Essay on the different Types of Microscope



Ultraviolet Microscope: The microscope employs ultraviolet rays for illumination of object. In this microscope lenses are made of fluoride, lithium fluoride or quartz instead of glass.

This microscope is used for quantitative and qualitative determination of cell components which absorb ultra violet rays. For example, as nucleic acid absorbs ultraviolet rays those places in a cell containing nucleic acid appear darker under this microscope than the other regions. Bright-field Microscope: Light is allowed to transmit through specimen in culture.

The contrast is given through staining the specimens. This requires fixing the cells (not alive). Dark-field Microscope: This microscope was developed by Zsigmondy (igo5).Here; all those objects which do not refract light are viewed against a dark background. Light is directed towards the specimen at an angle.

Then a special condenser having an opaque disc is used to transmit only the light reflected from the specimen. These results in a dark field, in which all objects with different refractive indices, are seen as brilliantly illuminated bodies against a dark background.

Polarizing Microscope: Polarizing light is used to analyze cell structure and cell inclusions. The polarized light vibrates only in one direction and cellular components are easily visible as bright objects against dark background.

Phase Contrast Microscope: This is a compound microscope where the light waves form differences in contrast and brightness so that different cell components are distinguished. This microscope was invented by Zernike in 1935 who was awarded Nobel Prize in 1953. Here living cells can be studied without fixing or staining them. In this case visible light is the source of illumination.

The beam of light splits and passes through different components of the cell having different refractive indices. As a result phase variations occur. These phase variations are visible.

Fluorescent Microscope: Fluorescent stains like quinine sulphate, rhodamine and auramine are used so that a set of filters in the microscope transmits only the light that is emitted from fluorescently stained molecules or tissues.

The source of illumination is U.V. light and the fluorescent bodies absorb U.V. light and emit visible light. This microscopy was developed by Coons in 1945.

X-ray Microscope: X-rays having shorter wavelength are used so as to' have greater resolving power. The structural details of molecules can be analyzed by it. Structural details of molecules like DNA, proteins are resolved by this microscope. X-ray diffraction analysis reveals molecular configuration.

Confocal Microscope: Laser beam is focused to a point and scanned across the specimen in two directions.

This results information of clear images of one plane of the specimen. The images of other plane of the specimen are excluded so that, the final images do not blur. Fluorescent dyes may be used to enhance the images.

Cell Fractionation

Cell fraction is a procedure of separating organelles from the cytosol of the cell and also from each other. The first step involves homogenization of tissues or cells. Homogenization means breaking down of cell membrane to release its contents. This is done by grinding the tissues/cells in a grinder or blender for 1-2 minutes.

The tissues/ cells are kept in a homogenizing medium containing about 0.2 M sucrose during the process of homogenization. The sucrose creates a medium with an osmotic pressure similar to that within organelles. This prevents diffusion of water into the organelles. As a result the organelles never burst.

The cellular organelles like nucleus, mitochondria, endoplasmic reticulum etc. differ in size. So, if they are subjected to centrifugation, different organelles should sediment at different rates. So differential centrifugation procedure is employed for a rough fractionation of the cytoplasm contents.

The organelles also differ from each other in specific gravity. As a result they float at different levels in a density gradient. After differential centrifugation further purification can be done by density gradient centrifugation.

Differential Centrifugation

"The centrifugal force is proportional to the radius of the centrifugal head and to the square of the angular velocity." Hence a relatively smaller head is used in a centrifuge machine and the head is allowed to rotate at high speed. A head of approximately 10cm in diameter rotating at a speed of 40, ooorpm (rotation per minute) produces a force of about 10000g (gravity).

The centrifuge tube containing the tissue homogenate is held at an angle to the axis of rotation to keep the path of the particles in solution as short as possible. Different sub cellular fractions sediment at the bottom of the centrifuge tube.

The clear liquid part contained in the tube is called supernatant that contains other sub cellular components not yet sedimented.The sediment at the bottom of the tube is called pellet.

On a single centrifugation, a mixture of components is obtained. In order to get pure fractions, the precipitate or pellet is to be centrifuged repeatedly.

Density Gradient Centrifugation

Different organelles have different buoyant densities because of different proportions of proteins and lipids in them. In density gradient centrifugation sucrose solutions of varying densities are taken in a centrifuge tube.

The cell homogenate is allowed to centrifuge in this tube containing sucrose density gradient. After centrifugation the organelles float at different density level in the tube depending upon their individual buoyant density. From each layer materials are carefully removed and examined to establish the sedimentation position of individual organelle.

Then from that layer purified organelles are obtained. In sucrose density gradient, densities of sucrose increase from the top to the bottom. After centrifugation the less dense components float at the top and dense particles remain at the bottom parts of the tube. Each sub cellular component forms a specific zone or layer.