Microscope

Presented by: Dr Avinanda Biswas

1st year PGT of Microbiology
Malda Medical College and Hospital
Introduction:
• Microscopy refers to the use of a specialized instrument called
“microscope” to view objects and area of objects that cannot be seen
with naked eye so that their characteristics are readily observable.

 It plays pivotal role in the laboratory for detection of micro-organism

directly in clinical specimen and in the characterization of organisms
grown in culture.
History:
 1590: Hans Janssen developed first microscope.

 1609:
• Galileo Galilei
• developed first compound microscope.

 1620: Christian Huygens, another Dutchman developed a simple 2 lens ocular system.
 1676:
• Anton Van Leeuwenhoek used a single lens microscope
constructed by him.

• He was the 1st scientist who observed bacteria & other

micro-organisms with single lens microscope.

• He is the “father of microscopy”.

Properties of Microscope:
A good microscope should have following properties:
I. Good Resolution:
• Resolution power refers to the ability to produce separate images of closely placed
objects.
• The resolution power of;
o Unaided human eye -0.2mm(200µm)
o Light microscope- 0.2µm
o Electron microscope- 0.5nm

• Resolution depends on refractive index of the medium.

• Oil has higher refractive index ,it enhances the resolution power of microscope.
II. Good Contrast:
• Improved by staining of the specimen.

• When the stain binds to the cells, the contrast is increased.

III. Good Magnification:

• Achieved by use of lenses.

• There are two types of convex lenses which is used.

i. Ocular lens with a magnification power of 10X.

ii. Objective lenses: Scanning(4x), Low power(10x), High power(40x) and Oil
immersion(100x)
Types Of Microscope:

 Bright field or Light microscope

 Dark field microscope
 Phase contrast microscope
 Fluorescence microscope
 Electron microscope
Bright field or
Light microscope

• It forms a dark image

against a brighter
background.
 Light microscope….

Principle :
• The rays emitted from the light source pass
through the iris diaphragm and fall on the
specimen.

• The light rays passing through the specimen

are gathered by the objective and a
magnified images formed.

• This images is further magnified by ocular

lens to produce the final magnified virtual
image.
Light microscope….
 Light microscope….

Gram-positive bacteria observed under oil Gram-negative bacteria observed under oil
immersion appear purple. immersion appear pink.
 Light microscope….

 ADVANTAGES:
• Used to view live or stained cells

• Simple setup

 DISADVANTAGES:
• Staining may destroy the specimen or introduce artifacts.

• Resolution is limited to 200nm .

DARK FIELD MICROSCOPE
• Produces a bright image of the object against a dark background.

• enhances the contrast of unstained bodies.

• Principle :
• It has condenser with central opaque area that blocks
light from entering the objective lens directly & has a
peripheral annular hollow area which allow the light pass
through and focus on the specimen obliquely.

• Only the light which is reflected by specimen enters the

objective lens whereas unreflected light does not enter
the objective as a result specimen is brightly illuminated
but the background appears dark.
USES OF DARK GROUND MICROSCOPY:
o Useful in demonstrating
• Treponema pallidum
• Leptospira
• Campylobacter jejuni
• Endospore

Treponema pallidum in Dark-field microscope

 DARK FIELD MICROSCOPE….

 ADVANTAGES:
• Used to identify the living unstained cells and thin bacteria like spirochetes.

 DISADVANTAGES:
• Specimen needs to be strongly illuminated which can damage delicate
samples.
PHASE CONTRAST MICROSCOPE:
 This microscope visualizes the unstained living cells by creating
difference in contrast between cell and water.

 Principle:
• Use of Condenser - similar to that of dark field microscope.

• Light passes through a cell, some light rays are bent due to variation in
density & refractive index within the specimen.
• The undeviated light rays strike a phase ring in the phase plate (optical disc
located in the objective) while deviated rays miss the ring and pass through
the rest of the plate.

• The background formed by undeviated light is bright while the unstained

object appears dark and well defined.
 PHASE CONTRAST MICROSCOPE…

 Advantages: Useful for studying

• Microbial motility
• Determining the measurement of living cells
• Detecting microbial internal cellular components

 Disadvantages :
• Phase ring limit the aperture which decrease the resolution.
• Not ideal for thick organism and particles
Naegleria fowleri under phase contrast
microscope
Fluorescence microscope
 It refers to microscope that uses fluorescence property to generate an
image.
 In this microscope, fluorescent dyes are exposed to UV rays, dyes are
excited and make this invisible rays in to visible rays.

