ASSIGNMENT

SUBJECT – BIOTECHNICS
TOPIC – INVERTED MICROSCOPE

Submitted By: Submitted To:

Anshul Kushwaha Dr. KIRAN SINGH
M.Sc, Semeseter – II
Department of Molecular and
Human Genetics, BHU.
 When we talk about the science behind something, we actually refer to the
macromolecular process behind that thing. So in order to study macromolecular things
that we cannot see with the naked eye we use some special type of optical instrument
that allows us to see and study those things. These macromolecular things are called
microscopic.
“The branch of science in which we examine microscopic things and structures
with the help of microscopes is called microscopy.”

Microscope:-
In ancient Greek language word Mikros means ‘small’ and word skopein means
‘to look’. This clearly tells us that the word 'microscope' is derived from the ancient
Greek language, which refers to an instrument that has the ability to show an object that
cannot be seen with the naked eye. This instrument creates an optical illusion by
increasing the optical reflection of that particular object. This reflection is an image of
that real object and is large enough to be seen.
In simple language the microscope is a set of lenses in a particular position.

Principle of simple optical microscope:-

Lenses have the property of enhancing the reflection of any object placed near
the lens with the help of optical illusion. A simple microscope works on the principle that
when a tiny object is placed within its focus, a virtual, erect and magnified image of the
object is formed at the least distance of distinct vision from the eye held close to the
lens. We use a set of lenses in a microscope so that the combined magnification of the
lenses give us a reflection that can be seen with the eye.
When light from a light source passes through a thin transparent object. Objective lens
produces a magnified ‘real image’ of the object. This image works as object for ocular
lens which is again magnified to isolate a magnified ‘virtual image’, which can be seen
with naked eye.

Classification of microscope: -
Classification of microscope is done on many basis, some of them are given below,

Classification on the basis of light source:

There are many microscopes that use different types of light sources instead of
simple light. Thus we can distinguish those microscopes according to the light source
used by them:

 Optical microscope - this kind of microscopes use visual spectrum of light to

produce image of the sample. These are simplest microscopes which can be
seen in every laboratory.

 Electron microscope - this use a beam of electron rather than light to generate
an image. These cannot be used for living organisms/cell.

 Scanning probe microscope - this scans the surface of sample with a probe
and this interaction is used to measure fine surface shape.

Classification basis on application: -

In this criterion of microscope classification, we focus on the use of
microscopes rather than light sources, such as the efficiency and work specificity of the
microscope.

 Biological microscopes have high magnification ranging from 50x to 100x. This
microscope uses sliced sample for observation. We cannot use sample in natural
state under these types of microscopes.

 Binocular (stereoscopic) microscopes have magnification ranging from 10x to

50x. Used to observe insects and minerals in their natural state.
Classification basis on structure: -
Microscopes can also be distinguished based on their structure, pattern of lens
and light source.

 Upright microscope where object is placed below lens, used to observe

specimens on slides.

 Inverted microscope observe target from below, used to observe cell culture
present in a dish.

Inverted microscope

The main limitation of the simple light microscope is that we only examine pre-
prepared slides, this requires us to prepare very thin slides of the object of interest,
which is not possible in certain cases, such as when we examine whole cell culture or
we have to study the cell in its natural environment.

A new type of instrument was needed to overcome this problem. Taking note of this, J.
Lawrence Smith, faculty member of Medical College of Louisiana invented a special
kind of light microscope in late 1850s. In this microscope he used a new kind of
arrangement of lenses and light source, instead of the classical light source and lens
pattern of light microscope. He placed the microscope's light source and condenser lens
above the sample stage pointing downwards, while keeping the objective and turret
below the sample stage.

The term “inverted” comes from its new arrangement of lens and light source
which is a reverse of the normal construction of a microscope, where the objective
lenses are found above the stage while the condenser and the light source are below
the stage. Hence this microscope is called 'inverted microscope'.

Principle of inverted microscope:-

The working principle of the inverted microscope is basically the same as that of
an upright light microscope. They use light rays to focus on a specimen, to form an
image that can be viewed by the objective lenses. However, in the inverted microscope,
the light source and the condenser are found on top of the stage pointing down to the
stage. With the objectives located below the stage and pointing upwards, it collects light
from the condenser magnifying the image, which is then sent to the ocular lens. Light is
reflected by the ocular lens through a mirror.
Role of component of inverted microscope:-
In an inverted microscope, the source for transmitted light and the condenser are
placed on the top of the stage pointing down toward the stage. The objective lens is
located below the stage pointing up. The cells are observed through the bottom of the
cell culture vessel. And therefore, instead of viewing the image from up, downwards,
with the inverted microscope you view the image from down, upwards.

It has similar components to those of other microscopes, the difference is the

arrangement of these components, which are placed in inverted positions. It has:
Stage – they have a large fixed stage able to hold large vessels like Petri plates.
Objective lens – movable lenses that are 4- 6 in the number & of different
magnification powers which move on a vertical axis for viewing of the specimen.
Dual concentric knobs – fine and coarse adjustment knobs for fine-tuning and
focusing the objectives to the specimen.
Nosepiece – this is a rotating turret that holds the objectives.
Condenser lens – It functions primarily to concentrate the light on the specimen.

Removable camera, fluorescent illuminators, scanners can also be temporarily

attached.

Advantages:-

In the case of large or heavy specimens such as those in metallurgical sampling,

a conventional microscope is useless. But the inverted model design is perfect for
analysis. This microscope is excellent for metallurgical sample, and for the observation
of living specimens or tissues. Therefore, the process of cell division can be
investigated by using this which is not possible using a conventional compound
microscope.

 Lower view of culture vessel: Most cells in a cell culture are submerged at the
bottom of the system, so it is good to observe the cell culture from below to study
the cell. This is the main advantage of the inverted microscope. It is commonly
used to study living cells, observing cells from beneath the cell culture apparatus.

 No need for slide preparation: It can be used to view cell tissues in their parent
vessel, which are larger than microscopic slides, which makes it better than an
upright microscope that only sees specimens in small microscopic slides.

 Large amounts of sample: This microscope due to the wide stage allows us to
see a larger sample of medium than ordinary microscope that use smaller
sample volumes on a glass slide under a cover slip.

 Sterility of samples: The inverted microscope has a wide stage, which favors
viewing the specimens in a glass tube and Petri plate and, therefore, maintains
the sterility of the specimen, leaving the specimen untouched.

 Natural environment for sample; This microscope maintains a more natural

environment for the specimen, thus, extending the life of the specimen. Long-
term research can be conducted looking at valuable life processes.

Disadvantages/limitations:-

 Costly: The engineering of manufacturing this type of microscope is more

expensive, making them expensive to purchase.

 Rarely available in the market: Inverted microscopes are not so well known
among manufacturers due to their high cost, which is the main reason behind the
rare availability.

 Thickness of glass vessels: It is difficult to view specimens through thick glass

vessels such as ordinary petri plates, thus the vessels used for culture should be
as thin as possible for microscope purpose.

 Optical quality of glass vessels: To meet the criteria for successful inverted
microscopy, the bottom of the culture vessel must have the highest optical
features.