STUDY OF MICROSCOPE - EXPERIMENT

AIM: To study the compound microscope.

Definition: Microscope is an instrument, used to see the magnified images of very small objects, which cannot be seen with a naked eye. A good microscope not only provides high magnification (enlargement of the image of the object), but also gives fair resolution (differentiation of neighbouring point as separate entities). Commonly used microscope in biological experiments is the compound microscope where magnification takes place at two places.

Description:

It consists of 3 parts.

1.      Support system-It comprise of base, stage and body tube.

2.      Illumination system- It throws light on the object for proper viewing. It comprises of light source, mirror, iris, diaphragm and condenser. The light source may be a plain or concave mirror or electrically illuminated by a tungsten filament lamp or a halogen lamp. Mirror and electric light source are generally interchangeable.

3.      Magnification system- This includes a set of lenses aligned in such a manner so that a magnified real image can be viewed. The objective is a set of lenses placed near the object. It partially magnifies the object, which can be observed through the eyepiece in a more magnified form.

Illumination system used in microscopes:

1.      Plain mirror- Use plain mirror when a fixed source of light is used

2.      Concave mirror- When sky light is used Concave mirror helps to converage the beam on to the condenser.

3.      Substage lamp interchangeable with mirror- Where there is no electricity or battery, mirror can be used

4.      Built in stage lamp(tungsten filament or Halogen lamp) with intensity adjustment

Light adjustment in a microscope:

While viewing an object, sometimes the objects has to brightly illuminated, on other occasions, less light is needed

I)       Less light rays on the object can be altered in 2 ways by means of condenser

·         Condenser can be moved upwards with the knob so as to make the object more brighter.

·         Condenser can be moved downwards to make the objects less brighter.

II)    Light illuminating the object may be adjusted with Iris Diaphragm. The diaphragm may be opened or closed to increase or diminish the light falling on the condenser here making the object more or less bright.

PARTS OF MICROSCOPE

Eye piece -To observe the magnified image of the objective.

Draw tube -To fit the eye piece inside.

Coarse adjustment knob -To bring the objective roughly.

Fine adjustment knob -To bring the fine adjustment of the specimen.

Body tube -Holds the objective at the bottom and eye piece at the top.

Revolving nose piece -Holds the objectives, which can be revolved.

Arm -To hold microscope while handling.                                  

Mechanical stage -Rectangular/square in shape, aperture at the center help in holding the specimen with spring clips provided.

Objectives -The magnification lenses showing the real inverted image. It is available in 6x, l0x, 45x & l00x.

Condenser –Condenses the light and allows parallel beam of light.

Mirror -Reflects the light through the condenser and aperture

Base - Foundation of the microscope.

FACTS AND FIGURES ABOUT MICROSCOPE:

1.      Magnifying power(MP):

M.P- magnification of objective x magnification of eye piece. eg. if you are observing an object on a slide using 10x objective and 5x eye piece then MP=10x5=50

Thus the object viewed is magnified 50 times.

2.      Resolving power of objective(R.P):

Resolving power of an objective is defined as the ability to separate distinctly 2 small elements of an object which are situated at a short distance apart R.P. can be measured by Numerical aperture (N.A) of an objective greater the N.A greater is the resolving power.

3.      Working distance:

The distance between the object and the objective is known as working distance.

The working distance decreases with increasing magnification. This means higher the power of objective, lesser the working distance.

4.      Focusing:

Focusing an object while viewing through an eye piece means adjustment of working distance this is done with the help of coarse adjustment and fine adjustment knob, coarse adjustment knob is rotated to bring the object in field of view and the fine adjustment knob is rotated to get a sharp image.

5.      Field of view:

The area of the object which one can view through the eye piece is the field of view. The field of view narrows as magnification increases.

6.      Objectives:

Different objectives used in microscopy are

4x- Also known as scanner, which is very low power and is used mainly, to bring a particular part of the object in the field of view.

10x- Low power objective, with the help of this one identifies the part to be observed in high power, this does not reveals much details.

40x-This is a high power objective to reveal finer details of the object. This is spring loaded, which means spring is fitted between the front and back lenses of the objective. This is to protect the front lens as the working distance is low in high magnification and the lens may touch the slide while focusing. The spring does not allow pressure on the front lens when it touches the slide.

100x-Oil immersion lens-This also is a spring loaded objective, requiring very low working distance and will give an image only when the object is immersed in Cedar wood oil. This oil is used as it has high refractivity and allows very high resolving power.

ELECTRON MICROSCOPE

For the maximum magnification to the tune of 200000 to 400000 times, electron microscopy is used now a days. In electron microscopy, beam of electrons is used instead of light waves to produce the magnified image. In case of light microscopy, the source of illumination is light bulb or natural light. But in electron microscopy tungsten filament with high voltage of 80kV is used instead of glass lenses the electromagnetic lenses are used in electron microscope and focusing is done by varying the current. In case of electron microscope is not observed by eye it is projected on to a photographic plate or screen.

Electron microscope is installed in dust free, vibration free, area without magnetic fields and in air conditioned room.

The material should be perfect dry for observation. Living organisms can not be observed. However it is very useful for understanding the ultra structure of viruses and different types of animal and plant cells. It is being used since 1940.

INSTRUCTIONS FOR THE USE OF MICROSCOPE

      1.   Microscope should be handled carefully as it is valuable instrument.

2.      Keep the microscope free from dust. Avoid touching the instrument with wet fingers.

3.      Do not allow any liquids to get on to the lenses or the stage.

4.      Place the microscope in an upright position at a convenient height facing the source of light being used.

5.      Ensure that the eyepiece, objectives, mirror and condenser are clean, removing any dust with a soft brush and lens tissue.

6.      Ensure that the coarse and fine adjustments and condenser focusing are working properly. If a mechanical stage provided, get acquainted with the   extent and directions of its motion. Adjust the mirror to reflect a good light into the body tube. If there is much more light in one part of the field than another, tilt the mirror gently until the most even illumination is obtained.

7.      Illuminate with the plane mirror when a sub stage condenser is used and with the concave mirror, when it is not in used.

8.      Confirm that the slide with the object to be examined and the cover slip are clean and dry. Place the slide on the stage with the specimen to be examined below the objective.

9.      While lowering the objective, watch from the side to avoid breaking the slide or damaging the objective. Using the low power, bring the object in to focus with the coarse adjustment. Finally, turn the   fine adjustment slowly till the focus is perfect and the object is seen clearly. Readjust the mirror slightly, if necessary, to secure maximum illumination. Rotate the nose piece and bring the high power objective in to position without touching the coarse adjustment. With the help of the fine adjustment, bring the object in to sharp focus. If a sub stage condenser is used, it must be accurately focused. 

10.  Do not use the high power unless the object is covered with a cover slip, care  must be taken to ensure that the lens does not touch the specimen at any time.

11.  Examine the slide carefully before drawing. Keep your note book on the right side of the microscope or at your left if you are left handed.

12.  After use, rotate the nose piece son that an empty position is over the stage. Clean the lenses with lens tissue or a piece of clean soft leather. A soft cloth dipped in xylol may be used to remove dirt, which is not easily wiped off with a cloth.

13.  Be sure always to clean the microscope before putting it aside. Always keeps an eye piece in the microscope to prevent dust getting into the body tube. Keep the microscope covered when not in use.