 Principle :
• Lights from source are passed through the excitation filter
• Excitation filter allows only rays of shorter wavelength like UV rays
(exciting rays), and blocking all other long wavelength rays.
 Fluorescence microscope…

• Exciting rays then get reflected by

dichromatic mirror so that they fall on the
specimen stained by fluorescent dyes.

• Fluorescent dyes absorbs the exciting rays,

gets activated and emits rays of higher
wavelength (visible rays).

• A barrier filter, near objective is used to

removes any remaining UV rays which could
otherwise damage the viewer’s eyes.
 Fluorescence microscope…

• Applications:
a) Auto fluorescence:
• Certain microbes directly get fluoresce when
placed under UV lamp e.g. Cyclospora

CYCLOSPORA oocysts on stool examination. The oocyst wall is autofluorescent.

b) Microbes coated with fluorescent dye:

• Acridine orange dye – Plasmodium / filarial
nematodes
• Auramine phenol- tubercle bacilli

Auramine O stain sputum smear showing TB BACILLI

 Fluorescence microscope…

c) Immunofluorescence:
• It uses fluorescent dye tagged immunoglobins to detect cell surface antigens or
antibodies bound to cell surface.

• two types: Direct and Indirect immunofluorescence test.

• It is similar to ELISA, but differs by some important features;

 Fluorescent dye is used instead of enzyme for labeling of antibody.
 It detects cell surface antigens.
 Used to detect antibodies bound to cell surface antigens, unlike ELISA which detects free
antigen or antibody.
 Fluorescence microscope…

 Principle:
• Absorbing high energy- shorter wavelength uv light rays by the fluorescent
compounds and in turn emitting visible light rays with a low energy-longer
wavelength.

• Fluorescent dye is used to conjugate the antibody and such labeled antibody can
be used to detect the antigens or antigen-antibody complexes on the cell
surface.

• Fluorescein isothiocyanate is commonly used.

 Fluorescence microscope…

 Applications

• Detection of autoantibodies(e.g. antinuclear antibody) in

autoimmune diseases.

• Detecting microbial antigens, e.g. rabies antigen in corneal smear.

• Detection of viral antigens in cell lines inoculated with the specimens.

Electron microscope (EM)
 It was invented by German Ernst Ruska in 1931.

 It uses accelerated electrons as a source of illumination.

 EM has a much better resolving power than a light microscope as

wavelength of electrons can be up to 100,000 times shorter than
that of visible light photons.
Differences between light microscope and electron microscope

Features Light microscope Electron microscope

o Highest magnification About 1000-1500 Over 100,000

o Best resolution 0.2 0.5nm

o Radiation source Visible light Electron beam

o Medium of travel Air High vacuum

o Specimen mount Glass slide Metal grid

o Type of lens Glass Electromagnet

Transmission electron microscope : (TEM)
• Most common electron microscope
• Specimen preparation:
• Fixation : cells are fixed by using glutaraldehyde or osmium tetroxide for
stabilization.
• Dehydration: specimen is then dehydrated with organic solvents (e.g. acetone
or ethanol)
• Embedding: specimen is embedded in plastic polymer and then is hardened
to form a solid block.
• Slicing: specimen is then cut into thin slices by an ultra microtome knife.
• Thin slices of the specimen are mounted on a metal slide(copper)
Principle
• Electrons (electron gun) pass through a magnetic
condenser & then bombard on thin sliced specimen

• Specimen scatters electrons passing through it then

electron beam is focused by magnetic lenses to form an
enlarged, visible image on a fluorescent screen.

• A denser region in the specimen scatters more electrons &

therefore appears darker in the image since fewer
electrons strike that area of the screen.
 Applications of TEM:
• It can reveal the details of flagella, fimbriae, and intracellular
structures of a cell.

