The bright field microscope or light microscope or compound microscopes is the most common and the most indispensable instrument in the biology laboratory. Basically the microscope has two types of parts.
The mechanical parts provide the structural framework of the microscope and support the optical parts.
The optical parts are involved in the magnification of the object. The following is a detailed description of the various parts of a light microscope.
Image Source: microscope.com
Base, Pillar and Inclination Joint:
ADVERTISEMENTS:
The base and the attached pillar serve to support the entire microscope. The base is generally ‘V (horse shoe) shaped or sometimes ‘U’ shaped. Usually the pillar and the base are made heavy in order to minimize vibration.
The inclination joint joins the arm of the microscope to the pillar. The joint permits the tilting of the microscope to any degree as desired by the observer.
Arm and body tube:
The arm is a slightly bent solid piece of iron which is attached at one end to the pillar by the inclination joint. The other end of the arm has the body tube to which the principal optical systems are attached.
ADVERTISEMENTS:
The body tube is a cylindrical Structure and at the point of attachment to the arm there is a graduated rack and pinion which helps in the movement of the body tube up and down.
Draw tube, revolving nose piece:
Fitting properly inside the upper end of the body tube is the draw tube, which may be drawn up. “The ocular lens fits into the draw tube.
The purpose of the draw tube is to adjust tube length so that the objective lens and the ocular are at a specified distance.
The revolving nose piece is a circular rotating disc attached to the body tube. Objective lenses are attached to this.
Adjustment knobs:
ADVERTISEMENTS:
The body tube can move up and down on the rack and pinion and the movement is regulated by two sets of knobs – the course adjustment knob and the fine adjustment knob.
The coarse adjustment knob brings in greater degree of movement while the fine adjustment knob is used for slight and fine adjustment of the body tube.
The adjustment knobs are meant to bring in the object into proper focus. While the range of the coarse adjustment knob is greater, that of the fine adjustment knob is limited.
Stage:
This is a small platform attached to the arm of the microscope. This is meant to keep the objects (on sides) for the purpose of observation.
It has a hole in the center exactly in line with the body tube and the condenser below to allow the light to pass through.
In most modern microscopes, the stage is fitted with a mechanical device called mechanical stage to help in the vertical and horizontal movement of the slide.
Illumination:
In order to collect and reflect the light beams there are two kinds of devices attached to the base of the microscope. These are – mirror or an artificial illumination (built in illumination).
The mirror collects the light either from a natural source (sun light) or an artificial source (electric light) and reflects the rays into the microscope.
The mirror has both sides reflective – one side is plain, the other side is concave. Usually concave side is used when natural light is focused and plain side is used while focusing artificial light.
In some of the compound microscopes there is provision of built in illumination (instead of a mirror) which has a devise to use 8 or 12 volt bulb and provide direct illumination to the stage.
Sub stage Condenser; Iris diaphragm:
The light either reflected by the mirror or by a built in source will be diffuse and does not have sufficient density in order to condense the light and focus it on to the object, there is a sub stage condenser.
This consists of a condensing lens. The condensing lens may be fixed or in some microscopes it can also move up and down for suitable adjustment on a rack and pinion. The condenser helps to focus all the light on the object.
Beneath the condenser there is an iris diaphragm which regulates the quantity of light entering into the condenser. The iris diaphragm may be closed and opened with the help of a lever to regulate the entry of light.
Optical parts:
There are two sets of optics or lenses in a compound microscope. These are eye pieces (ocular) and objectives.
Eye pieces (Ocular):
The ocular or eye piece is a short tube with two lenses, which fits into the upper part of the draw tube. The main function of the eye piece is to magnify the image of the object formed by the objective. Eye pieces are marked 5X, 10X, 15X etc.
The eye piece commonly used is the 10X Huygenian eye piece with monocular tube. These eye pieces are also referred to as negative oculars. The positive ocular or Ramsden eye pieces are constructed with convex surfaces of both lenses facing inwards.
Objective lenses:
These are attached to the revolving nose piece and are very important as they affect the quality of the image seen by the observer based on their type of construction objectives are classified into achromatic, apochromatic and fluorite.
The first one is simple in construction and less expensive, while the other two are complicated more expensive and used in costly microscopes these two are also corrected for most of the common aberrations that occur in the lenses.
The objectives are of the following four types based on their magnification – 1OX (Low), 40X (Medium), 60X (High) and l () OX (Oil immersion). Several microscope manufacturers use different colored rings to distinguish individual objectives.
They can also be distinguished based on their length; low power being the shortest and oil immersion, the longest.
