Microscopic techniques which are used in lab and it is very helpful in exam preapartion also and details study about each and every techniques.

1. MICROSCOPIC TECHNIQUES

2. TYPES
1. OPTICAL MICROSCOPY
- Conventional light microscopy
- Fluorescence microscopy
- Confocal microscopy
- Phase-contrast microscopy

3. Contd…
3. ELECTRON MICROSCOPY
Scanning electron microscopy (SEM)
Transmission electron microscopy (TEM)
Source: www.microscope .com

4. OPTICAL MICROSCOPY
 The optical microscope, often referred to as light
microscope, is a type of microscope which
uses visible light and a system of lenses to magnify
images of small samples.
 It was first created by ROBERT HOOKE in 1665.
 There are two basic types of optical microscopes:
simple microscopes and compound microscopes.
 A simple microscope uses a single lens for
magnification, such as a magnifying glass,
 compound microscope uses several lenses to
enhance the magnification of an object.

5. SIMPLE
MICROSCOPE
COMPOUND
MICROSCOPE

6. 2.COMPOUND MICROSCOPE
 A high power or compound
microscope achieves higher
levels of magnification than a
stereo or low power microscope.
 It is used to view smaller
specimens such as cell structures
which cannot be seen at lower
levels of magnification.
Essentially, a compound
microscope consists of structural
and optical components.
 Light is passed through the
sample (called transmitted light
illumination). Larger objects
need to be sliced to allow this to
happen efficiently
Source: www.microscope.com/education

7. IMAGES UNDER COMPOUND MICROSCOPE
Source: www.microscopeworld.com

8. 3. CAMERA LUCIDA
Helps in drawing microscope
images of objects on paper.
It works on simple optical
principle reflecting beam of light
through a prism and a plane
mirror.
 The microscopic image of the
object is reflected by the prism on
to the plane mirror and there from
the image is reflected on to the
plane paper.

The observer moves the pencil
on the lines of the image and
draws a correct and faithful figure
of the object on the paper.
Source: www.bgs.ac.uk.com

9. Source: www.research gate.com
A CAMERA LUCIDA OF HOLOTYPE OF FOSSIL PLANTS

10. 4. STEREO-MICROSCOPE
Source: www.microscope .com
 The stereo- or dissecting
microscope is an optical microscope
variant designed for observation
with low magnification (2 - 100x)
using incident light illumination (light
reflected off the surface of the sample
is observed by the user), although it
can also be combined with transmitted
light in some instruments.
It uses two separate optical
paths with two objectives and two
eyepieces to provide slightly different
viewing angles to the left and right
eyes.
In this way it allows a three-
dimensional visualization of the
sample.

11.  Great working distance and
depth of field are important
qualities for this type of
microscope, allowing large
specimens such as small
animals, plants and organs to
be viewed with most parts in
focus at the same time. In
addition to the ocular and
objective lens,
Stereomicroscopes typically
contain:
1. Focus wheel
2. Light source
3. Base
4. Ocular (eyepiece) lenses
Many stereomicroscopes also
have adjustable magnification.
USES
Source: www.edutrade .com

12. PHASE CONTRAST MICROSCOPE
• Converts phase shifts in
light passing through a
transparent specimen to
brightness changes in
the image.
• Details in the image
appear darker/brighter
against a background
• Colorless and
transparent specimen,
such as living cells and
microorganisms
Source: www.nobelprize.org

13. With regard to periodic movements,
such as sinusoidal waves, the phase
represents the portion of the wave
that has elapsed relative to the origin.
Light is also an oscillation and the
phase changes, when passing through
an object, between the light that has
passed through (diffracted light) and
the remaining light (direct light).
PRINCIPLE
 Transparent cells can be observed without staining them
because the phase contrast can be converted into
brightness differences.
- because it is not necessary to stain cells, cell division and
other processes can be observed in a living state.
USES Source: microscopeworld.com

14. FLUORESCENCE MICROSCOPE
 Fluorescence is the
property of some
atoms and molecules to
absorb light at a
particular wavelength
and to subsequently
emit light of longer
wavelength
 It is an optical
microscope that uses
fluorescence and
phosphorescence to
study properties of
organic and inorganic
substance
Source: microscopeworld.com

