PHASE CONTRAST MICROSCOPY | DARK FIELD MICROSCOPY AND ELECTRON MICROSCOPY

 

PHASE CONTRAST MICROSCOPE

ELECTRON MICROSCOPE

DARK FIELD MICROSCOPE

TABLE OF CONTENT:

Introduction, Definition, Principle, Component, Working, Application, Advantages & Disadvantages of Phase Contrast Microscopy.

Introduction, Definition, Principle, Component, Working, Application, Advantages & Disadvantages of Dark Field Microscopy.

Introduction, Definition, Principle, Component, Working, Application, Advantages & Disadvantages of Electron Microscopy.

Analytical Electron Microscopy [AEM].

  Scanning Electron Microscope [SEM]. 

Transmission Electron Microscope [TEM].

Scanning Transmission Electron Microscope [STEM].

PHASE CONTRAST MICROSCOPY:

INTRODUCTION OF PHASE CONTRAST MICROSCOPY:

Phase contrast microscopy, first described in 1934 by Dutch physicist Frits Zernike. It is optical microscopy technique. Phase shifts themselves are invisible, but based on brightness variation, it become visible. It is a contrast-enhancing optical technique that can be utilized to produce high-contrast images of transparent specimens, such as living cells (usually in culture), microorganisms, thin tissue slices, lithographic patterns, fibers, latex dispersions, glass fragments, and subcellular particles (including nuclei and other organelles). This microscope possible for biologists to study living cells & multiplication through cell division. 

PRINCIPLE OF PHASE CONTRAST MICROSCOPY:

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:

Þ By generating constructive interference between scattered & background light rays in regions of the fields.

Þ By reducing amount of background light that reaches the image plane.

When light passes through cells, small phase shifts occur, which are invisible to the human eye. In a phase-contrast microscope, these phase shifts are converted into changes in amplitude, which can be observed as differences in image contrast.

 

COMPONENTS / PARTS OF PHASE CONTRAST MICROSCOPY:  Phase-contrast microscopy is basically a specially designed light microscope with all the basic parts in addition to which an annular phase plate and annular diaphragm are fitted.

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

WORKING OF PHASE CONTRAST MICROSCOPY:  

·         Partially coherent illumination produced by the tungsten-halogen lamp is directed through a collector lens and focused on a specialized annulus (labeled condenser annulus) positioned in the substage condenser front focal plane.

·         Wavefronts passing through the annulus illuminate the specimen and either pass through undeviated or are diffracted and retarded in phase by structures and phase gradients present in the specimen.

·         Undeviated and diffracted light collected by the objective is segregated at the rear focal plane by a phase plate and focused at the intermediate image plane to form the final phase-contrast image observed in the eyepieces.

 

APPLICATIONS OF PHASE CONTRAST MICROSCOPY:  

HOW TO USE PHASE CONTRAST MICROSCOPE:

1.       Phase contrast microscope is used to produce high-contrast images of transparent specimens, such as living cells (usually in culture), microorganisms, thin tissue slices, lithographic patterns, fibers, latex dispersions,

2.      It used to study membrane permeability of the cells

3.      It helps to study cell division, phagocytosis.

4.      It used in the visualization of mitochondria, nucleus & vacuoles etc.

5.      It used in the visualization of living & unstained cells.

6.      It used to observe growth of living cells in plants.

 

ADVANTAGES OF PHASE CONTRAST MICROSCOPY:  

ADVANTAGES OF PHASE CONTRAST MICROSCOPE:

·         It provides the clear image of unstained cells. (Living cells can be observed in their natural state without previous fixation or labeling).

·         It provides high transparent contract images of the cells & more visible.

·         Examining intracellular components of living cells at relatively high resolution. eg: The dynamic motility of mitochondria, mitotic chromosomes & vacuoles.

·         Its cost is affordable.

·         It is widely applied in biological & medical & research.

·         It enhances prolong observation of living cells.

