- 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
|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.
- Methylene blue, crystal violet, hematoxylene, basic Fuchsin, +vely charged stains (cations) that bind to the –vely charged groups on proteins and nucleic acids.
- Eosin, Orange G, Aniline blue and Fast green, -vely charged stains (anions) that binds to the +vely charged groups on proteins and phospholipids.
- 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.
|Yeast under Dark field 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.
|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.
- 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.
- 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.
- 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|
- Monolayer technique: Used for studying macromolecules such as DNA and RNA.
- Thin sectioning: Ultra microtomes are used to study morphology of cells.
- Negative staining: Used to study small particles like viruses or macromolecules.
- Shadow casting/Heavy metal Shadowing: 3D structure of viruses, DNA molecules or collagen fibres etc.
- Tracers: To study biological process like phagocytosis, molecular transport across plasma membrane. Eg: gold, iron oxide
- Freeze-fracture: To study the molecular arrangement of biological membranes.
- Whole mounts: To study chromosomes
|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.
- 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.