Microscope

1 Introduction to MLT compiled by Waqtola C

 Learning Objectives
After completion of this session, the learners should be
able to:

 Describe the basics of microscope: optical systems, magnification,

resolution, its purpose

 List components of microscope

 Describe types of microscope

2 Introduction to MLT compiled by Waqtola C

 Definition of microscope
 is a magnifying equipment that uses different types of lens

 used to visualize minute objects (animate and inanimate), that

cannot be seen by our naked eye = magnification

 able to distinctly observe two adjacent points as separate and

distinct = resolution

 was invented by Anton van Leeuwenhoek

3 Introduction to MLT compiled by Waqtola C

 Working principle of the microscope
 Important properties of microscopy are: magnification & resolution

 The magnified image of the object (specimen) is first produced by a lens

close to the object called the objective
 This collects light from the specimen and forms the primary image.

 A second lens near the eye called the eyepiece (ocular) enlarges the
primary image converting it into one that can enter the pupil of the eye.

 The magnification of the objective multiplied by that of the eyepiece

gives the total magnification of the image seen in the microscope.

Objective Eyepiece Total

Magnification Magnification Magnification
 4X 10X 40X
 10X 10X 100X
 40X 10X 400X
 100X 10X 1000X
4 Introduction to MLT compiled by Waqtola C
 Objective Lenses
 Low power (10X) Objective
 Used for the initial scanning and observation in most
microscopic work.
 When using 10 X
 Close iris diaphragm
 Lower the condenser
 High-dry power (40X) Objective
 Is used to study un stained specimens such as stool and urine
sediments for more detailed examination.
 When using 40 X
 open the iris diaphragm half way
 raise the condenser half way

5 Introduction to MLT compiled by Waqtola C

 Objectives cont…
 Oil immersion (100X) Objective
 Routinely used for morphologic examination of blood films and
microbes

 An oil immersion lens requires that special grade of oil

(immersion oil) be placed b/n the objective and the slide

 The oil is used to increase the intensity of light by increasing

the refraction, oil has higher refractive index than water & air.

 When using 100 X

 open the iris diaphragm completely

 raise the condenser completely

6 Introduction to MLT compiled by Waqtola C

 Resolving power of the microscope
 Resolution may be defined as the ability to distinguish two closely
adjacent structural details as being actually separate and distinct.
 Resolution power of human eye is 0.25 mm ; light microscope is 0.25µm ;
electron microscope is 0.5 nm
 i.e the smaller the distance b/n the two points the larger is the resolving
power
 The increase in magnifying power is always linked to an increase in
resolving power.

 The higher the resolving power of an objective, the closer can be the fine
lines or small dots in the specimen which the objective can separate in
the image.

 d= λ/NA; where d distance λ wavelength NA numerical aperture

 The resolving power of an objective is dependent on what is known as
the numerical aperture (NA) of the objective.

7 Introduction to MLT compiled by Waqtola C

 Numerical Aperture
 The numerical aperture is a designation of the amount of light
entering the objective from the microscope field, i.e. the cone of
light collected by the front lens of the objective (an index or
measurement of the resolving power).

 It is dependent on the diameter of the lens and the focal length of

the lens.

8 Introduction to MLT compiled by Waqtola C

 Numerical Aperture
 Defined as the product of the refractive index of the medium outside the

lens (n) and the sine of half the angle of the cone of light absorbed by the
front lens of the objective (u) or
 Is a number that expresses the ability of a lens to resolve fine detail in an

object being observed

E.g.
o NA 0.25 on X10 objective
o NA 0.65 on X40 objective
o NA 1.25 on X100 objective

 The greater the N.A the greater the resolving power.

9 Introduction to MLT compiled by Waqtola C

 Working principle of an oil immersion objective

 When a beam of light passes from air into glass it is bent and when
it passes back from glass to air it is bent back again to its original
direction.

 This has effect on oil immersion objective and affects the NA of the
objective and consequently it’s resolving power.

 The bending effect on the objective can be avoided by replacing the

air between the specimen and the lens with oil, which has the same
optical properties as glass, i.e. immersion oil.

 The oil provides better resolution and a brighter image by collecting

extra oblique light.

10 Introduction to MLT compiled by Waqtola C

11 Introduction to MLT compiled by Waqtola C
 Types of microscope
 there are many types of microscopes

1. Light microscope = use light as a source of illumination

 Bright field microscope

 Dark field microscope

 Phase contrast microscope

 Fluorescence microscope

2. Electron microscope = use beam of electron

 Transmission electron microscope (TEM)

 Scanning electron microscope (SEM).

