Principles and application of light,

phase contrast, fluorescence

microscope

Presented by…

Thakor Maitri M.
M.Sc. (Botany)
Department of life sciences,
H.N.G.U., Patan.

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 Contents
 Introduction
 Light microscope
◘ Principle
◘ Applications
 Phase contrast microscope
◘ Principle
◘ Applications
 Fluorescence microscope
◘ Principle
◘ Applications
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 Introduction
 A microscope is an instrument which used to
see object that are too small to be seen by the
naked eye.
 The object of magnification of cells and their
components was achieved by the lenses of
various type or a combination of lenses which
could magnify the minute objects upto a
particular limit, and therefore so many lenses
were combined together to form an instrument
known as the microscope.
 In Greek : micros = small ; skopein = to see, to
3 look.
  Credit for the first microscope is usually given
to Zacharias Janssen, in Middleburg, Holland,
around the year 1595.
 Eyeglass crafts – Magnify object 3-10x.

Objective
Body tube

Eyepiece
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 The Light microscope

 The most important scientific tool for a student of

biology is the light microscope.

 The light microscope consists of various

components which gather light and redirect the
light path so that a magnified image viewed object
can be within a short distance.

 The student’s microscope or the compound

microscope of twentieth century is the microscope
of much improved and modified type.
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  Types – Simple dissecting M , compound M ,
stereomicroscopes.

6 Sources:
www.Biologydiscussion.Com/microscope/
 Principle of light microscope

 In the light microscope, light is produced from

either an internal or external light sources and
passes through the iris diaphragm, a hole
variable size which controls the amount of
light reaching the specimen.

 The main components of the compound light

microscope include a light sources that is
focussed at the specimen by a condenser lens.

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  The slide is held on the stage at 90° to the
path of light which next travels through the
specimen.

 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.
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 How does it work ?
 Light microscope are
compound microscope,
which means they contain at
least two lenses.

 Lenses are curved pieces of

glass or plastic that bend
rays of light and can magnify
objects, making them appear
bigger than they actually are.

 Light microscope shown here

magnifies an object in two Sources : dr-monsrs.n

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stages.
  Light from the mirror is reflected up through
the specimen, or object to be viewed, into the
powerful objective lens, which produce the
one magnification.

 The image produced by the objective lens is

than magnified again by the eyepiece lens,
which act as a single magnifying glass.

 The magnified image can be seen by looking

into the eyepiece lens.

 Important factor in light microscopy include:

Magnification, Resolution, Contrast.
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 Application of the Light microscope
 Light microscopes play a large role in today’s
biology. Handy in use.

 Biologists use the microscopes to observe

objects and details at a cellular level to learn
more about the building blocks of all
organisms.

 Microscopes are also used to observe real

time movement in cells and organisms.

 Lastly, microscopes are used in forensics to

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help solve many crimes.
  Microscopes provide the students with an
understanding of real cells and their
supporting structures.

 Also, microscopes provide students who are

inclined towards the medical field a more
intense look at the career choice and devlop
basic skills.

 Lastly, microscopes are used in biology to

study diseases like cancer and AIDS to help
diagnose the disease in patients and to help
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find a cure for them.
  Microscopes are used when studying light and
optics to learn how light refracts through
converging lenses and how a combination of
different lenses with varying focal lengths
affects the properties of the image.

 Often times, there will be human evidences left

on the crime scene.

 This allows forensic scientists to examine the

evidence under a microscope and match the
results with a database to find the culprit.
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  Minerologist’s also use light microscopy, typically
with a special preparation of a sample called thin
section.

 As the name imlies, thin section are very thin

slices of a rock. The sample needs to be thin
enough for light to travel through from the light
sources to the user’s eye.

 The thin section will allow the shape of different

crystal grains to be seen.

 The microscope can be used with different

techniques, like epifluorescences and phase
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contrast.
 The Phase contrast microscope

 Phase contrast microscopy first described in

1934 by Dutch physicist Fritz zernike, Whom
awarded by Nobel prize in physics in 1953.

 A phase contrast microscope makes it possible

by utilizing two characteristics of light,
diffraction and interference, specimens based
on brightness differences.