 

DISADVANTAGES OF PHASE CONTRAST MICROSCOPY:  

PHASE CONTRAST MICROSCOPE DISADVANTAGES:

·         It is only effective to observe individual cells.

·         To use phase-contrast the light path must be aligned.

·         It provides bright hollow surrounding the image.

·         Phase-contrast condensers and objective lenses add considerable cost to a microscope, and so phase contrast is often not used in teaching labs except perhaps in classes in the health professions.

·         Generally, more light is needed for phase contrast than for corresponding bright-field viewing, since the technique is based on the diminishment of the brightness of most objects.

 

DARK FIELD MICROSCOPY:

WHAT IS DARK FIELD MICROSCOPY:

WHAT IS DARK FIELD MICROSCOPE

INTRODUCTION OF DARK FIELD MICROSCOPY:

A 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.

A dark field microscopy is used to examine live micro- organisms that either invisible in the ordinary light microscope, cannot be stained by standard methods, or are so distorted by staining that their characteristics then cannot be identified. Instead of the normal condenser, a dark field microscope uses a dark field condenser that contain an opaque disc. The disc blocks light that would enter the lens directly, only the light is reflected off the specimen enters the objective lens. Because there is no background light, the specimen appears light against black background- the dark field. 

 

PRINCIPLE OF DARK FIELD MICROSCOPY:

HOW DOES PHASE CONTRAST MICROSCOPE WORK:

It creates contrast b/w the object & surrounding fields such that, the background is dark and the object is bright. The objective & ocular lenses are used in this microscope are same like ordinary microscope. However, a special condenser is used, which prevents the transmitted light from directly illuminating the specimen. Only oblique scattered light reaches the specimen & passes on to the lens & causing the bright objects against dark background.

 

COMPONENTS OF DARK FIELD MICROSCOPY: 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 OF DARK FIELD MICROSCOPY:

DARK FIELD MICROSCOPY USES:

1.       Useful for the demonstration of very thin bacteria not visible under ordinary illumination since the reflection of the light makes them appear larger.

2.      Used for diagnosis of syphilis.

3.      Viewing of bacteria, algae & blood cells.

4.      See hair line metal fracture.

5.      Darkfield is used to study marine organisms such as algae, plankton, diatoms, insects, fibers, hairs, yeast and protozoa as well as some minerals and crystals, thin polymers and some ceramics.

 

ADVANTAGES OF DARK FIELD MICROSCOPY:

1.       It is very simple & effective technique.

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

3.      It is ideal to study marine organism like diatoms, algae & plankton etc.

4.       It is used for research on live bacterium.

5.      The quality of images obtained from this technique is impressive.

 

DISADVATAGES OF DARK FIELD MICROSCOPY:

1.       The one of the main disadvantage of dark-field microscopy is the low light levels seen in the final image.

2.      If you used oil in the condenser then it is impossible to avoid air bubbles on slides.

3.      The image is prone to degradation & distortion.

4.      It needs an intense amount of light to work.

5.      The sample must be very strongly illuminated, which can cause damage to the sample. 

 

ELECTRON MICROSCOPY:

 

INTRODUCTION OF ELECTRON MICROSCOPY:

Ernst Ruska (1906-1988), a German engineer and academic professor, built the first Electron Microscope in 1931, and the same principles behind his prototype still govern modern EMs.

It is a special type of microscope having a high resolution of images, able to magnify objects in nanometers, which are formed by controlled use of electrons in vacuum captured on a phosphorescent screen.

Electron Microscopes uses a beam of accelerated & highly energetic electrons as a source of illumination to examine objects on a very fine scale. This examination brings the details about the observant. The wavelength is 100000 times shorter than visible photon light. It has higher resolving power than light microscope.

 

TYPES OF ELECTRON MICROSCOPE: Based on the working they are four types;

1.       Analytical Electron Microscopy [AEM].

2.      Scanning Electron Microscope [SEM]. 

3.      Transmission Electron Microscope [TEM].

4.      Scanning Transmission Electron Microscope [STEM].

 

Analytical Electron Microscopy [AEM]: 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.

