❑ PHASE CONTRAST MICROSCOPY, DARK FIELD

MICROSCOPY & ELECTRON MICROSCOPY

 PHASE CONTRAST MICROSCOPY:

‘Frits Zernike’ discovered this microscope. It used to visualize unstained living cells. It is optical
microscopy technique. Phase shifts themselves are invisible, but based on brightness variation,
it become visible.
This microscope possible for biologists to study living cells & multiplication through cell division.

 PRINCIPLE:

The principle based on the small phase changes in the light rays, induced by different thickness
& different refractive index by parts of object. Transform in different light intensity.
It is the translation of invisible phase shifts into visible differences of intensity. In this
microscope the image contrast is increased in two ways:

 By generating constructive interference between scattered & background light rays in

regions of the fields.
 By reducing amount of background light that reaches the image plane.

Apertures Condenser Objective Diffractive light (red)

Lamp

Collector lens condenser annulus specimen phase plate surrounding light image plane
  COMPONENTS:

It has light sources, condenser, objective lens system, ocular lens system annular diaphragm &
phase plate.

 APPLICATIONS:

01. It used to study membrane permeability of the cells

02. It helps to study cell division, phagocytosis.
03. It used in the visualization of mitochondria, nucleus & vacuoles etc.
04. It used in the visualization of living & unstained cells.
05. It used to observe growth of living cells in plants.

 ADVANTAGES:

01. It provides the clear image of unstained cells.

02. It provides high contract images of the cells.
03. Its cost is affordable.
04. It is widely applied in biological & medical & research.
05. It enhances prolong observation of living cells.

 DISADVANTAGES:

01. It is only effective to observe individual cells.

02. It provides bright holo surrounding the image.
 DARK FIELD MICROSCOPY:

Dark-field describes an illumination technique used to enhance the contrast in unstained

organisms. It works by illumination the sample with light.
This produce the appearance of dark, almost black & bright background object on it.

 PRINCIPLE:

It creates contrast b/w the object & surrounding fields so that back ground is dark. The
objective & ocular lenses are used in this microscope are same like ordinary microscope.
Only oblique scattered light reaches the specimen & passes on to the lens & causing the bright
objects against dark background.

Objective lens

Stages

Condenser lens
  COMPONENTS:

It has dark ground condenser that focuses only the oblique rays of light on to the specimens,
high intensity light lamp, a funnel stop that reduces the apparatus of the objective to less than
one.

 APPLICATIONS:

01. Used for diagnosis of syphilis.

02. See bacteria, algae & blood cells.
03. See hair line metal fracture.

 ADVANTAGES:

❑ It is ideal for viewing unstained & little absorbed objects.

❑ It is ideal to study marine organism like diatoms, algae & plankton etc.
❑ It is used for research on live bacterium.

 DISADVATAGES:

01. The image is prone to degradation & distortion.

02. It needs an intense amount of light to work.
03. If you used oil in the condenser then it is impossible to avoid air bubbles on slides.
  Electron Microscopy:

This microscope used a beam of accelerated electron as a source of illumination. The

wavelength is 100000 times shorter than visible photon light. It has higher resolving power than
light microscope. Based on the working they are four types;

- Analytical Electron Microscopy [AEM].

- Scanning Transmission Electron

Microscope [STEM].
- Scanning Electron Microscope [SEM].

- Transmission Electron Microscope [TEM].

 PRINCIPLE:

AEM is a type of microscopy for capturing information on the interaction b/w the incident
electron & the specimen. It is a tool for observing Nano-scale structure also. The chemical state
analyses in the micro-size observation areas.

TEM is a microscopy technique in which a beam of electron is transmitted through an ultra-thin

specimen, interacting with the specimen as it passed through it. It manly used in the cancer
research & virology.

STEM is a modified type of TEM. It uses the magnetic lenses to focus a beam of electron. The
image is formed y primary electron coming through the specimen.

SEM is a microscopy technique that produce images of a sample by scanning it with a focuses
beam of electron.
It is use in ultra-high vacuum, air & various liquid states. It is also used for the examination of
live specimen.
 Electron source
Anode

Condenser lens
Electron beam Condenser aperture
Scan coils Sample
Objective lens
Selected area
Intermediate lens
Sample projective lens
Secondary electron detector
Screen
(A). SEM [B].TEM

❑ APPLICATIONS:

01. Its ability to view structure of specimen at a higher resolution.

02. It is used for particle analysis or materiel characterization in a research laboratory.
03. It is used to explore the molecular nature & mechanism of disease.

 ADVANTAGES:

01. It is used to study the object of more than 0.2 micrometer.

02. It is used for cell metabolism.
03. It is used to study for micro structure of nature.
04. It is used for study of intracellular pathogens & viruses.
05. It is used to analysis of subcellular structure.
 DISADVANTAGES:

➢ Instrument is highly expensive.

➢ The electron microscopy is dynamic.
➢ The cooling system is needed.
➢ An electron microscope requires that all samples be viewed in a vacuum.