Downloaded 114 times
Uploaded byPulipati Sowjanya
Microscopy
Microscopy techniques allow observation of microscopic organisms. Light microscopy uses optical lenses and magnification up to 2000x, while electron microscopy uses electron beams and achieves magnification up to 400,000x. Different microscopy types are used depending on the sample and desired information. Brightfield microscopy images stained samples, while darkfield images unstained samples against a dark background. Fluorescence microscopy uses fluorescent dyes and phase contrast observes living unstained cells. Electron microscopy reveals ultrastructures but requires processed samples due to vacuum conditions.
More Related Content
Microscopy
- 1. MICROSCOPY Dr. Pulipati Sowjanya Professor& Head Dept. of Pharmaceutical Biotechnology Vignan Pharmacy College Vadlamudi, Guntur (Dt)
- 2. MICROSCOPY • Microscope wasinvented by Antony Van Leeuwenhoek in 17th century. • Required for the morphological studyof microorganisms. • USES - to magnify the image. - to achieve maximum resolution. - to provide sufficient contrast for observation
- 3. Resolution: the extentto which details in the magnified object are maintained. Resolving Power (RP) : the smallest distance by which 2 points can be separated and still be observed as 2 distinct / different points. RP (eye) – 200 µm RP (visible light) – 300 nm RP (electron microscope) – 0.1 nm DEFINITIONS
- 4. Types of Microscopes Inlight microscopy the magnification is obtained by optical lenses using light waves. This includes • Bright-Field microscopy • Phase contrast microscopy • Fluorescent microscopy Electron microscope – Uses beam of electrons to produce the image. The specimen samples can be examined by either transmission or scanning electron microscopy Microscopes are broadly categorized into light (optical) & electron depending upon magnification
- 5. Bright-Field Microscopy • Microorganismsdo not absorb much light, but when they are stained with dye, it increases their light absorbing ability. • This results in greater contrast & colour differentiation. • This type of microscopes produce about 1,000 to 2,000 magnification. • If magnifies greater than 2,000 the image becomes fuzzy. The microscopic field is brightly lightened and the microorganisms appear dark because they absorb some of the light.
- 6. Bright-Field Microscope A Bright-FieldMicroscope
- 7. Dark-Field Microscopy • Thelight microscope is equipped with a special kind of condenser that transmits a hallow cone of light from the source of illumination. • Most of the light directed through condenser does not enter the objective, but some of the light rays will be scattered if the transparent medium contains objects such as microbial cells. • The objects are brightly illuminated against dark background.
- 8. Dark-Field Microscopy • Thisdiffracted light will enter the objective & reach the eye; thus the object (microbial cell) will appear bright in dark microscopic field. • Dark-Field microscopy is valuable for the examination of unstained microorganisms suspended in fluid wet mount & hanging drop preparations. Dark-Field Microscope
- 9. Dark-Field Microscopy Treponema pallidum;a spirochete that causes syphilis Spirillum volutans; a very large bacterium with flagellar bundles Clostridium botulinum; a bacterium causes botulism
- 10. Dark-Field Microscopy • Veryuseful in finding extremely small, unstained and / or moving objects. • Organelles like cilia, flagella, vacuoles and cell nuclei can be clearly seen. USES
- 11. Fluorescence Microscopy • Suchsubstances are called fluorescent & the phenomenon is termed as fluorescence. • The microorganisms are stained with a fluorescent dye and then illuminated with blue light. • The blue light is absorbed and green light emitted by the dye. • Chemical substances absorb light but only some substances will emit light of a longer wavelength and a lesser energy content.
- 12. • Acridine orange: Orange • Auramine-Rhodamine : Yellow • Calcofluor white: White • Fluorescein Isothiocyanate (FITC) : Green FLUOROCHROMES Fluorescence Microscopy Fluorescence Microscopy
- 13. Fluorescence Microscopy • Thefluorescent dyes are combined with antibodies which are specific to microorganisms. • Antibodies to which fluorescent dye is attached are known as labelled antibodies. • The labelled antibodies are then mixed with a suspension of bacteria & examined by fluorescent microscopy. • The bacterial cell combined with labelled antibody will be visible in microscope,
- 14.