The oil immersion lens in used for very high magnification the lens is so named because a drop of a highly refractive liquid (refractive index same as glass) is added on the slide while viewing through an oil immersion objective.
The main functions of the objective lenses are -1. Concentration of light rays coming from the object, 2. Forming the image of the specimen and 3 Magnifying the image
Magnification:
A microscope is primarily used to enlarge or magnify the object that is being viewed which can not otherwise be seen by the naked eye. Magnification may be defined as the degree of enlargement of an object provided by the microscope.
Magnification by a microscope is the product of the individual magnifying ability of the oculars and the objectives for example if the ocular is 1 OX, and objective is 40X, the specimen is magnified 400 times.
If an oil immersion objective (100X) is used along with 1 OX ocular, the specimen is magnified 1000 times. The following factors play an important role in magnification.
(i) Optical tube length
(ii) Focal length of the objective lens
(iii) Magnifying ability of the ocular
Total magnification can be calculated as follows:
Total magnification = Optical tube length / Focal length of the objective X magnification of ocular.
Resolving power:
Theoretically if the magnifying power of the ocular and objectives are increased it should be possible to get higher and higher magnifications.
By using high powered lenses a magnification up-to 3000 can be obtained but the image will be blurred and details are not clear.
This is due to the fact that in a microscope not only the lenses, but the wave length of the illumination is also important and this decides the resolving power of the microscope.
Resolving power of a microscope is defined as its ability to distinguish between two particles situated very close. In a magnified image the object should not only be larger but the details should also be clear.
This is possible when a microscope has the ability to see two points situated very close as two distinct entities.
In other words, resolving power may be said to be the minimum distance at which two structural entities of an object can be visualized as discrete individual structures even in the magnified image.
(The above explanation may be illustrated clearly with a comparison with the human eye. Human eye functions on the same principal as that of light microscope i.e. we see objects because of the light reflected by them.
The human eye has a resolving power about 0.25 mm in the sense, two dots situated 0:25 mm (or more) apart can be seen as two dots; anything closer will look like a single dot).
The resolving power of a microscope depends on two factors – the wavelength of the light used for illumination and the numerical aperture (NA) of the objective.
In optical light (bright field) microscopes, the wave length of the light used falls in the visible range (400 – 750nm). Within this range if light of shorter wave length is used the resolution will be higher.
For example blue light has a shorter wavelength than red light. Greater resolution can be achieved by using a blue light as a source of illumination than a red light.
The second factor that decides the resolving power (RP) is the numerical aperture of the objective. NA is defined as the property of a lens that decides the quantity of light that can enter into it. It depends on two factors.
(i) Refractive index of the medium that fills the space between the specimen and the front of the objective lens and
(ii) The angle of the most oblique rays that can enter the objective lens (The more divergent or oblique rays that an objective can admit, greater is the resolving power).
NA can be mathematically calculated by the following formula
NA = n sin/Where
n – Refractive index of the medium angular aperture. This is defined as the angle between the most divergent rays passing through the lens and optical axis of the lens. Rays cannot enter the objective if their angle of divergence from the normal rays (straight rays) is greater than half the angular aperture.
Using NA, the resolution power of the microscope can be calculated as follows.
RP = Wave length of light / 2 x NA
For instance if a yellow light of wave length 580nm with NA of 1.0 is used RP will be as follows: RP=580/2×1 =290nm
If a blue light is used the RP will be still higher (i.e. it can resolve objects smaller than 290nm).
Limit of resolution:
This may be defined as the shortest distance between two objects when they can still be distinguished as two separate entities. Highest resolution in a light microscope is possible with the shortest wave length light (visible) and with the objective having highest NA. This can be expressed as follows:
d=X/ 2NA
Where d = resolution and X = wave length. For instance if we use green light (520nm wave length) and an objective with 1.3 NA the resolution may be calculated.
From the above it can be calculated that the smallest particle that can be seen by light microscope is the one not smaller than 0.2 mm in dimension.
Working Principle (higher magnification) of Oil Immersion Lens:
When an objective lens (less than 100X) other than oil immersion is used it is referred to as a day objective; in the space between the specimen and the front of objective there is only air. Air has a refractive index of 1.0.
Rays whose angular divergence from the center of the optical axis is greater than half the angular aperture cannot enter the objective lens and are lost. If a drop of oil (Cedar “wood oil) is placed in between the specimen and the objectives lens.
Many ways whose angle of divergence is more, also can enter the objective lens as the refractive index of oil about 1.5. Thus NA of the objective is increased which results in better resolution and higher magnification.