15.  The specimen is
illuminated with light of a
specific wavelength which
is absorbed by the
fluorophores, causing
them to emit light of
longer wavelengths
 The illuminated light is
separated from the much
weaker emitted
fluorescence through the
use of a spectral emission
filter
PRINCIPLE
DICHROTIC
FILTER
Source: www.pnta.com

16. USES
• Especially useful in
the examination of
biological samples:
• Identify the
particular molecules
in complex structure
(e.g. cells)
• Locate the spatial
distribution of
particular molecules
in the structure
• Biochemical
dynamics
Source: projects .ncsu. edu
Source: pinterest.com

17. CONFOCAL MICROSCOPE
 Works by passing a
laser beam through a
light source aperture
which is then focused
by an objective lens
into a small area on
the surface of sample
 An image is built up
pixel-by-pixel by
collecting the emitted
photons from the
fluorophores
(fluorescent molecule)
in the sample. Source: www. aperturegames.com

18. PRINCIPLE
The CLSM works by
passing a laser beam
through a light source
aperture which is then
focused by an objective
lens into a small area on
the surface of your
sample and an image is
built up pixel-by-pixel
by collecting the
emitted photons from
the fluorophores in the
sample
Source: Molecular expressions, science optics and you

19. USES
 It is being exploited to
study a wide range of
pharmaceutical systems
including phase-separated
polymers, colloidal systems,
microspheres, pellets,
tablets, film coatings,
hydrophilic matrices, and
chromatographic
stationary phases, detecting
molecules.
CONFOCAL LASER SCANNING MICROSCOPY for detection of reactive nitrogen species in
olive leaves. In control plants, the localization of endogenous NO by CLSM with DAF-2 DA showed
an intense green fluorescence in vascular tissues (xylem and phloem) and a smaller intensity in the
upper and lower epidermal cells . Under salt stress the green fluorescence was homogenously
intensified in all cell types. Source: www. sciencedirect.com

20. ELECTRON MICROSCOPY
 An electron microscope uses a
beam of accelerated electrons
as source of accelerated
electrons as a source of
illumination.
 Electron microscopes have a
high resolving power than
light microscopes and can
reveal the structure of small
objects.
 It offers unique possibilities to
gain insight into
Structure
Topology
Morphology
Composition of materials
Source: en.wikipedia.org

21. TYPES OF ELECTRON MICROSCOPES
2. SCANNING
ELECTRON
MICROSCOPE
1. TRANSMISSION
ELECTRON
MICROSCOPE

22. 8.1.TRANSMISSION ELECTRON MICROSCOPE (TEM)
 Transmission electron
microscope is a microscopy
technique whereby a beam of
electrons is transmitted
through an ultra thin
specimen, interacting with
the specimen as it passes
through.
 An image is formed from the
interaction of electrons
transmitted through the
specimen; the image is
magnified and focused on to
an imaging device.
Source: www.slideshare.net.com

23. PRINCIPLE
Source: www.slideshare.net.com

24. USES
Source: www.gettyimages. com
The main application of
transmission electron
microscopy is to provide high
magnification images of
internal structure of a sample.
TEM can visualize the
complexity of the cells and
show cellular structures.
 It can identify small
organelles and determine the
structural difference or change
in tissues under different
conditions

25.  A scanning electron
microscope is a type of
microscope that produces
images of a sample by
scanning it with a focused
beam of electron.
 The electrons interact
with atoms in the sample,
producing various signals
that contain information
about samples surface
topography and
composition.
Source: www.alamy.com
8.2. SCANNING ELECTRON MICROSCOPE

26. SEM

27. Source: www.madrat.com

28. USES
 Use for the live specimen
examination.
 Use for the visualization of
intra cellular changes.
 Use for 3D tissue imaging.
For investigation of virus
structure
 Can analyze surface fractures,
provide quantitative chemical
analysis and identify crystalline
structures
Source: www.slideshare.net.com