Tools And Techniques Of Cell Studies

TOOLS AND TECHNIQUES OF CELL STUDIES MICROSCOPY 1. The microscope is the first powerful tool used in biological studies. 2 Microscope is an instrument for magnifying minute objects. 1. Polarizing Microscope (i) Polarization microscopy is useful mainly for viewing highly ordered objects such as crystals or bundles of parallel filaments. (ii) The mitotic spindle, made up of microtubules cannot be resolved by light microscope, but can be studied with polarizing microscope. (iii) This microscope has two rotatable polarizing devices called polarizer and analyzer. 2. Ultraviolet Microscope (i) This microscope uses the UV-rays of shorter wavelengths (1500-3500 Å) (ii) As the glass lenses cannot transmit radiations of wavelengths below 4000 Å, an ultraviolet microscope uses a lens composed of fused quartz, calcium fluorite or lithium carbonate. (iii) This microscope is useful in the qualitative and in some cases quantitative determination of nucleoproteins. 3. Fluorescence Microscope (i) The first 'fluorescence microscope' was described by Lehmann in 1911. (ii) Electron microscope uses a focused beam of electrons to produce an enlarged image of an object. (iii) This microscope uses UV light with higher wavelengths (3500-4000 A) stimulation on cell components stained with fluorescent dyes. 4. Electron Microscope (i) M.Knoll and E. Ruska. two German scientists invented the electron microscope in 1932 (ii) In this microscope the source of light is a beam of electrons to produce an enlarged image of an object. (iii) In this microscope the source of light is a beam of electrons of a shorter wavelength of about 0.005 nm. (iv) Electron microscope is an ideal instrument for studying cellular ultrastructure. (V) Two major types of electron microscope are employed in biological studies the transmission electron microscope (TEM) and scanning electron microscope (SEM). (vi) The TEM is the most commonly used electron microscope. scope. (vii) The resolution power of a TEM is 10 Å whereas its magnification is 1 to 2 lac times. (viii) In order to be observed under the electron microscope, the specimen must first be dehydrated and then placed in a vacuum. (ix) The scanning electron microscope is an instrument that is especially useful for examining the surface of a specimen. (x) The resolution power of a SEM is about 50 Å and its magnification power is usually around 20,000 times. (xi) A relative newcomer to the field of microscopy is the high voltage electron microscope (HVEM) 5. Bright Field Light Microscope (i) The most common microscope, used by all students of science, is the bright field light microscope. (ii) Resolution power is the ability of an optical system to distinguish images of objects within its field of view. (iii) The resolution power of the human eye is 0.1 mm (100 um) and the light microscope is 0.2 um. (iv) A compound microscope (bright field light microscope) has two lenses systems, the ocular lens (eyepiece) and the objective lens. (V) A compound light microscope used visible light (4000-7000) Å wavelength). (vi) The maximum magnification possible by a light microscope is about 2000 times. 6. Darkfield Microscope (Ultramicroscope) (i) The sample is viewed only with oblique rays, it is particularly useful for studying the suspensions of bacteria. (ii) One sees only those light rays that are scattered from objects, the images appear bright on a black background. 7.Phase-contrast Microscope (i) Zernicke (1935) introduced the principle of phasecontrast microscopy for which he was awarded the Nobel Prize. (ii) Phase-contrast microscopy enhances the contrast between cells and their environments and between internal organelles and their surroundings. (iii) Phase-contrast microscope is used to study living or unstained cells. (iv) This microscope enables one to see mitochondria, mitotic chromosomes, nucleoli, and other organelles quite clearly in living cells. (8) Interference Microscope (i) The interference microscope is based on the same principle as the phase-contrast microscope. (ii) This microscope has wider applications, it is relatively easy to vary contrast and to obtain colour effects. (iii) Interference microscope can be used to make quantitative determinations such as the determination of dry weight. (iv) This microscope has a highly accurate optical balance, unlike the phase-contrast microscope. Landmarks in Microscopy Inventor/Scientist Year Contribution Country Conrad Gesner 1558 1590 Switzerland Hans Janssen and Zacharias Janssen Galileo Galilei Netherland Italy 1660 Athanasius Kircher 1658 Germany Marcello Malpighi Italy 1661 Robert Hooke 1665 1674 England Antoni van Leeuwenhoek Netherland Made magnifying lenses to observe a group of protists called forminifera Constructed the first compound microscope with a magnification power between 10x and 30x, called "flea-glasses" Made microscopes with greater magnifying powers to study facets in the compound eyes of insects A treatise on microscopy was published by him and he made a microscope with thirty-two times magnification power. First to use microscopy to examine and describe thin slices of animal tissues Built several compound microscopes of his own and examined a thin slice of dried cork discovering 'cells'. Made microscopes with magnification up to 300x and first to observe sperm, bacteria, protozoa, rotifers, and hydra. E.Abbe 1877 Netherland Zigmondy Designed ultramicroscope using UV-rays. 1900 M. Knoll and E.Ruska 1932 Oil immersion objectives having a resolution of 0.25 micron were produced. Germany Produced one of the first transmission electron microscopes (TEM). F.Zernicke Designed phase-contrast microscope for viewing unstained living cells Max Haitinger Developed fluorescence microscopy 1935 1935 Metric Units of Length 1 Angstrom 10 -10 meter 1 Nanometer (nm) 10-9 meter 1 Micrometer (µm) 10-4 meter 1 Millimeter (mm) 10-3 meter 1 Centimeter (cm) 10-2 meter CELL FRACTIONATION 1. This method involves the homogenization or destruction of cell boundaries by different mechanical or chemical procedures followed by the separation of the sub cellular fractions according to mass, surface tension and specific gravity. 