LIGHT MICROSCOPE

Presented by- Harit

Kumar
Moderated by- Dr. Shinu P.

MICROSCOPE
An ancient Greek word
Mikrs = small
Skopen = to look or see
Not to be seen by naked eye.
The science of investigating these objects that are not
seen by naked eye -Microscopy.

HISTORY
1590 Two Dutch eye glass makers, Zaccharias Janssen and
son Hans Janssen used multiple lenses placed in tube.

1665 Robert Hooke looked sliver of cork through

microscope lens and noticed some "pores" or "cells" in it.

 1674 Anton van Leeuwenhoek -simple microscope & the first

person to describe bacteria. Magnification up to 300.

1893 - August kohler gave key principle of sample illuminationKohler illumination, is center to achieve theoretical limits of light
microscopy.

 1931- Ernst Ruska - Transmission Electron Microscope.

1932- Phase Contrast Microscope- Frits Zernike.

1980- Scanning Electron microscope - Gerd Binnig and Heinrich Rohrer.

Last decades of 20th century- development of fluorescent microscope.

TERMS

Resolution
Ability to distinguish (resolve) two close-together points as
separate.

Contrast
Differences in intensity between two objects, or between an
object and background
Magnification
Degree of enlargement.

Total Magnification
Magnification of objective Power of Eye-piece

Numerical Aperture
Relates to extent to which light is concentrated by condenser
and collected by objective.

DIFFERENT TYPES OF MICROSCOPES

Simple
Dissection

Compound

Electron

Transmission Scanning

Light Dark-field
Fluorescent

Phase-Contrast

Difference b/w Simple, Compound &

Electron Microscope
Simple Microscope

Compound
Microscope

Electron Microscope

Source
of Source
of
illuminationDay
illuminationDay
light
or electrical light.
Only one glass
Two sets of glass
lens for magnifying
lenses
for
objects.
magnifying
objects.
Total magnification
is by one lens.
Total magnification
is
the
Uses- Botany lab
for studying plant
multiplication
of
anatomy.
eyepiece
and
objective
magnifications.
Uses- Studying the
structure
of
different objects.

Source
of
illuminationBeam of electrons.
Instead
glass
lenses,
electron
optical
lenses
used.
Magnification
is
very high up to
10,000,00.
UsesStudying
ultrastructures.

COMPOUND
COMPOUND
MICROSCOPES
MICROSCOPES
LIGHT MICROSCOPES
LIGHT MICROSCOPES

PRINCIPLE OF LIGHT
MICROSCOPE

COMPONENTS OF
MICROSCOPE

Eyepiece
Body Tube
Revolving Nosepiece
Objective Lens
Stage Clips
Diaphragm
Light

Arm
Stage
Coarse Focus
Fine Focus
Base

COMPONENTS OF
MICROSCOPE

Supporting
Illumination
Adjustment
Magnification

A. Supporting System :-

Revolving Nose-piece

Limb

Mechanical Stage
Stage

Foot

B. Magnification System :Eye piece lens

Objective lens

 Eye-Piece lens -

Magnifying power of Eye-Piece is marked on it.

Magnification of image is as follows :
Magnifying power
Magnification
4
6
10

4 times
6 times
10 times

 Objective lens
Magnifying power of objective is engraved on sleeves of lens:

Magnifying power Magnification

10
10 times
40
40 times
100
100 times
Third objective is oil immersion
Numerical aperture -engraved on sleeves next to Magnification :
Numerical Aperture
Objective
0.30 10
0.65 40
1.30 100

Total Magnification
Laboratory Microscope provides magnification of :
(Eye-piece Objective)
Magnification
104
1010
1040
10100

= 40 (Scanner)
= 100 (Low Power)
= 400 (High Power)
=1000(Oil Immersion)

Total

COLOR BANDS ON
OBJECTIVE LENS
Color band is a quick reference to the objective
magnification :
-

4x
10x
40x
100x

=
=
=
=

Red
Yellow
Light Blue
White

C. Illumination System
Day light
Electrical light

Mirror
It reflects rays from light source on to the
object. Two sides are Concave side and other
is Plane side.

