Objectives

 

1. To understand microscope to observe cellular components(Plant Cell)

 

2.  Constructing  your own remote trigger able microscope

 

 

 

Microscope

 

The invention of microscope has opened a new dimension in science. The purpose of a microscope is to magnify and thereby study an object or organisms which are too small to be seen by naked eye. Anton Van Leeuwenhoek , a fabric merchant of Holland first invented the microscope and so known as ‘Father of microscopy’. It is necessary to understand the way in which the lenses bend and light focusing to know how a light microscope works. Most of the works in biology depends on the study of the structural details of the cells. Cells are defined as the basic building blocks of all organisms, and without microscope one would be not able to explain even a simplest biological process. The basic microscope system used in the laboratory is the light microscope in which visible light passes directly through the lenses and specimen. The light microscope employs visible light to detect small objects, is probably the most well-known and well-used research tool in biology.  Usually all modern microscopes are compound microscopes in which the magnified image formed by the objective lens is again enlarged by additional lenses with different magnification. Objective is the most important part of microscope which will produce a clear image rather than a magnified one. Resolution is an important factor for the functioning of objective. Resolution is the capacity of a lens to distinguish between small objects which are close together.

 

Light Microscopy

 

Light microscope uses the properties of light to produce an enlarged image. It is the simplest type of microscope. Based on the simplicity of the microscope it may be categorized into:

 

1.  Simple microscope
2. Compound microscope

 

 

Simple microscope

 

It uses only a single lens lens such as a magnifying glass or hand lens. Most of these are double convex or plan convex lens. The developments of advanced techniques for grinding and shaping lenses allowed professionals such as Hans Janssen and Anton van Leeuwenhoek to develop simple microscopes which advanced the study of biology significantly.

 

Compound Microscope

 

The common light microscope used in the laboratory is termed as a compound microscope. It consists of two types of lenses (ocular and objective), that functions to magnify an object under analysis. The lens which is closest to the eye is called ocular lens (eye piece), where as the lens closest to the object is known as objective lens. Basically, the objective lens provides the primary magnification which is multiplied (compounded) by the eye piece. The first magnified image obtained with objective lens, is again magnified by the eye piece to give a virtual inverted image. Compound microscopes are usually heavier, larger and more expensive than simple microscopes due to the increased number of lenses used in construction.

 

 

 

Parts of a Microscope

 

It consists of mainly three parts:

                                                                                                                                                                 

1. Mechanical part - base, c-shaped arm and stage.
2. Magnifying part - objective lens and ocular lens.
3. Illuminating part - sub stage condenser, iris diaphragm, light source.
 

 

Mechanical part

 

1. Base: It helps in holding the various parts of microscope. It also contains the light source.
2. C-shaped arm: It is used for holding the microscope. And which is connected the eyepiece to the objective lens.
3. Mechanical stage: It is a rigid platform on which specimen to be viewed is placed. It has an aperture at the centre to permit light to reach the object from the bottom. The object on the slide can be moved either sideways or forward and backward with the help of the positioning knobs.

 

Magnifying part

 

1.  Eyepiece (Ocular lens):

It is the lens where the final image of the object is viewed. Usually; these lenses have a magnification of either 10X or 15X.

 

2. Objective lens:

There are three types of objective lens: 4X (scanning objective)

• 10X (Low power objective lens).
• 40X (High power objective lens).
• 100X (Oil immersion objective lens).

 

 

 

3. Illuminating part    

 

1. Sub stage condenser:

 

It is seen below the stage and made up of a system of convex lenses which focus light from illuminating sources and is used to condense light towards the object. Lowering the condenser diminishes illumination whereas raising the condenser increases the illumination.

 

 

2. Iris diaphragm:

 

It is seen immediately below the condenser and operated by small lenses which protrude to one side. Opening and closing of iris diaphragm controls the light reaching the object.

 

 

3. Light source:

Light source is situated at the base of the microscope. It is controlled by an ON /OFF switch and a lamp rheostat. Tungsten-halogen lamps are highly reliable light source used in the light microscope. It generates a continuous distribution of light across the visible spectrum.

 

Adjustments Knobs in the Microscope

 

Coarse Adjustment Knob

 

Objective lenses can be moved towards or away from the specimen by using this coarse adjustment knob.

 

Fine Adjustment Knob

 

It is used to fine tune the focus on the specimen and also used to focus on various parts of the specimen. Commonly one uses the coarse focus first to get close and moves to the fine focus knob for fine tuning.

 

Importance of Microscope in Cell Staining:

 

Cells are the basic structural and functional units of every organism. All the living cells can be classified into two distinct types – prokaryotic and eukaryotic cell. The word eukaryotic means” true nucleus” and the word prokaryotic means “before nucleus”; reflecting the fact that prokaryotic cells are evolved before eukaryotic cells. Organisms of the domain bacteria and archae consist of prokaryotic cells. Protests, fungi, animals, and plants all consist of eukaryotic cells. The actual staining process involves immersing the sample in dye which is then followed by rinsing and observation. Sometimes additional step such as heating might be required. Many dyes require the use of a mordant. Mordant are chemical compounds that form an insoluble precipitate with the stain. These chemicals are mainly used to intensify the color given by the cell. The application of stains to  specimens enables investigators to observe the morphology and arrangement of individual cells using a microscope.  Stains enhance the contrast in the microscopic image. They are chemical agents that have the ability to adhere strongly to certain molecules present in cells (both prokaryotic and eukaryotic) and tissues.  Cell staining is a technique that can be used to visualize cells and cell components under a microscope. By using different stains, one can preferentially stain certain cell components, such as a nucleus or a cell wall, or the entire cell.
 

 

Plant cell:

 

Plant cells are eukaryotic cells whose cells have membrane bound nuclei and organelles. They are generally larger than animal cells. Plant cells contains some organelles which are not present in animal cells include cell wall, central vacuole and plastids such as chloroplasts. The plant cell wall located outside the cell membrane. The cell wall is more rigid because of the presence of cellulose and lignin. The cell wall gives shapes to the cells, supports and protects the cell. The plasma membrane is present in the plant cell regulates the uptake and exit of molecules and ions. In addition to the cell wall and plasma membrane, plant cells have the tonoplast which encloses the vacuole.

Plant cells contains membrane bound nucleus. DNA in the plant cell is located within the nucleus. Plant cells contain many membrane bound organelles or other tiny cellular organelles that carry out specific functions for the cellular respiration and growth. Plant cells possess chloroplast, which impart green color to the plants, and will help them for utilizing the sunlight.  Basically a plant cell is classified into three types.

 

•  Parenchyma cells: help in storage, photosynthesis-support and other functions.

 

• Collenchyma cells: present during the time of maturity and have only a primary wall.

 

• Sclerenchyma cell: Helps in the mechanical support.
   

Remote Triggering Light Microscope

 

Remote triggering light microscope is a bit complex process. So here we have limited the controls to the microscope. In our experiment a user can control only coarse focus knob. Objective lenses can be moved towards or away from the specimen by using this coarse adjustment knob. For this experiment we half fixed 10X (Low power objective lens). User could not able to switch the objective lens. The mechanical stage, Eye piece and slides are already fixed here. To conducting experiment user want to adjust coarse focus knob. After connecting the device adjust knob to get clear image of the cell.

 

Coarse adjustment knob

 

 

The slide we have fixed here is safranin stained tissue of a plant . They are classic choices for study in early levels of biology lab work. It is easily obtained and provides a clear view of cell structures.