Microscopy

Mrs. Pradnya Wadekar Bapat
Asst. Professor
ABCP , Sangli

Microscope:
 Microscope may be defined as an optical instrument
consisting of a lens or combination of lenses for
making an enlarged or magnified image of a minute
object.
 The science dealing with aspects of microscope is
called as Microscopy
Mrs. Pradnya Wadekar Bapat, Asst. Prof, ABCP Sangli

Classification of Microscope:
Depending upon no of
lenses
Simple Compound

Depending
upon no of
eyepiece
Monocular binocular

Depending upon light source
Light/optical
microscope
Brightfield Dark
field
Fuorescence
Phase
contrast
Electron
microscope
TEM SEM

Bright field microscope
 The ordinary microscope is called as a bright field
microscope.
 It forms dark image against bright background.

Compound Microscope

 The sub stage condenser is mounted beneath the stage and
focuses a cone of light on the slide.
 The curve upper arm holds body assembly to which
nosepiece and ocular lens or eyepiece are attached.
 The nose piece holds three or five objective lenses of
different magnifying power.
 These lenses can be rotated to position any objective
beneath the body assembly.
 Ideally the microscope should be parafocal.
 Parafocal-
Image should remain in focus when objective are changed
 For bright field microscopy staining of organism is required

WORKING OF COMPOUND MICROSCOPE
 Light is transmitted and focused by mirror and
condenser.
 Focused light illuminate the object or specimen.
 The refracted light is collected by an objective
where primary image of the object is formed, it is
real, inverted enlarged image of the object.
 The eyepiece further magnifies this primary
image into virtual, erect enlarged image, this is
the final image that lies above the stage.

How Image is formed
•Image is created by objective and
ocular lenses working together.
•Light from illuminated specimen
is focused by the objective lens
creating enlarged image within
the microscope.
•The ocular lens further modifies
the primary image.
Total magnification is calculated
by magnification by objective
multiply by magnification by
eyepiece.
Ex : 45x X 10x =450x

APPLICATIONS
 Observation of morphology of microorganisms.
 Detection of cell structures.
 Observation of intracellular structures.
 Observation of motility.
 Measurement of size.
 Observation of blood smears.

Advantages
 Bright field compound microscopes are commonly used to view
live and immobile specimens such as bacteria, cells, and tissues.
 For transparent or colorless specimens, however, it is important
that they be stained first so that they can be properly viewed
under this type of a microscope.
 Staining is achieved with the use of a chemical dye. By applying it,
the specimen would be able to adapt the color of the dye.
Therefore, the light won’t simply pass through the body of the
specimen showing nothing on the microscope’s view field

Parts of compound microscope and its
functions:
 Support system: stage, base and body tube
 Illumination system: mirror, iris diadhragm, condesor
 Magnification system: eyepiece, objective

Ocular:
 Functions: magnifises real image of object as formed
by objective
 Corrects some defects of objective
 Used for observation of image
 Different types of eyepieces are used depending
upon the kind of objective located on microscope-
 Huygenian ocular
 Ramsden/ positive ocular
 Compensating ocular/ positive ocular

Types of ocular:

 Huygenian ocular:
 Plain surfaces of lenses are facing upward
and diaphragm is situated in between
leses
 These are also called as negative occular
as the focus occurs within the eyepiece
 The “F” (field lens) lens collects rays from
as wide fieldof image as possible and
focuses at or near the “d”

Rmasden/ posive ocular
 The convex surfaces of E and F
are facing inward
 The d is present below F
 Used for micrometry usually
 More accurate results the
huygenian

Compensating ocular/ positive ocular
 Are used with specific
objective
 Consist of 3 lens system as
lower lens contains two
concave surfaces

objectives
 Magnifies the real image of the object
 Units the light at the point of the image
 Gathers the light rays coming from any point of the
object
 Types-Achromatic, Flurite & Apochromatic

 Apochromatic objective represents the highest
degree of optical perfection
 Apochromatic objectives are always used with
compensating eyepiece and proper centered
condenser

 Most microbes are
observed with
achromatic or
apochrmatic objectives
used for oil immersion
 They increase the cone
of rays that enter the
objective
Nair=1, n glass=1.5 & noil=1.5

Condeser
 Also called substage condeser as it is situated between
mirror and object
 A good condenser sneds the light through the object
under an angel that is sufficiently large to fill the
aperature of back lens of objective

 Abbe condenser: used for general microscopy, simple
and good light gathering property
 Disadvantage: spherical and chromatic abberation
 Variable focus condenser: lower lens is flexible, similar
to Abbe when lower lens is raised
 Achromatic lens: chromatic and spherical abberations
are corrected hence used in photomicrography

Dark field Microscopy

 Dark field microscopy allows viewer to observe living
unstained cell and organisms simply by changing the
way in which they illuminate the object.
 A hollow cone of light is focused on the specimen in
such a way that unreflected and unrefracted rays do
not enter the objective.
 Only light that has been reflected or refracted by the
specimen forms the image
 The field surrounding specimen appears dark while
the object brightly illuminated.
 The dark field microscope can revel considerable
internal structure in larger eukaryotic
microorganism.
Dark field Microscopy

How image formed in dark field microscopy

Advantages
 The advantage of darkfield microscopy also
becomes its disadvantage: not only the
specimen, but dust and other particles scatter
the light and are easily observed
 For example, not only the cheek cells but the
bacteria in saliva are evident.
 The dark field microscopes divert illumination
and light rays thus, making the details of the
specimen appear luminous.

 Dark field light microscopes provides good
results, especially through the examination of
live blood samples.
 It can yield high magnifications of living bacteria
and low magnifications of the tissues and cells of
certain organisms.
 Certain bacteria and fungi can be studied with
the use of dark field microscopes.