12 Introduction to MLT compiled by Waqtola C
 Bright field microscope

 is a light compound
microscope, which is routinely
used in medical labs of
hospitals and/or health centers

 uses light rays to produce a

dark image against a bright
background.

 used to view fixed and live

specimens, that have been
stained with basic stains which
gives a contrast b/n the
image and the image
background.

13 Introduction to MLT compiled by Waqtola C

 Principle of Brightfield Microscope

 a uniform beam of the illuminating light pass thru specimen of focus

 differential absorption and refraction of the light beam then produce a

contrasting image

 the specimens are stained to introduce color for easy contracting

characterization

 the colored specimens will have a refractive index that will differentiate
it from the surrounding, presenting a combination of absorption and
refractive contrast

 the specimen which is placed on a microscopic slide is viewed under

LPF, HPF, or oil immersion or/and covered with a coverslip

14 Introduction to MLT compiled by Waqtola C

 Advantages of Brightfield
 is simple to use with few adjustments involved while viewing the
image

 can be used to view both stained and unstained.

 the optics of the microscope do not alter the color of the

specimen.

 the microscope can be adjusted and modified for better viewing

such as
 installing a camera, to form a digital microscope
 the way image illumination is done such as by
 use of fluorochromes on the specimen
 viewing under a dark environment, forming a darkfield microscope

15 Introduction to MLT compiled by Waqtola C

 Disadvantages of brightfield
 it has low contrast hence most specimens must be stained for them to be
visualized.

 use of oil immersion may distort the image

 the use of coverslip may damage the specimen

 staining may introduce extraneously unwanted details into the specimen or

contaminate the specimen

 it is tedious to stain the specimen before visualizing it under microscope

 needs a strong light source for magnification and sometimes the light
source may produce a lot of heat which may damage or kill the specimen

16 Introduction to MLT compiled by Waqtola C

 Dark field microscope

 is similar to the ordinary light

microscope; however, the condenser
system is modified so that the
specimen is not illuminated directly.

 the condenser directs the light

obliquely so that the light is deflected
or scattered from the specimen,
which then appears bright against a
dark background

 Living specimens may be observed

more readily with darkfield than with
brightfield microscopy

17 Introduction to MLT compiled by Waqtola C

 Principle of Darkfield
 the light enters a special condenser (the most essential part of the
dark-field microscope) which has a central blacked-out area so that the
light cannot pass directly to enter the objective

 the dark field microscope removes the dispersed light so that only the
scattered beams hit the sample

 this microscope uses reflected light instead of transmitted light used in

the ordinary light microscope.

 the only light entering the eye comes from the micro-organisms
themselves, no light entering the eye directly from light source, the mo
are seen brightly illuminated against a black background, like stars in a
night sky.

18 Introduction to MLT compiled by Waqtola C

 Advantages of Darkfield Microscope
 is a very simple yet effective technique

 is well suited for uses involving live and unstained biological samples,
such as
 Treponema palladium; Borreliae in blood; Microfilariae in blood

 the quality of images obtained from this technique is excellent

 the techniques are almost entirely free of artifacts, due to the nature of
the process.

 dark field can be achieved by making modifications to microscope

19 Introduction to MLT compiled by Waqtola C

 Limitations of Darkfield Microscope

 The main limitation is the low light levels seen in the final image

 The sample must be very strongly illuminated, which can cause damage
to the sample.

20 Introduction to MLT compiled by Waqtola C

 Phase contrast microscope

 is an optical microscopy technique

that converts phase shifts in the
light passing through a
transparent specimen to bring
brightness of the image

 Unstained living cells absorb

practically no light.

 Poor light absorption results in

extremely small differences in the
intensity distribution in the image;
this makes the cells barely visible
in a brightfield microscope.
21 Introduction to MLT compiled by Waqtola C
 Principle of Phase contrast Microscopy

 When light passes through cells,

small phase shifts occur, which
are invisible to the human eye.