- 15. Phase Contrast Microscopy •It is a valuable microscope for studying living unstained cells. It is widely used in applied & theoretical biological studies. Phase Contrast Microscopy
- 16. Phase Contrast Microscopy •It uses a conventional light microscope fitted with a phase- contrast objective system and makes it possible to distinguish unstained structures within a cell. • Principle: Light passing through one material and into another material of slightly different refractive index or thickness will undergo a change in phase. • Advantage : since staining is not involved, live organisms can be observed.
- 17.
- 18. Electron Microscopy • Inventedby Knoll & Ruska in 1936. • Uses electrons in place of light. • Electrons are focused by electromagnetic field. • Image is formed on a fluorescent screen or is taken on a photographic material. • Resolving power is 100,000 times more than light microscope.
- 19. Electron Microscopy • 1.Transmission EM ( TEM ) 2. Scanning EM ( SEM ) • SEM allows the study of cell surfaces with greater contrast & higher resolution than TEM. Disadvantages : • Only dead & dried objects can be examined, since the medium is vacuum. • Cell morphology is distorted. Types of Electron Microscopy
- 20. Electron Microscopy Transmission ElectronMicroscopy • It provides tremendous useful magnification because of much higher resolution obtained by short wavelength of the electron beam and magnifies the specimen. • The electron microscope uses electron beams & magnetic fields to produce the image, whereas the light microscope uses light waves & glass lenses. • The electron microscope produces a wavelength of 0.05A0. It is possible to resolve objects as small as 10 A0.
- 21. Electron Microscopy Transmission ElectronMicroscopy • The resolving power of the electron microscope and it produces magnification upto 4,00,000 times. • The specimen should be prepared as extremely thin dry film and is introduced into the instrument at a point between the magnetic condenser and the magnetic objective. • The magnified image may be viewed on a fluorescent screen.
- 22. Electron Microscopy Scanning ElectronMicroscopy • The specimen is subjected to a narrow electron beam which rapidly moves over the surface of the specimen. • This causes the release of a shower of secondary electrons & other types of radiation from the specimen surface. • The intensity of these secondary electron depends on the shape & the chemical composition of the irradiated object. • The secondary electrons are collected by a detector which generates an electronic signal. • Signals are scanned in a manner of a television system to produce an image on a cathode tube.
- 23. Electron Microscopy Scanning ElectronMicroscopy • The scanning electron microscope lacks the resolving power obtainable with the transmission electron microscope but has the advantage of revealing a striking three dimensional picture. Limitations: • The specimen to be examined is kept in a chamber that is under very high vacuum. So the cells cannot be examined in live state. • The drying process alters some morphological characteristics. • Low penetrating power of electron beam that requires very thin section of specimen to observe the internal structures of the cell.
- 24. Comparison of differenttypes of Microscopy Type of microscopy Max. magnification Appearance of specimen Useful applications Bright field 1,000-2,000 Specimens stained or unstained. Bacteria stained and appear as color of stain Gross morphological features of bacteria, yeasts, molds, algae & protozoa Dark field 1,000-2,000 Generally unstained, appears bright or lightened in an dark back ground Microorganism that exhibit some characteristic morphological features. Ex.: Spirochetes Fluorescence 1,000-2,000 Bright & colored based on color of the fluorescent dye Diagnostic techniques reveals identity of organisms. Phase contrast 1,000-2,000 Varying degrees of darkness Examination of cellular structures in living cells. Ex: yeasts, algae, protozoa & some bacteria Electron 2,00,000 – 4,00,000 Viewed on fluorescent screen Examination of viruses & ultra structures of microbial cells