reveal fine details of cell structure, for which resolution- is even more important than magnification.

Magnification is the ratio of image size to the specimen size.

Resolution power: it is the ability of a microscope to distinguish between two different points.

Resolving power is the ability of magnifying instrument to distinguish two objects that are close together.

Limit of resolution is the minimum distance between two points that allows for their discrimination as two separate points. 

                                       Limit of resolution  1/ resolution power

The limit of resolution of the light microscope is approximately 0.2 µm; two objects separated by less than this distance appear as a single image. This theoretical limitation of light microscopy is determined by two factors:

a.       The wavelength (λ) of visible light

b.       The light gathering power of the microscope lens (N.A)

 

ABBE EQUATION::

Limit of resolution = λ * 0.61 / N.A

 Where, (N.A = numerical aperture, amount of light falling on the objective lens = n sin O)

(n = refractive index of the medium , O is the angle of cone formed by falling light).

So lower the wavelength, better would be the resolution power.

Several different types of light microscopy are routinely used to study various aspects of cell structure. However, in many case, the cells are stained first with dyes that react with the proteins and nucleic acids in order to enhance the contrast. Prior to staining, fixatives are used to stabilize and preserve their structures. Such staining procedures kill the cells. Therefore such microscopy techniques are not suitable for the experiments in which the observation of living cells is desired.

Optical variation of the light microscope can be used to enhance the contrast between light waves passing through regions of the cell with different densities. The most common method for this is phase contrast microscopy.

In bright field microscopy, transparent structures (like nucleus) have little contrast because they absorb light poorly. Poor light absorption results in extremely small differences in the intensity distribution in the image. This makes the cells barely, or not at all, visible in a bright field microscope. However, light is slowed down as it passes through these structures so that its phase is altered compared to light that has passed through the surrounding cytoplasm. Phase contrast microscopy convert these differences in phase to differences in contrast ,or we can say that :

 it is an optical microscopy technique that converts phase shifts in the light passing through a transparent specimen to brightness, thereby yielding improved images of live, unstained cells.

It was first discovered by Frits Zernike, in 1934.

Phase shift:     (refractive index + change in direction of light wave)

Working principal of an ordinary microscope:

In an ordinary microscope, the object is viewed due to differences in color intensities of the specimen. To create the color intensities, the specimen is first stained with suitable dyes which fill impart specific color. In an ordinary microscope, the contrast is obtained when the light rays pass through a stained specimen because different stains absorb different amounts of light. These differential absorption properties of stained specimen modify the intensity or amplitude of the light waves transmitted by different regions of the cells and this ultimately creates contrast in the image. Thus, staining is essential to create contrast in an ordinary microscope. Moreover, the unstained specimen cannot be observed through an ordinary microscope.

 

Working principal of phase contrast:

The phase contrast microscopy is based on the principle that small phase changes in the light rays, induced by differences in the thickness and refractive index of the different parts of an object, can be transformed into differences in brightness or light intensity. In simple terms, phase contrast microscopy is the translation of invisible differences of intensities.  In phase contrast microscope condenser have a annular stop which produces a hollow cone of light. As the light passes through the cone some light rays are bending due to variation in density and thickness of the specimen. Deviated light is focused to form the final image. Background formed by undeviated light is bright and unstained objects appear dark and well defined.

INSTRUMENTATION:

Light that passes through thicker parts of the cell is held up relative to the light that passes through thinner parts of the cytoplasm. It requires a specialized phase condenser and phase objective lenses.

Each phase setting of the condenser lens is matched with the phase setting of the objective lens. These are usually numbered as Phase 1, Phase 2 and Phase 3, and are found on both the condenser and the objective lens.

Parts of phase contrast microscopy:

The phase contrast microscope is similar to an ordinary compound microscope in its optical composition. It possesses a light source, condenser system, objective lens and ocular lens. It differs from the normal microscope in having two additional components:

(a)  The annular diaphragm:

·         Situated below the condenser

·         Made up of circular disc having a circular annular groove.

·         Helps to create a narrow, hollow cone or ring of light to illuminate the object

 

(b)  The Phase plate:

·         It is also called diffraction plate or phase retardation plate.

·         Located at the back focal plane of the objective lens.

·         The phase retarding components are coated on this plate.

·         It is a transparent glass disc with one or few channels.

·         The channel is coated with a material that can absorb light but cannot retard it.

