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