 In a phase-contrast microscope,
these phase shifts are converted
into changes in amplitude, which
can be observed as differences
in image contrast

22 Introduction to MLT compiled by Waqtola C

  In a phase-contrast microscope, the light path
consists of
 Light sources
 Annular diaphragm
 Condenser
 Specimen stage
 Objective lens
 Phase plate
 Ocular lens

 This annular diaphragm blocks most of the

light from the illuminator. Hence it produces a
hollow cone of light

 Hollow cone of light is focused on specimen

before reaching the objective lens

23 Introduction to MLT compiled by Waqtola C

  Light travelling directly from the illuminator (surrounding wave)
passes through one part of the phase plate whereas light
reflected, refracted or scattered by the specimen (diffracted
wave) passes through another part of the phase plate

 This causes surrounding wave to be out of phase with that of

diffracted wave

 It results in destructive interference that ultimately brings

contrast where sample appears dark against a bright background

 Generally, structures that differ in refractive index will differ in

levels of darkness

24 Introduction to MLT compiled by Waqtola C

  illumination produced is directed through a
collector lens and focused on a specialized
annulus positioned in the condenser

 Wavefronts passing through the annulus

illuminate the specimen and either pass
through undeviated or are diffracted and
retarded in phase by structures and phase
gradients present in the specimen

 Undeviated and diffracted light collected by

the objective is segregated at the rear focal
plane by a phase plate and focused at the
intermediate image plane to form the final
phase-contrast image observed in the
eyepieces.
25 Introduction to MLT compiled by Waqtola C
26 Introduction to MLT compiled by Waqtola C
 Fluorescence microscope
 Uses the principle of fluorescence to
generate bright image in dark background

 Fluorescence is the re-emission of light of

larger wavelength when a fluorophore
compound is excited with light of smaller
wavelength (UV light) having higher energy.

 Stokes shift is the distance between the excitation

and the emission λ max (wavelength).

 Very small stroke shift creates overlap of excitation and

emission wavelengths and thus makes it difficulty of
detection of emitted fluorescence distinguished from the
excitation light, therefore selecting a fluorophore compound
which could have larger strokes shift is critically important

27 Introduction to MLT compiled by Waqtola C

 The light path of fluorescence microscope consists of

 Light source  Objective lens

 Excitation filter
 Dichroic mirror

 Dichroic mirror
 Emission filter

 Objective lens
 Ocular lens

 Sample
 Detector

28 Introduction to MLT compiled by Waqtola C

  Light source could be xenon arc lamp, mercury vapor lamp, LED, or
LASER light

 Excitation filter is used to select the excitation wavelength of light from

a light source

 Dichroic mirror is used to allow light of certain wavelength to pass

through while reflecting light of other wavelengths

 Light of the excitation wavelength is used to illuminate the specimen

(stained with fluorochrome stains) through the objective lens

 Emission filter is used to transmit the emission range of the fluorophore

while filtering out the entire excitation range of fluorophore

 The emitted light is then focused to the detector by the ocular lens
29 Introduction to MLT compiled by Waqtola C
 Advantages and limitations
 Advantages  Limitations

 Highly sensitive & specific  Photobleaching: fluorophores lose

their ability to fluoresce as they
 Different molecules can be stained
are illuminated
with different colors, hence allows
multiple molecules to be tracked  Phototoxicity: fluorescent
simultaneously molecules tend to bring toxic
effects on cells
 Used for both in vitro and in vivo
imaging  Only allows observation of
specific structures w/c are labeled
for fluorescence

 Some examples of tests using fluorescence microscope in clinical labs include:

 Examination of sputum and c.s.f for AFB using an auramine staining technique
 Examination of acridine orange stained Trichomonas viginalis flagellates

30 Introduction to MLT compiled by Waqtola C

 Electron Microscope

 uses a beam of accelerated electrons as a source of illumination instead

of light which is used in light microscope

 electrons are generated from heated tungsten filament , or electron gun

 high energy accelerating voltage (about 20kv) is used to focus the electrons
into a straight line passing through the sample (ultra thin, 200 times thinner
than those used in optical microscope) in air tight vacuum chamber.

 specimen holder is an extremely thin film of carbon or collodion held by a

metal grid

 final image is projected on a fluorescent screen

31 Introduction to MLT compiled by Waqtola C

  has much better magnification with excellent resolution than light
microscope because electrons have very low wavelength as compared
to light (0.2nm vs 500nm)

 the smaller the wavelength the better is the resolution; d= 0.61λ/NA

 uses electromagnetic lenses (condenser, objectives and ocular) instead

of glass lenses in light microscopy

 two types EM exists: Transmission EM and Scanning EM

32 Introduction to MLT compiled by Waqtola C

 Transmission EM (TEM)
 The light path of electron microscope is very similar to the light microscope
except the use of electron as source of illumination and use of
electromagnetic lenses. The path consists of
 electron source = focused as a single beam using high energy voltage
 condenser = electromagnetic
 sample
 objective lens = electromagnetic
 ocular lens = electromagnetic
 detector

