Fundamental Microscopy

Microscope:- In Fundamental Microscopy, the microscope may be defined as an optical instrument, consisting of a lens or a combination of lenses, for making enlarged or magnified images of minute objects.

Why is a microscope needed?

To study microorganisms.

The human eye is unable to see an object less than 0.1mm size placed at a distance of 25 cm.

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Simple microscope/single microscope

Consists of only one lens or magnifying glass held in a frame, usually adjustable, stand for conveniently holding the object to be viewed, and a mirror for reflecting light.

Compound Microscope 

It differs from a simple microscope in having two sets of lenses, one known as objective and the other as an eyepiece, mounted in a holder commonly known as a body tube.

History of Microscope

The study of biology was based on visual evidence till the mid-17th century.

The use of magnifying lenses for the study was not very powerful.

In 1590, Zacharius Janssen( a Dutch spectacle maker) used two magnifying lenses for magnifying images. (The probably first record of a combination of lenses i.e. compound microscope).

Later such compound microscopes were improved and used by Galileo (1610) and Robert Hooke(1660).

Robert Hooke had seen and drawn a white mold, looked at microorganisms, and documented them in his book “Micrographia” in 1665.

Antony Van Leeuwenhoek (1632-1723) a Dutch linen merchant, opened the world of microorganisms to human civilization.

Skilled in the art of blowing and fine metalwork.

Made minute lenses and observed a great variety of objects such as saliva, leaves, and blood circulating in the tail of a salamander.

Given credit as first to see and describe bacteria.

Worked with a simple microscope, with one lens, ground by himself and fixed between 2 copper and silver plates.

Made around 543 microscopes of such types.

The focal distance was 1.0 – 2.5nm, magnification around 270X, and resolving power of 1.4 μm.

Saw many creatures and named “animalcules”, which we now known as Protozoa and bacteria and is often called the Father of Microbiology.

The credit of the development of the microscope cannot be attributed to one person only.

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Some Basic Terms In Microscopy

Projection distance – The distance between the virtual image and the eyepoint is known as the projection distance, usually around 250mm.

Depth of focus – It depends on the numerical aperture(NA) and the magnification and is inversely proportional to both. The depth of focus is the maximum distance from which the objective can be moved along its axis (up or down) without causing blurring of the image.
Standard tube length /Mechanical tube length: The length of the body tube of the microscope. It is normally 160 mm.

Magnification – degree of enlargement.

Resolving power – This is defined as the capacity of the microscope to distinguish between two points lying very close to each other.

Limit of resolution – This is defined as the minimum distance at which two objects lying close together appear as two distinct entities. Limit of resolution = 0.61λ / N.A. ( Numerical Aperture ).

Where 0.61 is a constant representing the minimum detectable difference in contrast; λ is the wavelength of light (illumination); N.A. is the Numerical Aperture(light gathering capacity)

Working distance – This is the distance between the specimen and the front lens of the objective when the specimen is in sharp focus. It varies with the type of objective in use(10x,45x,100x).As the magnification increases working distance decreases.

Refractive index(R.I.) – It is a measure of how greatly a substance slows the velocity of light. The direction and magnitude of bending are determined by the RI of the two media forming the interface.

Types Of Microscopy

1) Bright Field Microscopy
2) Dark Field Microscopy
3) Phase Contrast Microscopy

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