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Microscopic Techniques

Cell is the basic unit of life. The extremely minute size of this wonderful structure remained an obstacle for cytologists until the invention of microscope. Our exploration into the interiors of cell is undoubtedly the contribution of microscopes. The advancement in the field of microscopy has contributed much in understanding cell better and better.

microscope
Key Points for Your Exams 

Limit of resolution: The smallest distance that may separate two points on an object and still permit their observation as distinct separate points.

Limits of resolution
  • Human eye: 0.1 mm or 100 um
  • Light microscope: 0.2 um or 2000 A0
  • Scanning Electron Microscope SEM: 5-10nm or 50-100 A0
  • Transmission electron microscope: 0.5 nm or 5 A0

Different types of microscopes

Light microscope: uses beam of light focused using glass or optical lenses

Bright field microscope: for fixed cells
Yeast under Bright field microscope
Yeast under Bright field microscope

  • Stains are chemicals that can selectively attach toa particular molecules of particular cellular structures and helps to distinguish from other parts of the cell.
  • Eg: eosin and methylene blue binds to proteins and Fuchsin binds to DNA.

Stains
Basic stains: 
  • Methylene blue, crystal violet, hematoxylene, basic Fuchsin, +vely charged stains (cations) that bind to the –vely charged groups on proteins and nucleic acids.
Acid stains: 
  • Eosin, Orange G, Aniline blue and Fast green, -vely charged stains (anions) that binds to the +vely charged groups on proteins and phospholipids.
Cytochemical stains: 
  • Here enzyme reaction catalyses the production of a coloured precipitate from a colourless precursor.
  • Ex. Peroxisomes can be visualised by using attaining catalase enzyme.
  • Acid Fast stain: for Mycobacteria detection.
Dark field microscopy

Yeast under Dark field microscope
Yeast under Dark field microscope
primarily used in microbiology for detection of bacteria especially suspension of bacteria.
The image appears bright on a dark or black background.

Phase contrast microscopy

Yeast under Phase contrast microscope
Yeast under Phase contrast microscope
  • uses the principle of different densities and refractive indices of different parts of cells.
  • Application: to observe unstained and living cells (mitotically dividing cultured cells), structure and movement of large organelles like mitochondria, nucleus in living cells.
Immuno fluorescence microscope
Yeast under Immuno fluorescence microscope
Yeast under Immuno fluorescence microscope
  • Here a light microscope has the capacity to detect light emitted by a fluorescent compound. Fluorescent compounds which absorbs light at one (excitation) wavelength and then emits light at longer (emission) wave length.
  • Example: Rhodamine that emits red light, Fluorescein that emits green light.
  • Application: Observing movements inside living cells.

Confocal Scanning Microscope
  • An advanced form of Immuno fluorescence microscope
  • Application: Produces clear images of cells or larger specimens. Normally the images are combined by a computer to provide a 3 dimensional image. 
Electron Microscope
  • Beam of electrons are focussed using electromagnetic lenses. Specimens should be fixed, dehydrated and mounted in vacuum as electrons are scattered by air molecule.
  • Disadvantages: Cannot view living specimens.
Transmission Electron Microscope (TEM)
  • Thousand times magnification than light microscope. Excellent for viewing internal details. Ultra thin sections are required.
  • Methods of specimen preparation-
  • Fixation-Dehydration-Embedding-Sectioning-Mounting-Staining and viewing
  • Commonly used stains:  Heavy metals such as gold and osmium tetroxide.
Rabies virus under Transmission Electron Microscope
Rabies virus under Transmission Electron Microscope
  1.  Monolayer technique: Used for studying macromolecules such as DNA and RNA.
  2. Thin sectioning: Ultra microtomes are used to study morphology of cells.
  3. Negative staining: Used to study small particles like viruses or macromolecules.
  4. Shadow casting/Heavy metal Shadowing: 3D structure of viruses, DNA molecules or collagen fibres etc.
  5. Tracers: To study biological process like phagocytosis, molecular transport across plasma membrane. Eg: gold, iron oxide
  6. Freeze-fracture: To study the molecular arrangement of biological membranes.
  7. Whole mounts: To study chromosomes 
Scanning Electron Microscope (SEM)
 Avian flu virus under Scanning Electron Microscope
 Avian flu virus under Scanning Electron Microscope
  • To study the surface topography (surface details) of a specimen. Less resolution than TEM. Unsectioned specimens are fixed and coated with a thin layer of a heavy metal (platinum).SEM produces 3D image.


Cryo electron Microscope
  • Used to study the interior of 3D structures (ex: viruses) with much higher resolutions. A very thin section is rapidly frozen and the sample is kept at -160 0C in the vacuum of microscope. No fixing, staining or drying in this technique.
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