2. Cell organelles can be separated by the methods of differential and gradient centrifugation. 3. The isolation of individual organelle from cell homogenate is achieved through differential centrifugation. 4. For biochemical analysis of mitochondria, the cells must be subjected to cell fractionation. 5. Theodor Svedberg won the Nobel Prize (1926) for chemistry the development of analytical ultracentrifugation. 6. The sedimentation coefficient is measured in Svedberg units (S) which is proportional to molecular weight of sedimenting particles. X-RAY DIFFRACTION 1. This technique is based on the diffraction of radiations when they enter small obstacles. 2 X-ray diffraction is one of the most important tools in molecular biology and ultrastructure. 3. This permits biologists to determine the orientation of molecules and to recognize the atomic organization. 4. X-ray diffraction is a good technique to determine the three-dimensional positions of the atoms in the molecules like nucleic acid and protein. AUTORADIOGRAPHY 1. Autoradiography is the direct use of radiations in the study of biological molecules. 2. Autoradiographic techniques can be used to trace the path of carbon in photosynthesis 3. Autoradiography involves the incorporation of radioactive substances into the molecules and the detection of their presence by photographic techniques 4. The most useful radioactive isotopes employed in autoradiography are beta emitters ELECTROPHORESIS 1. Electrophoresis is a technique in which charged molecules are placed in a medium and exposed to an electric field. 2. The charged molecules migrate through the medium at different rates, according to charge, length and other properties. 3. Electrophoresis is useful for the analysis of numbers and types of components in a mixture as well as for isolating and purifying molecules. 4. Protein electrophoresis is used to detect amino acid variations in hundreds of human proteins. 5. Proteins with slight variations in their amino acid sequence, will migrate at different rates through electrically charged gels. CHROMATOGRAPHY 1. Chromatography is a technique employed to separate different molecular species. 2. This technique is based on the principle of differential migration of components of a mixture resulting in their separation. 3. Chromatography is particularly used by cell biologists interested in isolating and purifying materials ranging from plant pigments to amino acids. 4. Chromatographic methods are useful for preparative as well as analytical purposes 5. Major types of chromatography comprise ion exchange chromatography partition and adsorption chromatography, gel chromatography, etc. CYTOLOGIC STAINING 1. Dyes are usually classified as acidic and basic. 2. Acidic stain is generally used for staining the cytoplasm particularly proteins, e.g. acid fuchsine, Congo red, janus green, orange G, and methyl blue. 3. Basic dye is used for staining the nucleus, particularly nucleic acids, e.g., basic fuchsine, crystal violet, methyl green, and safranin. VITAL STAINING 1.This involves the staining of structure in living cells, either in the body(in vivo) or in preparation (in vitro). 2 The three most widely used vital stains are janus green B. neutral red and methylene blue. 3 Janus green B is a vital stain for mitochondria, it is used for the determination of cytochrome oxidase activity in the cell. 4. Neutral red is a vital stain used to stain plant vacuoles. 5. Methylene blue is employed for staining the dividing cells. HISTOCHEMISTRY AND CYTOCHEMISTRY 1. Histochemistry is the analysis of the chemical constituents of a tissue 2. Cytochemistry deals with the chemical characterization and localization of substances or groups of substances in the cell. 3. Faulgen reaction is a specific test for establishing the presence of DNA. 4. Faulgen nuclear reaction has been developed by R. Faulgen and H. Rossenbeck in 1924. 5. Faulgen reaction involves the mid acid hydrolysis of DNA exposing aldehyde group and subsequent staining with Schiff’s reagent (Leucobasic fuchsine). Common Histochemical techniques Detection of proteins Mercuric bromophenol blue Ninhydrin Schiff Millon reaction Diazonium reaction Sakaguchi test Detection of carbohydrates Best carmine Periodic acid-schiff (PAS) Alcian blue PAS Sudan black B Detection of lipids Sudan black B Nile blue sulphate Oil red O Plasma reaction Detection of nucleic acids Azure B (DNA and RNA) Methyl green (DNA) Pyronin G/Y (RNA) Feulgen reaction (DNA) Stains for Chromosomes 1. Acetocarmine (0.5% carmine in 45% acetic acid) 2. Acetic orcein (1% orcein in 45% acetic acid) 3. Acetic lacmoid 4. Leucobasic fuchsine 5. Crystal violet 6. Carbol fuchsine 7. Gomori’s haematoxylin Stains for Plant Cell Wall Substances 1. Cellulose 2. Pectin Substance 3. Lignin 4. Cuticular Substance Zinc Chloride (Chlorzine iodide) Ruthenium red Phloroglucinol hydrochloride Sudan III