 Condenser
- Situated between mirror and stage.
- Function - To bring rays of light to common focus on
objective to be
examined.

Condenser

Open Diaphragm

Closed Diaphragm

Diaphragm
- Found within the condenser
- Function- To reduce or increase the amount of light.

D. Adjustment System
Coarse adjustment Screw Used to achieve an
approximate focus.
Coarse adjustment Screw
Fine adjustment screw

Fine adjustment Screw Used to bring the objective into

perfect focus, moves the
objective very slowly.

 Condenser adjustment and Centering

Screw
Used to adjust condenser and there are about 3
screws which are used to center condenser exactly
in relation of objective.

Iris diaphragm lever

 Mechanical stage controls

Consists of two screws :
- One move it backward and forward
- Other is meant to move it left or right.

LIGHT MICROSCOPES
Instrument use
Visible light
&
Magnifying lenses

Magnifying lenses two set of lenses - objective and

eye piece.

STEPS OF USING
MICROSCOPE
Mount the specimen on the stage
Optimize the lighting
Adjust the condenser
Think about what you are looking for
Focus, locate, and center the specimen
Adjust eyepiece separation, focus
Select an objective lens for viewing
Adjust illumination for the selected objective lens.

HOW TO FOCUS THE

OBJECT????
A. Use of low power
objective (5 or 10):

Adjust condenser to lowest position.

Lower the objective until just above the slide preparation.
Raise objective by using coarse adjustment screw, until clear image is observed.

Distance b/w front lens of objective & objective slide (when image is
focused) :

OBJECTIVEWORKING DISTANCE
10
5.0 6.0 mm
40
2.5 1.5 mm

B. Use of high power objective (40) :

Adjust condenser half way down.

Iris Diaphragm should be half open & Illumination light - low
Move objective clockwise to 40 objective lens.
Now raise objective to get clear image. Also use fine adjustment screw.

Video of setting up of Microscope at 10 & 40 :

hjhjk.mp4

Use
(100) :
C.

of

Oil

Immersion

Objective

Adjust condenser to uppermost position. Open iris

diaphragm fully.
Also fully open the illumination light.
Place tiny drop of oil immersion on dry stained
preparation.
Lower objective, until it is in contact with oil.

Distance b/w front lens of objective & objective slide (when

image is focused) :
OBJECTIVEWORKING DISTANCE
100
0.5 0.2 mm

Video of setting up of Microscope at 10 & 40:

Harit1.mp4

APPLICATIONS OF LIGHT
MICROSCOPE
A. In examining Wet Mounts at 40:
Condenser half way down.
Diaphragm also half open.
Illumination light also low.

Wet mount showing pus cells

Wet mounts

Microscope position while observing

B. In examining Stained Smears at

1000:
Condenser is totally up.
Diaphragm is fully open.
Illumination system is also high.

Gram Positive Cocci

Gram Negative Bacilli

Microscope position while Observing smears

HOW TO CARRY
MICROSCOPE???

Right way

Wrong Way

When moving microscope, always carry it with both hands. Grasp

arm with one hand & Place the other hand under the base for support.

CARE & MAINTENANCE OF

MICROSCOPE
Good preventive
includes:

maintenance

and

care

Regular cleaning of oculars and objectives

Avoid damaging oculars and other optics with eye
make-up or other debris
Careful handling to avoid abrupt motions
Protect from direct sunlight, high temperature,
humidity, dust and vibration
Use Linen cloth to clean the lenses.
Cover when not in use with vinyl or plastic dust
cover

Cleaning of Microscope
A. Cleaning the objective: Dry objective - Use linen cloth.
Oil immersion objective- Remove oil with absorbent paper
Use Xylene and dry cloth.( Careful xylene
can
cause removal of objective cement)

B. Cleaning Eye-piece:

Upper eye piece Soft cloth

Lower field lens- Paint brush

C. Condenser & Mirror:

Condenser- Linen cloth moistened with xylene.

Mirror- Soft cloth moistened in alcohol.

D. Stage:

Stage- Not with xylene removes black paint.

Clean with cloth impregnated with petroleum jelly.

CLEANING OF
MICROSCOPE

THANKYOU