 Heavy atoms in the sample absorb more electrons but light atoms absorb
less and thus transmit more; ie the result of this varying absorbance result
in image formation in black and white

 Magnetic field helps to maintain the beam of electron in air tighter vacuum
chamber
33 Introduction to MLT compiled by Waqtola C
34 Introduction to MLT compiled by Waqtola C
 Scanning EM (SEM)
 Help to visualize the sample in 3D shape and details of the cellular structures
with greater magnification and resolution

 Little difference from TEM in that anode and scan coil exists. Anode is situated
between the source and the condenser whereas scan coil is b/n the condenser
and objective lens.

 Anode (positive electrode) is used to align negatively charged electrons in a

single beam focusing through the specimen

 Once the beam hits the sample then scattered electron based on the difference
of depth is diffracted and then detected by CMOS detector to form 3D image

 more electrons escape from the higher surface of the sample versus low electron
escape from lower side of the surface of the sample creates differential image which is
then captured by the CMOS sensor and projected as a 3D image, i.e., presence of the
different surface components of the sample
35 Introduction to MLT compiled by Waqtola C
 Applications of EM
 used to investigate the ultrastructure of a wide range of biological and
inorganic specimens including microorganisms, cells, large molecules,
biopsy samples, metals, and crystals.

 Industrially, electron microscopes are often used for quality control

and failure analysis.

 Modern EM produce electron micrographs using specialized digital

cameras and frame grabbers to capture the images.

 Science of microbiology owes its development to the electron

microscope. Study of microorganisms like bacteria, virus and other
pathogens have made the treatment of diseases very effective.

36 Introduction to MLT compiled by Waqtola C

 Advantages and limitations of EM
 Advantages  Limitations

 Very high magnification and  The live specimen cannot be observed

incredibly high resolution
 Requires the specimen to be dried and cut
into ultra-thin sections before observation
 Material rarely distorted by
preparation
 As the EM works in a vacuum, the specimen
should be completely dry.
 It is possible to investigate a
greater depth of field  Expensive to build and maintain

 Diverse applications  Image artifacts resulting from specimen

preparation.

 This type of microscope is a large,

cumbersome extremely sensitive to vibration
and external magnetic fields.

37 Introduction to MLT compiled by Waqtola C

 Major parts of Light microscope

A. Frame work of the microscope: This includes:

 An arm (stand): - The basic frame of the microscope to which the

base, body and stage are attached.

 A stage: - the table of the microscope where the slide or specimen

is placed.

 A foot or base: - is the rectangular part up on which the whole

instruments rest.

38 Introduction to MLT compiled by Waqtola C

 B. Focusing system: This encompasses
 Coarse and fine focusing adjustments

 Course adjustment: - controlled by a pair of large knobs positioned

one on each side of the body. Give rough image.

 Fine adjustment: - moves the stage so slowly to give clear image

 Condenser adjustments: -
 focused usually by rotating a knob to one side of it to move the
condenser up or down.

 the condenser aperture is adjusted by the iris diaphragm, which is found

just below the condenser.

 the principal purpose of the condenser is to condense the light required

for visualization.

39 Introduction to MLT compiled by Waqtola C

C. Magnification system: comprises

Objectives: -
 components that magnify the image of the
specimen to form the primary image.
 For most routine laboratory work 10x, 40x
and 100x (oil immersion) objectives are
adequate.
Eyepiece:-
 the upper optical component that further
magnifies the primary image and brings the
light rays to a focus at the eye point.
Multi head microscope
 It consists of two lenses mounted at the
correct distance.
 It is available in a range of magnifications
usually of 10x, 15x and sometimes as high as
20x.
 N.B: Based on their number of eyepiece,
microscopes can be classified as monocular,
binocular, trinocular, multi head microscopes

40 Introduction to MLT compiled by Waqtola C

 D. Illumination system

Condenser and iris

 Condenser is a large lens with an iris diaphragm. The condenser
lens receives a beam from the light source and passes it into the
objective.

 The iris is a mechanical device mounted underneath the Condenser

and controls the amount of light entering the condenser.
Mirror
 Mirror is situated below the condenser and iris.

 It reflects the beam of light from the light source up wards

through the iris into the condenser.