 It requires additional specialized structure

annular diaphragm and phase contrast ring.
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16 Sources: www.biologydiscussion
.com/microscope/phase contrast-
 Principle of Phase Contrast Microscope

 It based on the wavelength (nature) of light rays

and the fact that light rays can be in phase or
out of phase.

 Different shade of grey are distinguished to our

eyes due to differences in amplitude of light
rays.

 PCM converts invisible small phase changes

caused by the cell component in to visible
intensity changes.
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  In a Phase contrast microscope, one set of
light rays comes directly from the light
sources.

 The other set comes from light that is reflected

or diffracted from a particular structure in the
specimen.

 The images differences in refractive index of

cellular structure. Light passes through thicker
parts of cell is held up relative to the light that
passes through thinner parts of cytoplasm.
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  How does it work ?

 Light that does not interact with the speciman

is collected by the objective passes through
the phase ring, and is regarded exactly ¼
wavelength.

 The Phase shifted is not detectable by the eye

so the resulting image on the image plane in
the microscope appears as a normal bright
background.

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  Light passing through one material & into
another material of slightly different refractive
index or thickness will undergo a charge in
phase.

 This charge in are translated into variations in

brightness of the structures.

 Natural light vibrates in many directions but

polarized light only one direction.

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21 Sources :
https://www.bwoptics.com/newsend2asp
 Application of Phase Contrast microscope
 Most commonly used to provide contrast of
transparent specimens such as living cells or
small organisms.

 Useful in observing cells cultured in vitro during

mitosis.

 Phase contrast enables visualization of internal

cellular components.

 It’s used in examination of growth, dynamics, and

behaviour of a wide variety of living cells in cell
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culture.
  It applied for equipment from the study of
the living biological specimens, medical
applications, study of live blood cells, and
other biological and science applications.

 It’s used in diagnosis of tumour cells.

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 The Fluorescence microscope

 This microscope additionally requires an

excitation filter, a barrier and a dichromatic
mirror, fluorescent stain.

 Fluorescent microscope is much the same as a

conventional light microscope with added
features to enhance its capabilities.

 A specific wavelength of light is used to excite

fluorescent molecule in specimen. Light of
higher wavelength is then imaged.
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  It is also used to visually enhance 3-D features
at small scales.

 This is achieved by using powerful light

sources, such as lasers, that can be focused to
a pinpoint.

 This focusing is done repeatedly throughout

one level of a specimen after another.

 Most often an image reconstruction program

pieces the multi level image data together into
a 3-4 D reconstruction of the targeted sample.
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26
Sources :Zeiss-
 Principle of the Fluorescence microscope

 When certain compounds are illuminated with high

energy light, they then emit light of a different ,
lower frequency. This effect is known as
Fluorescence.

 In most cases the sample of interest is labelled

with a fluorescent substance known as a
fluorophore and then illuminated through the lens
with the higher energy sources.

 Often specimens show their own characteristic

auto fluorescence image, based on their chemical
makeup.
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  The key feature of fluorescent microscopy is
that it empoys reflected rather than transmitted
light, which means transmitted light techniques
such as phase contrast and DIS can be
combined with fluorescent microscopy.

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  How does it work ?

 The specimen is
illuminated with light of
a specific wavelength
(or wavelengths) which
is absorbed by the
fluorophores, causing
them to emit longer
wavelength of light (or a
different colour then the
absorbed light ).

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 Application of Fluorescence microscope

 Fluorescence microscopy is a critical tool for

academic and pharmaceutical research,
pathology, and clinical medicine.

 This method is used for demonstration of

naturally occurring fluorescent material and
other non- fluorescent substances or micro-
organisms after staining with some
fluorescent dyes. e.g.; Mycobacterium
tuberculosis, amyloid, lipids, elastic fibers etc.

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  Imaging structural components of small
specimens, such as cells.

 Conducting viability studies one cell

populations (are they a live or dead ?).

 Imaging the genetic material within a cell (

DNA & RNA ).

 Viewing specific cells within a larger

populations with techniques such as FISH.

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 1) Biophysics
Author : Vasantha pattabhi ,
N. Gautham.
Edition : 2009 ( Second )

2) Basic Biophysics For Biologist

Author : M. Daniel
Edition : 2003

3) WWW.Slideshare.net
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