Lecture four

Microscope
Outline
Introduction
Purpose/use of Microscope
Types of Microscope
Basic Parts Components and Functions of
microscope
Working principle of Microscope
Safety for microscope
Troubleshooting of microscope
Preventive maintenance
Repair of microscope
Record history for microscope
Summary
Introduction
Laboratory information enables physicians
and other healthcare professionals to make
appropriate evidence-based diagnostic or
therapeutic decisions for their patients.

Clinical laboratory services are the most cost

effective, least invasive source of the objective
information used in clinical decision-making
PURPOSE/USE OF MICROSCOPE

The main purpose of microscope is to view objects that are

not visible to the naked eye.

 Laboratory microscope uses to diagnose diseases like

malaria, roundworms, tuberculosis, dysentery, crypto
sporidium giardia
TYPES OF MICROSCOPE
Microscope can be classified based on
 purpose for which it is intended,
 Image making physical principles,
 area of application and versatility.

Basically microscope classified as follows

LIGHT MICROSCOPES
 Simple Microscope
 Compound microscope
 Dissection or Stereo Microscope:

ELECTRON MICROSCOPE
 Transmission Electron Microscope (TEM)
 Scanning Electron Microscope (SEM)

FLUORESCENCE MICROSCOPE
DARK FIELD MICROSCOPE
Simple Microscope
A simple glass magnifier is used. (Convex lenses
(thicker in the center than the periphery)). The
specimen or object could be focused by use of the
magnifier placed between the object and the eye

A simple microscope is one that uses a single lens

for magnification, such as a magnifying glass while
a compound microscope uses several lenses to
enhance the magnification of an object.
 Compound microscope
 It is type of microscope which most widely used.

 It consisted of two convex lenses aligned in series [Link] object

glass (objective) closer to the object or specimen, and an
eyepiece (ocular) closer to the observer’s eye with means of
adjusting the position of the specimen and the microscope
lenses.

 The compound microscope achieves a two-stage magnification.

 The objective projects a magnified image into the body tube of

the microscope and the eyepiece further magnifies the image
projected by the objective (more of how this is done later).

 Can magnify up to 2000x

Light path of compound Microscope
Principle of compound
microscope
Light is produced from either an internal or
external light source and passes through the
iris diaphragm, a hole variable size which
controls the amount of light reaching the
specimen.

The light then passes through the condenser

which focuses the light onto the specimen.

The slide is held on the stage at 90 degrees to the path of

light which next travels through the specimen.
Principle of compound microscope

The objective lens magnifies the image of the specimen

before the light travels through the barrel of the
microscope.

The light finally passes through the eyepieces lens and

into the viewer’s eye which sends impulses to the brain
which in turn interprets the image.
Image formation in compound
Microscope
Dissection or Stereo
Microscope
It is type of light microscope contains lens in
different angles that provides a three
dimensional viewing of objects for complete
diagnosis.
 Typically uses light reflected from the
surface of an object rather than transmitted
through it

 It doesn’t have very strong magnifying

power like the compound microscope, but
can be very useful in studying of dissection
parts of living organisms. It is used mainly in
the field of medical science including
Dissecting Microscope

 A simple, binocular, light microscope

 Low magnification about 4 X

 Does not flip image

 Good for seeing 3 dimensional features

 Used during dissections(classification) and to see

fairly large, opaque (non-transparent) objects

Electron Microscopes
 Most modern microscopes

 Use a beam of electrons to make

the object extremely bright
 Use magnetic Lens

 Extremely high magnifications

are possible up to 1,000,000x

 Used to see extremely small

details within a cell

 They are applicable used to

observe very small objects like
viruses, DNA, parts of cells.
Scanning Electron Microscope (SEM)

Scans the surface of a specimen with a

beam of electrons
Creates a 3d image
Magnify up to 200,000 times
Transmission Electron
Microscope (TEM)
Beam of electrons transmits through the entire
specimen
If electrons can pass through the specimen it
creates a light and dark image
Thicker parts of the specimen are darker than
thinner parts
Total magnification is 1,000,000 x
FLUORESCENCE
MICROSCOPE
 A fluorescence microscope is an optical microscope
that uses fluorescence to study properties of organic
or inorganic substances.

 Fluorescence microscopy is capable of imaging the

distribution of a single molecular species based solely
on the properties of fluorescence emission.

 Fluorescence Microscopy is a special form of light

microscopy.

 It uses fluorescence to highlight structures in fixed

and living biological specimens instead of using
absorption, phase or interference effects.
FLUORESCENCE MICROSCOPE…
Fluorescent dyes:- (Fluorophore)A fluorophore is a
fluorescent chemical compound that can re-emit light
upon light excitation.

A light source :-Four main types of light sources are

used, including xenon arc lamps or mercury-vapor
lamps with an excitation filter, lasers, and high- power
LEDs.