Condensers:
 Commonly used
 Suitable for objects that
don’t require the highest
magnificaton
 Can be used for dark field
microscopy by inserting dark
field stop

 Cardiode Condeser:
 Designed to be used with oil
immersion objective and
intense light source and
specially for colloidal
solutions and suspensions
 The NA of objective must not
be greater than that of the
condenser
 Quarts slide and cover slip is
used instead of glass

IMP terms in microscopy:
 Magnification: degree of
enlargement
 Working distance:
Each turn of fine
screw=0.1mm
No of turns required to bring
sharp focus=6
Working distance=6*0.1

 Resolving power: ability to distinctintly separate two
small elements in structure of an object that are
short distance apart
It is expressed quantitatively
as microscope’s limit of
resolution LR
 Numerical aperature
NA=nsinϴ

 Higher N.A
 Better light
generation
 Better Resolution
 Shorter the
Wavelength
 Better Resolution.

Phase is the position of a point in time (an instant) on
a waveform cycle.

Phase difference is the difference, expressed in degrees or
time, between two waves having the same frequency and
referenced to the same point in time.

Phase shift is any change that occurs in the phase of one
quantity, or in the phase difference between two or more
quantities.

PHASE CONTRAST MICROSCOPY:

Fluorescence microscopy

INTRODUCTION
 Electron microscope is a type
of microscope that uses a particle
beam of electrons to illuminate a specimen
& create a highly-magnified image.
 Co-invented by Germans, Max Knoll
and Ernst Ruska in 1931

 Electron microscopes have much greater resolving
power than light microscopes & can obtain much
higher magnifications of up to 2 million times, while
the best light microscopes are limited to
magnifications of 2000 times.
 Can be used to study the Topography, Morphology,
Composition & Crystallographic Information.

 Extremely thin slices of the microbial specimen are needed
as electrons can be easily absorbed and scattered by the
solid matter.
 The specimen must be 20-100nm thick 1/50 to 1/100 the
diameter of typical bacteria
 Such low thickness can be obtained by fixing cells on plastic
support & cutting them properly.
 For this cells are fixed using chemicals like glutaradehyde &
dehydrated using organic solvents like acetone. After
complete dehydration specimen is soaked in liquid un-
polymerised epoxy plastic & finally plastic is hardened.
 Thin sections of this are prepared using glass or diamond
knife
 Such specimen is further prepared staining with heavy metal
stains e.g. Lead citrate & Uranyl acetate as electron
scattering is a funcion of atomic weight.
KEY FEATURES

TYPES
 There are 2 types of electron
microscopes:
 Scanning electron microscope
 Transmission Electron Microscope:
 Process is carried out under vaccum
to avoid friction and e can move
freely.
 The "Virtual Source" at the top
represents the electron gun,
producing a stream of
monochromatic electrons. The usual
potential is around 10000 – 15000V.
 Thirmioinc gun as source of e

 Electron source consists
of e source and magnetic
lesnes
 Condensor lens, objective
lens and projection lens
are magnetic lenses
which are formed by
passing constant current
through coil of wire
enclosed in anion shield
 Magnetic lenses help to
control the path of
electrons
 Viewing port and
fluorescent screen is also
there

 The e emitted by e
source are accelerated
by high potential
 Condensor magnetic
lens concentrates these
e
 E get partially absorbed
by the object
 Objective lens forms
enlarged image of the
object
 This magnified image
serves as virtual object
for projecting magnetic
lens and produces final
image of the object

TEM
 The e- stream is focused
to a small, thin, coherent
beam by the condenser
lenses 1 & 2.
 The beam is restricted by
condenser
aperture knocking out
high angle electrons.
 The beam strikes the
specimen and parts of it
are transmitted.

TEM
 This transmitted portion is focused by the objective lens into an
image and magnified.
 The projection lens further magnifies the image and projects on
fluorescent screen or photographic screen
 And the image is visible on fluorescent screen or photographic
screen
 The degree of electrons by specimen is related to number and
mass of atoms that lies in the electron path
 Since most of the biological matter constituents are low
molecular weight, the contrast can be enhanced by staining with
salts of heavy metals such as uranium or tungsten.

A TEM micrograph is shown of a slice
through RBCs. The center RBC shows the
characteristic concave disk shape.
English: Transmission electron micrograph of L-
form (cell wall deficient) bacteria derived from
Bacillus subtilis.
PC:ww.wikipedia.com
PC-Electron Microscopy of Human
Blood Cells John F.
Lesoine, University of Rochester

SEM:
•Buid up by Van Ardene
•The specimen is
subjected to narrow
beam of e- which rapidly
moves over the surface of
specimen
•release of sec e- sec
are collected by detector
which generates
electronic signal
•These signals are then
scanned in manner of
television to produce an
image on a cathode ray

SEM
 Gives three diamension view of object
 The surface topography is revealed with clarity and
depth
SEM image of a red
blood cell and a white
blood cell stacked on
top of the red blood
cell. It is possible that
the RBC is being eaten
by the growth that is
wrapping onto its
surface.
PC-Electron Microscopy of Human Blood Cells John F. Lesoine, University of Rochester

The porous cell may be eating the cell that is standing
up. Notice how the structure of the porous cell is similar
to the structure which is wrapping around the RBC.
PC-Electron Microscopy of Human Blood Cells
John F. Lesoine, University of Rochester

The clotting process is shown with RBCs red, the clot as
yellow and the activated platelet, orange, is found near the
bottom of the clot.
PC-Electron Microscopy of Human Blood Cells
John F. Lesoine, University of Rochester

Limitations:
 Vacuum is used during imaging, hence cells can’t be
in living state
 Drying may change some of the morphological
characters
 Due to low power of electron beam thin sections
are required
 NA of electron microscope lens is very small

Microscopy

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