·         The other portion (except channels) of the phase plate is coated with light retarding materials such as magnesium fluoride.

·         Phase plate helps to reduce the phase of the incident light.

 

WORKING::   How Contrast is created in Phase Contrast Microscopy?

The unstained cells cannot create contrast under the normal microscope. However,

When the lights pass through an unstained cell, it encounters regions in the cells with different refractive indexes and thickness. When light rays pass through an area of high refractive index, it deviates from its normal path and such a light ray experiences phase change or phase retardation. Light rays pass through the area of less refractive index remain undeviated (no phase change).

The difference in the phases between the retarded and un-retarded light rays is about 1/4 of original wave length (i.e., λ/4). Human eyes are NOT able to detect this minute changes in the phase of light and thus, such small phase changes do not create any contrast in the image.

The phase contrast microscope has special devices (annular diaphragm and phase plate), which convert these minute phase changes into amplitude changes or brightness changes so that a contrast difference can be created in the final image. This contrast difference can be easily detected by our eyes.

 

In phase contrast microscope, in order to get contrast, the diffracted waves have to be separated from the direct waves. This separation is achieved by the sub-stage annular diaphragm.

 

The annular diaphragm illuminates the specimen with a hollow cone of light. Some rays (direct rays) pass through the thinner region of the specimen and do not undergo any retardation and they directly enter into the objective lens. The light rays passing through the denser region of the specimen get retarded and they run with a delayed phase than the undeviated rays. The retardation of the phase of light is about 1/4 of the λ of the incident light. Both the retarded and unretarded light has to pass through the phase plate kept on the back focal plane of the objective to form the final image.

The phase plate is designed and positioned in such a way that the retarded light rays will pass through the area of phase plate where light retarding materials are coated. When the 1/4 (or λ/4) retarded light is passed through this region of phase plate, it is further retarded by 1/4 (or λ/4). With this, the final change or retardation will be- 1/4λ + 1/4λ = ½λ (or λ/4 + λ/4 = λ/2). The unretarded rays will pass through the channels of the phase plate and their phase is not altered by the phase plate.

When the unretarded and 1/2λ (or λ/2) retarded light are recombined (at the focal point), a negative or destructive interference is created because the crest and trough of the unretarded and retarded light rays will cancel each other. With the destructive interference, the image of the specimen appears darker against a bright background. On the other hand, if the undeviated light rays are passed through the phase regarding material, the two rays will be in the same phase and the result will be a positive or constructive interference.

In constructive interference, the image of the specimen becomes brighter against a dark background. Thus in phase contrast microscopy, the combination of destructive and constructive interferences creates high contrast in the final image.

 

       

RESULTS:

Applications:

·         Phase contrast is by far the most frequently used method in biological light microscopy.

·         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.

·         It is an established microscopy technique in cell culture and live cell imaging. It can be used, when high resolution is not required.

·         When using this inexpensive technique, living cells can be observed in their natural state without previous fixation and labeling.

·         It helps to study the biological processes and cellular events such as cell division, phagocytosis, etc.

·         Phase contrast was the traditional choice for imaging cell movement and behavior of cells growing in tissue culture such as chromosomal and flagellar movements.

·         For producing the high contrast images of :

ü  Microorganisms

ü  Fibers, thin tissue slices,

ü  Sub cellular particles

Advantages:

§  Provide the clear image of unstained cells.

§  Avoid damages of the cells due to chemical preparation and staining.

§  Provide high contrast images highlighting the fine details of the cells.

§  The optical construction is relatively simple.

§  As phase contrast is ideal for thinner samples, therefore an inverted microscope system can be used. This provides the additional advantage of more working space.

§  Uses a conventional light microscope fitted with a phase-contrast objective and phase-contrast condenser.

 

Disadvantages/limitations:

§  To use phase contrast the light path must be aligned.

§  Generally more light is needed for phase contrast than for corresponding bright field viewing, since the technique is somehow based on the diminishment of the brightness.

§  Images generated using Phase contrast microscope often produces a bright halo around the outline of details that have a high phase shift. This is due to the partial or incomplete separation of direct or deviated rays. This makes it hard to see the boundaries of details.

§  It is only useful for viewing individual cells or thin layer of cells. It does not work well with thick specimens as these can appear distorted.

§  The resolution of phase images can be reduced due to the phase annulus limiting the numerical aperture of the system.