The excitation filter:-The exciter is typically a band

pass filter that passes only the wavelengths absorbed
by the fluorophore, thus minimizing the excitation of
other sources of fluorescence and blocking excitation
light in the fluorescence emission band.
 FLUORESCENCE MICROSCOPE…
 The dichroic mirror A dichroic filter or thin-film filter is a
very accurate color filter used to selectively pass light of a
small range of colors while reflecting other colors.

 The emission filter. The emitter is typically a band pass

filter that passes only the wavelengths emitted by the
fluorophore and blocks all undesired light outside this
band – especially the excitation light.

 By blocking unwanted excitation energy (including UV and

IR) or sample and system auto fluorescence, optical filters
ensure the darkest background.
FLUORESCENCE
MICROSCOPE…
FLUORESCENCE MICROSCOPE…
Rabies specimen

22
 Dark Field Microscopy
 Used to examine live microorganisms that either invisible in
the ordinary light microscope, cannot be stained by standard
methods, or are so distorted by staining that their characteristics
then cannot be identified.

 Samples causing them to appear brightly lit against a dark,

almost purely black, background.

 Dark background/field with the specimen being the only one

illuminated.

 Most microscopes have the potential to do dark field microscopy

such as compound .
23
Dark Field Microscopy…
Instead of normal condenser, dark field microscope
uses dark field condenser that contain an opaque disc.

The disc blocks light that would enter the lens directly,
only the light is reflected off the specimen enters the
objective lens.

 Because there is no background light, the specimen

appears light against black background- the dark field.
Dark Field Microscopy…
Light source: Light bulb from a microscope

 Condenser type: Specialized to block most

light from the source; contains an
annular/patch stop which disperses the light in
various directions, resulting to a ―cone of
light‖

Image formed: Dark background with

illuminated specimen; may be inverted or not
depending on microscope used
25
 Dark Field Microscopy…
Total Magnification: Can range from those
of compound microscopes (10x to 1000x)

26
Dark Field VS bright field microscope
image

27
 Parts-of-the-microscope-and-functions

Basic components of microscope can be listed as

 Optical components(objective, condenser, eyepiece
and collector lens)

 Mechanical components(arm, base, stage, course and

fine adjustment knobs, nose piece& observation
tubes)

 [Link]

28
Monocular , Binocular and Trinocular
SAFETY
Wear Protective Clothing .

 lab coat

 pair of safety glasses and a pair of disposable gloves

Carry with two hands, to prevent damage to the

microscope

 Place one hand on the arm of the microscope and place

the other hand underneath the base of the microscope.

This method will give the microscope the most support.

Carry with two hands
Safety…
Do Not Touch the Lens
 This could damage the functioning of the microscope

Do Not Look into the Light

 If you are using a microscope with a mirror, never use

direct sunlight as a light source. This could cause eye

damage when looking into the microscope. If you are
using a microscope with a light source , do not look
directly into the light. This could cause eye damage as
well. Also, remember to turn off the light of the
microscope when it is not in use.
Safety…
Be Cautious Handling Slides
Always be careful when handling glass slides and cover
slips.
 If the slide or cover slip breaks, use protective gloves to
clean up the broken glasses. This will help prevent cuts
and contamination from slide contents.
Storing
Protect optical parts of the microscope from dust, dirt and
fungus
Always cover the microscope with the supplied dust cover
when not in use
Store in a dry place
Do not use the microscope in direct sunlight. Sunlight can

33
influence the quality of the specimen imaging.
Safety …
Electrical safety

To avoid electric shock or damage to the instrument,

unplug the microscope before replacing the bulb

Turn off and unplug the microscope before moving

Allow the halogen bulbs to cool before touching.

Halogen bulbs become extremely hot and may cause

burns if touched

Use only recommended halogen or fluorescent bulb

Cleaning solutions and
solvents:
Refer to manufacturer’s guide to select appropriate
organic solvent

Use Ethyl ether-alcohol, alcohol, or lens cleaner solution

for cleaning of lenses.(unless you get the manufacturers
guide)

Cleaning materials

Use Lint-free cotton gauze pads

Use Lint-free cotton swabs

Use Lens paper and blower

Cleaning process
a. Eyepiece
Check removing dust before wiping lens.
Clean with a cotton swab moistened with lens
cleaning solution.
Clean in a circular motion inside out.
Wipe with dry lens paper.
Repeat cleaning and drying if required.
 Cleaning process…
Objectives

You can clean objectives while attached to microscope

Moisten the lens paper with the cleaning solution

Wipe gently the objective in circular motion from inside out

Wipe with dry lens cleaning paper

 Cleaning process…

Microscope Stage

Wipe the microscope stage using the cleaning

solution by soft cloth

 Thoroughly dry the stage.

 Repeat above steps, if required.

38
Cleaning process…
The Microscope Body

Unplug the microscope from power source

 Moisten the cotton pad with a cleaning agent

 Wipe the microscope body to remove dust, dirt, and

oil

 Repeat the above steps if required

 Cleaning process…
The Condenser and Auxiliary Lenses

Unplug the microscope from power source.

