Cell Biology, Microscopy, and Analytical Techniques
Comprehensive Scientific Guide | Educationized Science Series
Brief History of Cell theory
Cell is the basic unit of structure and function of all living organisms; it means that cells are the building blocks of complex multicellular organisms. We now know about cells because of the cell theory. The cells Hooke observed through a simple microscope were actually cell walls left behind after the death of the real cells.
So, the first person who observed living, moving cells was Anton van Leeuwenhoek (A Dutch shopkeeper and Crafter of lenses). After building his own powerful microscope in the 1670s, he was able to observe single-celled organisms such as bacteria and sperm cells, which he collectively called animalcules (1).
In 1830s Matthias Schleiden (a botanist) and Theodor Schwann (a zoologist) put forward a revolutionary idea known as “cell theory”, which was supported by many other scientists later on.
Postulates of Cell theory
Following are the main points of cell theory:
- All living organisms are composed of one or many cells.
- All new cells arise from by the division of preexisting cells.
- Cell contain the heredity material of an organism which is passed from parent to daughter cells. All metabolic processes take place within the cells.
Size of Cell
Following comparisons will help you to get an idea about how small the cells could be. Smallest known cells are of a group of tiny bacteria known as mycoplasmas (2). Some of these spherical shaped bacteria are as small as 0.2 μm in diameter (where 1μm=about 0.000039 inches), with a total mass of 10-14 grams, equals to that of 8,000,000,000 hydrogen atoms.
Each human is composed of about 30,000,000,000,000 cells. On the other side some cells are as large as 130 mm-170 mm, which is the size of egg of Ostrich (largest cell in the world).
In short, cells vary in their sizes; but most of the cells, even being larger than atoms (average size of an atom is 1 nanometer), are still very small to be seen with naked eyes, thus to study such small structures the field of microscopy emerged.
Microscopy verses Microscope?
Microscope (micro = small and scope = to look at) is an instrument that magnifies (enlarge) objects and area of objects in order to examine them clearly, while Microscopy is a technical field of using a microscope. Similarly, Micrographs are the images that are taken using microscope. So, by knowing about microscope, we can use them to study cells and its organelles.
Difference between light and electron microscope
| S. No | Characteristics | Light Microscope | Electron Microscope |
|---|---|---|---|
| 1 | Magnification | From 1000 to 2000 X | From 1,000,000 to 10,000,000 X |
| 2 | Resolution | 250 nm | 0.5 nm |
| 3 | Source of image formation | Visible light rays | Electron beam |
| 4 | Image focused by | Glass objective lens | Electromagnetic objective lenses |
| 5 | Image viewed through | Glass ocular lens | Fluorescent screen |
| 6 | Specimen placed on | Glass slide | Copper mesh |
| 7 | Organism may be | Live | Always dead |
| 8 | Specimen requires special stain or treatment | Not always | Yes |
| 9 | Color of image | Colorful | Black and white |
8. Techniques used in Cell Biology
Microscopy along with various techniques provide valuable contribution to our understanding of cells as well as to study the functions performed by cellular organelles. By applying these techniques, we can isolate and examine various cell components. Some of the most common techniques used in cell biology are:
Profile of Stains
Profile of permanent stain
| Stain | Final Color | Suitable for |
|---|---|---|
| i. Aniline blue | Blue | Fungal hyphae and spores |
| ii. Borax carmine | Pink | Nuclei, Obelia colony |
| iii. Eosin | Pink / Red | Cytoplasm / Cellulose |
| iv. Feulgen’s stain | Red / Purple | DNA (particularly during cell division) i.e., chromosomes |
| v. Leishman’s stain | Red / Pink /Blue | Blood cells |
| vi. Methylene blue | Blue | Nuclei |
| vii. Safranin | Red / Purple | Nuclei, lignin & plant tissues |
Profile of Temporary stain
i. Aniline Sulphate: Yellow | Lignin
ii. Iodine solution: Blue-black | Starch
iii. Schultz’s solution (Chlor-Zinc-iodine): Yellow / Blue / Blue or Violet | Lignin, Cutin, Protein / Starch / Cellulose
12. Chromatography
An analytical technique used for separating a mixture of chemical substances into its individual components is known as chromatography or capillary analysis. One type of molecules can be separated from others by means of chromatography. The separation may depend on a range of chemical and physical properties of the molecules, such as solubility and molecular mass.
Basic principle of chromatography
The stationary phase may be a porous solid (such as silica, alumina etc) while mobile phase may be some solvent or a gas.
Types of chromatography
a. Paper chromatography: In this type of chromatography, the mixture is spotted near one end of a paper strip and then dipped into a specific solvent, which moves through the paper by capillary action carry the molecules with it. Similarly, a thin layer of silica may also be used instead of paper. It is used for the separation of photosynthetic pigments, sugar, amino acids and oligosaccharides etc.
b. Column chromatography: This technique involves the mobile phase flowing over a supporting matrix held in a glass or metal tube. It is used for removal of impurities and in the purification of compounds.
13. Electrophoresis
A technique used to separate macromolecules in a fluid or gel, based on their charge, binding affinity and size under the influence of different electric fields. Anaphoresis is the electrophoresis of negative charged particle whereas cataphoresis is the electrophoresis of positive charge ions. This technique is used to separate DNA, RNA or Protein molecules based on their size and electric charge.
Basic principle of electrophoresis
When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge.
Factors affecting the speed of molecules
- The amount of charge: i.e. greater is the charge faster will the movement of and vice versa.
- The size of molecules: i.e. smaller is the molecule faster will be its movement and vice versa.
14. Spectrophotometry
Spectrophotometer is a device that measures the number of photons (intensity of light) absorbed after it passes through sample, while spectrophotometry is a technique used to measure the change in percentage transmission of light (optical density) using spectrophotometer. The cell mass is directly proportional to the optical density. The determination of amino acids by spectrophotometric analysis generally depends upon absorption exhibited by their colored derivatives.
Basic principle of spectrophotometer
The photometer measures the amount of light, that passes through the sample, and delivers a voltage signal to the display. If the absorbing of light change, the voltage signal also changes.
15. Microdissection & 16. Micrometry
Microdissection
It refers to a variety of techniques in which microscope is used to aid the process of dissection of a cell and its organelles, such as:
- Chromosome microdissection: use of fine glass needle under a microscope to remove a portion from a complete chromosome.
- Laser microdissection: use of a laser through a microscope to dissect selected cells.
- Laser capture microdissection: use of a laser through a microscope to cause selected cells to adhere to a film.
Micrometry
It refers to the measurement of dimensions of microscopic objects under a microscope. Micrometers are two micro-scales used in micrometry, namely ocular micrometer and stage micrometer.
Basic principle of micrometry: While measuring a microscopic object, one of the scales is placed in eye piece (eye piece graticule) or ocular micrometer and other on the stage (stage micrometer). The micrometer has equal spaced division. Before using the eye piece micrometer, you will have to find out the real width of each unit on the scale at each magnification. In other words, you will have to calibrate the micrometer. This can be done by replacing the specimen with the stage micrometer, and using this to measure eye piece units at each magnification.
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