Scanning Electron Microscope [SEM]: SEM is a microscopy technique that produce images of a sample by scanning it with a focuses beam of electron. SEM focuses on the sample’s surface and its composition. It is use in ultra-high vacuum, air & various liquid states. It is also used for the examination of live specimen. Scan a gold-plated specimen to give a 3-D view of the surface of an object which is black and white. Used to study surface features of materials, cells and viruses. Scanning Electron microscope has resolution 1000 times better than Light microscope.

Transmission Electron Microscope [TEM]: 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.

TEM Accelerated using a positive electrical potential. Focused by metallic aperture and Electro magnets. Interactions occur inside the irradiated sample which are detected and transformed into an image.  Scanning electron microscopy allows for higher magnification and better resolution than standard light microscopy.

Since the sample is bombarded with electrons rather than light, the level of detail in a smaller area is much greater than a light microscope.

Scanning Transmission Electron Microscope [STEM]: STEM is a modified type of TEM. It uses the magnetic lenses to focus a beam of electron. The image is formed by primary electron coming through the specimen.

 

PRINCIPLE OF ELECTRON MICROSCOPY:

Electron microscopes use signals arising from the interaction of an electron beam with the sample to obtain information about structure, morphology, and composition.

1.     The electron gun generates electrons.

2.    Two sets of condenser lenses focus the electron beam on the specimen and then into a thin tight beam.

3.    To move electrons down the column, an accelerating voltage (mostly between 100 kV-1000 kV) is applied between tungsten filament and anode.

4.    The specimen to be examined is made extremely thin, at least 200 times thinner than those used in the optical microscope. Ultra-thin sections of 20-100 nm are cut which is already placed on the specimen holder.

5.    The electronic beam passes through the specimen and electrons are scattered depending upon the thickness or refractive index of different parts of the specimen.

6.    The denser regions in the specimen scatter more electrons and therefore appear darker in the image since fewer electrons strike that area of the screen. In contrast, transparent regions are brighter.

7.    The electron beam coming out of the specimen passes to the objective lens, which has high power and forms the intermediate magnified image.

8.   The ocular lenses then produce the final further magnified image.

 

COMPONENTS / PARTS OF ELECTRON MICROSCOP:

Electron Microscope has the following components:

1.       Electron Gun

2.      Electromagnetic Lenses

3.      Condenser Lens

4.      Objective Lens,

5.      Specimen Holder

6.      Image Viewing and Recording System.

7.      Fluorescent Screen.

8.     Camera for Recording the Image.

                                                                   

 

APPLICATIONS OF ELECTRON MICROSCOPY:  

WHAT ARE ELECTRON MICROSCOPE USED FOR:

WHAT IS ELECTRON MICROSCOPE USED FOR:

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

2.      It is used for particle analysis or materiel characterization in a research laboratory.

3.      It is used to investigate the ultrastructure of a wide range of biological and inorganic specimens including microorganisms, cells, large molecules, biopsy samples, metals, and crystals.

4.      It is used to explore the molecular nature & mechanism of disease.

5.      It is used for study of intracellular pathogens & viruses.

6.      It is used to analysis of subcellular structure.

 

ADVANTAGES OF ELECTRON MICROSCOPY:

1.       It has very high magnification & Incredibly high resolution

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

3.      It is used for cell metabolism.

4.      It is used to study for micro structure of nature.

5.      Material rarely distorted by preparation.

 

DISADVANTAGES OF ELECTRON MICROSCOPY:

1.       Instrument is highly expensive.

2.      The electron microscopy is dynamic.

3.      The cooling system is needed.

4.      The live specimen cannot be observed.

5.      An electron microscope requires that all samples be viewed in a vacuum.

6.      As the penetration power of the electron beam is very low, the object should be ultra-thin. For this, the specimen is dried and cut into ultra-thin sections before observation.

7.      Require train person for handling.

8.     As the EM works in a vacuum, the specimen should be completely dry.

 phase contrast microscope pdf

electron microscope definition