In this presentation, I have described the Principle and Application of Light, Phase contrast, Fluorescence.

1. MICROSCOPY :-
PRINCIPLE AND APPLICATION OF
LIGHT,
PHASE CONTRAST,
FLUORESCENCE.
-By
Narottam V. Priyadarshi

2. MICROSCOPY
 Microscopy is the technical field of
using microscopes to view objects and areas of
objects that cannot be seen with the naked eye.
 “Micro” refers to “tiny” and “Scope” refers to “view or
look at”.
 Microscopes are instruments designed to produce
magnified visual or photographic images of objects
too small to be seen with the naked eye and so they
can be studied.

3. CLASSIFICATION OF MICROSCOPE
 Depending on number of lenses
i. Single microscope
ii. Compound microscope
 Depending on number of eyepiece
i. Monocular microscope
ii. Binocular microscope
 Depending on source
i. Light or optical microscope
ii. Electron microscope

4. TYPES OF MICROSCOPE DEPENDING ON SOURCE
 Light microscope
1. Bright field Microscope
2. Dark field Microscope
3. Phase contrast Microscope
4. Fluorescence Microscope
 Electron microscope
1. Scanning tunneling microscope
2. Transmission electron microscope

5. LIGHT MICROSCOPY
 Optical or light microscopy involves passing visible
light transmitted through or reflected from the
sample through a single or multiple lenses to allow
a magnified view of the sample.
 The resulting image can be detected directly by the
eye and/or imaged on a photographic plate or
captured digitally.

6. BRIGHT-FIELD
Bright-field image of leaf

7. BRIGHT-FIELD
 Bright-field microscopy is the simplest microscope
of all the types of optical microscopes.
 Sample is illuminated using white light from bottom
side as shown in Figure and the image is observed
from above.
 The contrast in the specimen is caused by
absorbance of some of the transmitted light in
dense areas of the sample.
 The typical appearance of a bright-field microscopy
image is a dark sample on a bright background
and hence the name.

8. APPLICATION OF BRIGHT-FIELD
 Bright field microscopy is very simple to use with
fewer adjustments needed to be made to view
specimens.
 Some specimens can be viewed without staining
and the optics used in the bright field technique
don’t alter the color of the specimen.
 It is adaptable with new technology and optional
pieces of equipment can be implemented with
bright field illumination to give versatility in the tasks
it can perform.

9. DARK FIELD
Microscope image under
dark field imaging
condition

10. DARK FIELD
 Figure shows the light path in a dark-field
microscope. This mode is best for observing the
pale objects.
 Only light rays scattered by specimen enter
objective lens and the specimen appears light
against dark background.
 The contrast increases, which enables observation
of more details as shown in the Figure , which are
not seen in the bright background.
 This mode is used to observe living, unstained
preparations.

11. APPLICATION OF DARK-FIELD
 A dark field microscope is ideal for viewing objects
that are unstained, transparent and absorb little or
no light.
 You can also use dark field in the research of
live bacterium, as well as
mounted cells and tissues.
 It is more useful in examining external details, such
as outlines, edges, grain boundaries and surface
defects than internal structure.

12. PHASE CONTRAST
(a) bright-field and (b) phase contrast microscopes.

13. PHASE CONTRAST
 Phase contrast microscopy is an optical microscopy
technique that converts phase shifts in light passing
through a transparent specimen to brightness
changes in the image.
 Phase shifts are invisible, but become visible when
shown as brightness variations.
 The basic principle to make phase changes visible
in phase contrast microscopy is to separate the
illuminating background light from the specimen
scattered light (see Figure). This makes up the
foreground details, and to manipulate these
differently.

14. PHASE CONTRAST
 Light rays in phase produce brighter images, while
light rays out of phase produce darker images.
 Contrast is created because light waves are out of
phase.
 Enhances the contrast between intracellular
structures having slight differences in the refractive
index.
 Used to examine living organisms or specimens
that would be damaged/altered by attaching them
to slides or staining. Excellent way to observe living
cells.

15. APPLICATION OF PHASE CONTRAST
 Small unstained specimens such as a living cell can
be seen.
 It makes Highly Transparent objects more visible.
 Examining Intracellular components of living cells at
relatively high resolution.
 example:- The dynamic motility of Mitochondria, mitotic
chromosomes & vacuoles.
 It made it possible for Biologists to study living cells
and how they proliferate through cell division.

16. FLUORESCENCE MICROSCOPE

17. FLUORESCENCE MICROSCOPE
 Specimen is illuminated with light of a specific
wavelength.
 Light is absorbed by the fluorophores causing an
emission of longer wavelengths.
 The illumination light is separated from weaker
emitted fluorescence through the use of a spectral
emission filter.

18. APPLICATION OF FLUORESCENCE
MICROSCOPE
 Imaging structural components of small specimens
such as cells
 Conducting viability studies on cell populations (are
they alive or dead)
 Imaging the genetic material within a cell (DNA and
RNA).
 To differentiate different types of cells

19. THANK YOU…