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.
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: 5-10nm or 50-100 A0
- Transmission electron microscope: 0.5 nm or 5 A0
Different Types of Microscopes
🔬Bright field microscope: for fixed cells
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| 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
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| 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
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| 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
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| 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 (EM)
- Beam of electrons are focused using electromagnetic lenses. Specimens should be fixed, dehydrated and mounted in vacuum as electrons are scattered by air molecule.
- Disadvantages: Cannot view living specimens.
Learn more: Light Microscope vs Electron Microscope
🔬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.
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| 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
Application of TEM:
1. Magnification of biological and non biological samples up to resolution of 600,000X
2. Generation of information about the ultra structure of cell membranes , intercellular junctions, cell organelles, extra cellular matrix, etc.
3. Identification of the morphology of viruses, such as pox viruses, phages, papovirus etc.
3. Elucidation of the structure of bacterial flagella, fimbriae etc.
🔬Scanning Electron Microscope (SEM)
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| 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.
Learn more: SEM vs TEM
Application of SEM:
1. Generation of high resolution images of the object to reveal spatial variations in chemical composition
2. Detection of phases based qualitative chemical analysis , or on the analysis of crystalline structure
3. Precise measurement of the very minute features of the object down to 50 nm in size.
🔬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.
Practice Questions :
1. Which one of the following microscopic techniques provides a 3-dimensional perspective of live, unstained and transparent specimens obtained from the wild?A) Confocal microscopy
B) Fluorescence microscopy
C) Phase contrast microscopy
D) Differential interference contrast (Nomarski) microscopy
Ans: D) Differential interference contrast (Nomarski) microscopy
2. Electron microscope can provide magnification upto:
A) 40X
B) 4000X
C) 40000X
D) 400000X
Ans: D) 400000X
3. Which fluorescent dye is used to stain bacterial nucleic acids in florescence microscopy
A) Auramine
B) Rhodamine
C) Cyanoditolyl tetrazolium chloride
D) Acridine orange
Ans: B) Rhodamine
4. In the table given below, column I shows differnt microscopy techniques and column II shows major uses of the different microscopy techniques. select
the correct combination
A) 1-a, 2-b, 3-f, 4-e, 5-d, 6-c
B) 1-a, 2-b, 3-c, 4-d, 5-f, 6-e
C) 1-a, 2-b, 3-e, 4-d, 5-c, 6-f
D) 1-a, 2-b, 3-f, 4-d, 5-c, 6-e
Answer: B) 1-a, 2-b, 3-c, 4-d, 5-f, 6-e