Clean using lint-free cotton swabs moistened with

lens cleaning solution.

 Wipe with dry swabs.

40
Troubleshooting
Before beginning troubleshooting and maintenance of
microscope prepare and consider the following:

Ask the end users and clients about the problem

Collect operational and maintenance manual

Refer the past record history of the equipment end

users

Wear Personal Protective Equipment

Prepare the necessary tool kits, and measuring

instruments
Troubleshooting…
Connect the power cord to the socket out let

Check that the light intensity knob is at the minimum

level

Make the power supply ON.

Gradually maximize the light intensity knob

Check loose components or parts and damaged wires

Smell if burnt insulation occur

Troubleshooting…
If the bulb does not give light:
 Disconnect the power cord from the socket outlet

Check the main power supply with multi meter

Check the power cord function with continuity test

techniques
Check if the fuse is burn out

Check if the bulb is burn out

Check the circuit board components fails

Troubleshooting…
PROBLEM PROBABLE CAUSE SOLUTION

The lighting The electrical Check and repair the

system is not system shows electrical
producing voltage errors. System. Connect the
uniform microscope
light. through a voltage
stabilizer.
The Connect the plug to
microscope’s the outlet.
connector to the If any of the elements
wall outlet is are
slack (loose) defective, replace it.
The bulb is badly Reinstall the bulb.
installed and is
not making good
Troubleshooting…
The sample is The diaphragm’s Open the
poorly iris is almost diaphragm’s iris
illuminated. closed. until
the lighting is
adequate.
The condenser is Bring the
very far (very condenser closer.
low).
The condenser’s Clean the
lenses show dust condenser.
or fungal growth. Remove the dust
with a brush.
Remove the fungi
with a lens
cleaning solution.
Maintenance /Repair
REPAIR
Fix the problems which encountered in power cord.

Replacing the fuse:

Unplug the microscope from power source

Replace the burnt fuse as recommended

Replacing circuit board components:

 Replace individual components if possible

 If not possible replace the entire circuit board

Maintenance…
Replacing the bulb:
 Unplug the microscope from power source

 Find location of bulb

 Follow manufacturer’s instructions to remove the bulb

 Use tissue paper or an appropriate device to remove the bulb

from the microscope

 Check the model number on the bulb to ensure the use of

correct replacement bulb.

 Replace the bulb by holding it with lens paper or an

appropriate device.
 Never touch the bulb with your fingers.
 Adjustment / Maintenance…
If poor Image Quality encountered

Clean objective and lens

Clean scope( eye piece) thoroughly

Check oil, replace if any contamination or haze is

visible

Ensure the slide is thoroughly dry before applying oil

 Adjustment / Maintenance…

If Uneven Illumination encountered:

 Ensure nosepiece is rotated to position where it

clicks into place

 Centre condenser
 Correctly focus the lamp to achieve a uniform
level of brightness in the field of view

 Check to see bulb is correct model

49
Adjustment / Maintenance…

If Constant Refocusing occurs:

 Be sure slide is flat on stage

 Clean stage and underside of slide

 Be sure microscope is on flat and leveled surface

 Adjustment /
Maintenance…
Surging or Flickering (fluctuating) Light occurs:
 Loose plug or connection

 Clean bulb contacts (Unplug from power source)

 Assure halogen pins are in full contact

 Bulb needs replacement

 Check to see if cord is damaged - Replace, if any

wear and tear is visible.

 If voltage supply is erratic, use a voltage stabilizer

51
 Adjustment /
Maintenance…

If excessive contrast in the image occur:

Check the diaphragm’s iris of the

condenser is fully closed.

Open the iris of the diaphragm slightly.

52
Record history
 A service or maintenance history provides a record of the
work done on each individual item over time, and keeps the
records all in one place.

 This provides a reference where the specific problems of a

machine or item/area can emerge.

 The complete service or maintenance history is required in

the maintenance workshop.

 Because technicians can see what the recurring problems

are with equipment and what work has already been done
on the machine.

 Key facts from the service history need to be linked to the

equipment inventory.
Summary

Microscope is used to see objects that are too small for naked

eye.
 Laboratory microscope uses to diagnose diseases like malaria,
roundworms, tuberculosis, dysentery, crypto sporidium giardia

 Microscope can be basically classified as: Light

Microscope(simple , compound, dissection or stereo), Electron
microscope(TEM, SEM), Florescence microscope and dark field
microscope

Basic components of microscope can be listed as optical

components(objective, condenser, eyepiece and collector lens)
Summery….
Mechanical components(arm, base, stage,
course and fine adjustment knobs, nose piece&
observation tubes)

When operating microscope, we have to make

sure that we followed all the safety procedures.

 During troubleshooting and maintenance of

microscope we have to prepare operational and
maintenance manual and all the necessary tools
and follow all the stapes according to the
manual
55
 The end!

56