• Virus detection: it can detect viruses either directly from clinical

specimen or from tissue culture or after adding specific antiviral
antibody to the specimen
Scanning electron microscope (SEM)
 Used to examine the surfaces of microorganisms.
 It has a resolution of 7 nm or less.
 Its resolution is 1000 times better than light microscope.
 It provides a valuable combination of high resolution imaging, elemental
analysis, and recently, crystallographic analysis.
 FEATURES TEM SEM

o Electron beam Broad, static beam Beam focused to fine point;

Sample is scanned line by line

o Voltage needed Ranges from 60-300,000 volts Accelerating voltage much lower

o Interaction of beam electrons Specimen must be very thin Wide ranges of specimen allowed

o Imaging Electrons must passed through & Information needed is collected

be transmitted by the specimen near the surface of specimen

o Image Rendering Transmitted electrons are Beam is scanned along the surface
collectively focused by the objective of sample to buildup the image
lens & magnified to create a real
image
Other Microscopes And its uses…
Confocal Laser Scanning Microscope
 It is an advanced design of fluorescence microscope.
 It uses a laser beam to illuminate a specimen whose image is then
digitally enhanced for viewing on a computer monitor

 It uses point illumination and a pinhole in an optically conjugate plane to

eliminate out-of-focus signal and to get a better resolution of the
fluorescent image.
 Uses of confocal microscope:
• Observing cellular morphology in multilayered specimen.
• Diagnosing Ca cervix.
• Evaluation & diagnosis of basal cell carcinoma of skin.
INVERTED MICROSCOPE:
• As name suggest, this microscope
has its component placed in an
inverted order as that of light
microscope

• Here, light source & condenser lens

are placed above the specimen
stage while the objectives are
found below the stage.
 Inverted microscope…

• Uses:
• It is used in diagnostics fungal culture
• Used for diagnosis of nematology extraction specimens to observe
nematodes
• Used to observe living microbial cells found at the bottom of lab vessels

• Advantages:
• It has wide stage that favors it to view specimens in glass tubes & petri plates
• Can also be used to view and study cells in large amounts of the medium.
• It can also be used to view the cell tissues in their original vessel.
STEREO MICROSCOPE OR DISSCETING MICROSCOPE…

 This is a type of digital optical microscope designed with a low

magnification power by use of light reflected from surface of the
specimen.
 Its primary role is for dissection of specimens and qualitatively analyzing
the dissected samples.
 Other uses are;
• Studying the topography of solid samples.
• For microsurgical procedures.
POLARISING LIGHT MICROSCOPE:
 It is a technique where polarized light is used in microscope to evaluate
composition and three dimensional structure of anisotropic specimen.

 Commonly evaluated materials are

• Gout crystals
• Amyloid
• Suture material
• Bone
• Striated muscle
ATOMIC FORCE MICROSCOPE

It works by scanning a probe over the sample surface, building up a

map of the height or topography of the surface as it goes along.
It doesn’t need of any focusing, illumination, depth of field.
It also have height information that make it simple to quickly measure
the height , volume , width of any feature in the sample.
It physically feels the sample’s surface with a sharp probe , building up
a map of the height of sample surface.
It provides single atomic level structure so provide high resolution .
AFM Instrument
The main components of AFM are :
1. Microscopic stage – Moving AFM tip, sample holder, force sensor .
2. Control electronics – optical microscope, vibration controller.
3. Computer- the control electronics usually takes the form of a large
box interfaced to both the microscope stage and the computer.
Applications of microscopy in diagnostic microbiology

i. Rapid preliminary organism identification by direct visualization in patient

specimens.

ii. Rapid final identification of certain organisms by direct visualization in

patient specimens.

iii. Detection of different organisms present in the same specimen.

iv. Detection of organisms not easily cultivated in the laboratory.

v. Determination of an organism’s clinical significance; bacterial contaminants
usually are not present in patient specimens at sufficiently high numbers
(3105 cells/mL) to be seen by light microscopy.

vi. Pre-culture information about which organisms might be expected to grow

so that appropriate cultivation techniques are used.

vii. Evaluation of patient specimens for the presence of cells indicative of

inflammation (i.e., phagocytes) or contamination (i.e., squamous epithelial
cells).
viii.Determination of which tests and methods should be used
for identification and characterization of cultivated
organisms.

ix. A method for investigating unusual or unexpected laboratory

test results.
Microscope care:
Proper care and maintenance of microscope is important.

 Handle with care

 Keep lenses clear of slides
 Clean lenses
 Care of the bulb
 Store
 Thank
you…