 The mirror is used to reflect ray or electrical light

41 Introduction to MLT compiled by Waqtola C

 E. Sources of illumination
Day Light –

 A Microscope must not be used in direct sun light.

 Ordinary daylight may be sufficient for some work.

Electric light

 An ordinary 60-watt pearl electric bulb placed about 18 inches from the
microscope is sufficient for most routine work.

 Quartz halogen (quartz iodine) are very good light sources because they
give excellent white illumination and do not blacken like ordinary tungsten
lamps.
 Many microscopes are now provided with correctly aligned built-in sources
of illumination, which use tungsten or quartz halogen lamps operating on
6,8 or 12 volts through variable transforms.
42 Introduction to MLT compiled by Waqtola C
 Filters : Light filters are used in the microscope to:
 Reduce the intensity of light

 Increase contrast and resolution

 Adjust the color balance of the light to give the best visual effect

 Provide monochromic light

 Absorb light

 Transmit light of selected wavelength

 Protect the eye from injury caused by ultra-violet light.

43 Introduction to MLT compiled by Waqtola C

44 Introduction to MLT compiled by Waqtola C
45 Introduction to MLT compiled by Waqtola C
 Routine use of the microscope

 A microscope must always be used with gentleness, care and the

following should be noted.

1. Place the microscope on a firm bench so that it does not vibrate.

 Make sure that it is not be exposed to direct sun light.

 The user must be seated at the correct height for the convenient use of
the microscope.

2. Select the appropriate source of light.

3. Place the specimen on the stage, making sure that the underside of
the slide is completely dry.

46 Introduction to MLT compiled by Waqtola C

 4. Select the objective to be used. It is better to begin examination with 10x
objective.

5. Bring the objective as close as possible to the slide preparation

6. Adjust the light source until the illumination of image is at its

brightest.

7. Focus the condenser.

8. Adjust the aperture (opening) of the condenser iris according to the

specimen being examined.

47 Introduction to MLT compiled by Waqtola C

  The wider the condenser aperture, the brighter will be the specimen
and the smaller be the details, which can be resolved.

 The smaller the aperture, the greater will be the contrast.

 Certain specimens, example stained and mounted specimens give

little glare illuminated image with fine detail.

 Other specimens, like urine, unstained cerebrospinal fluid and saline

mounted fecal specimens give much glare and require a reduced
source of light to increase contrast.

48 Introduction to MLT compiled by Waqtola C

 9. Examine the specimen by systematically moving the slide with the mechanical
stage. N.B: The image of the specimen will be up side down and will move in
the opposite direction to the side.

10. For a higher magnification, swing the 40x objective into place.
 Focus the 40x objective, using the fine adjustment.

 If for any reason the image is not visible, lower the objective until it is
nearly but not quite touching the specimen.

 Then looking through the eyepiece, focus up wards with the fine adjustment
until the image comes into view.

11. For the highest magnification, add a drop of immersion oil to the specimen
and swing the 100x oil immersion objective into place, then open the iris fully
to fill the objective with light. Example. Stained blood smear, acid-fast stain,
etc
49 Introduction to MLT compiled by Waqtola C
 Care, Cleaning and Repair of microscope
 The microscope is one of the most expensive and delicate instruments.
 Good microscopy practice includes:

I. Daily cleaning and quality control(QC) check

a) Using a clean cloth, wipe any dust from stage and other surfaces of
microscope

b) Using lens tissue clean dry objective.

 Clean 100X objective with tissue dampened with xylene.
 Never use alcohol to clean the oil because it will dissolve the cement holding the
lens.

c) Carry out a QC check to ensure the lenses are completely clean.

50 Introduction to MLT compiled by Waqtola C

 II- Care when using the microscope

1. Do not force any mechanism.

2. Check stage and under side of the specimen re DRY and CLEAN.

3. Cover wet preparation with cover slip.

4. Use non-drying oil immersion.

5. Put eyepieces that are mot in use in closed container.

6. Always lift and carry the microscope well supported with hands.

7. Protect the microscope from dust, moisture and direct sunlight.

51 Introduction to MLT compiled by Waqtola C

 III- At the end of the Days

 Turn the switch off.

 Clean using a soft tissue.

 Do not leave the objective of eyepiece open.

 Decontaminate the stage with 70% alcohol dampened cloth.

 Cover with its dust cover.

52 Introduction to MLT compiled by Waqtola C

53 Introduction to MLT compiled by Waqtola C