Types and applications of Microscopy.ppt
- 1. Types and applications of microscopy in cell biology
- 2. Microscopes The optical microscope, often referred to as the "light microscope", is a type of microscope which uses visible light and a system of lenses to magnify images of small samples. Optical microscopes are the oldest and simplest of the microscopes. There are non-optical microscopes, which require chemical or ion staining of non-living samples, and can magnify exponentially greater than the optical microscope.
- 3. Other types of optical microscope the inverted microscope for studying samples from below; useful for cell cultures in liquid; the student microscope designed for low cost, durability, and ease of use; the research microscope which is an expensive tool with many enhancements; the fluorescence microscope the phase contrast microscope
- 4. Inverted microscope An inverted microscope is a microscope with its light source and condenser on the top, above the stage pointing down, while the objectives are below the stage pointing up. The light source in this microscope uses a 12V 1000 watt hydrogen lamp and the wavelength can be changed automatically. Inverted microscopes are useful for observing living cells or organisms at the bottom of a large container (e.g. a tissue culture flask) under more natural conditions than on a glass slide, as is the case with a conventional microscope.
- 7. Disadvantages of conventional microscope A traditional light microscope requires that the specimen be placed on a glass slide, typically under a cover slip. This usually means removing a small sample from the culture and placing it in the artificial environment created by the slide and cover slip. The temperature and oxygen content of the sample may change quickly from that of the culture as a result. Further, the organisms will be under increased pressure and in an unnaturally confined space as a result of the cover slip. Also, the sample will quickly dry out unless repeatedly replenished with water. The loss of water by evaporation and the periodic adding of water may change the salinity of the sample frequently. These changes impose severe stress on microorganisms that can affect their behavior and/or kill them in a short time.
- 8. Inverted Microscope Inverted microscopes makes possible to observe microorganisms in a large container under more natural conditions. Because of its configuration, You can place an entire culture or large sample in a relatively large container such as a petri dish and look at the entire contents of the container under more natural and less stressed conditions. Such a sample may sustain life over a much longer period. Disadvantage : Costly
- 9. Fluorescence microscope A fluorescence microscope is a light microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption.
- 10. Fluorescence microscope In most cases the sample of interest is labeled with fluorophore and then illuminated through the lens with the higher energy source. The illumination light is absorbed by the fluorophores (now attached to the sample) and causes them to emit a longer lower energy wavelength light. This fluorescent light can be separated from the surrounding radiation with filters designed for that specific wavelength allowing the viewer to see only that which is fluorescing.
- 11. Fluorescence microscope In most cases, a component of interest in the specimen is specifically labeled with a fluorescent molecule called a fluorophore (such as green fluorescent protein (GFP), fluorescein or DyLight 488).
- 12. Fluorescence microscope Typical components of a fluorescence microscope are the light source (xenon arc lamp or mercury-vapor lamp), the excitation filter, the dichroic mirror (or dichromatic beamsplitter),. The filters and the dichroic are chosen to match the spectral excitation and emission characteristics of the fluorophore used to label the specimen.
- 13. Principle of Fluorescence microscope The basic task of the fluorescence microscope is to let excitation light radiate the specimen and then sort out the much weaker emitted light from the image. First, the microscope has a filter that only lets through radiation with the specific wavelength that matches the fluorescing material. The radiation collides with the atoms in the specimen and electrons are excited to a higher energy level. When they relax to a lower level, they emit light. To become detectable (visible to the human eye) the fluorescence emitted from the sample is separated from the much brighter excitation light in a second filter. This works because the emitted light is of lower energy and has a longer wavelength than the light that is used for illumination.
- 15. Fluorescence microscope Most fluorescence microscopes in use are epifluorescence microscopes (i.e. excitation and observation of the fluorescence are from above (epi) the specimen). These microscopes have become an important part in the field of biology Fluorophores lose their ability to fluoresce as they are illuminated in a process called photobleaching. Special care must be taken to prevent photobleaching through the use of more robust fluorophores or by minimizing illumination.
- 17. Phase-contrast microscopy Phase contrast microscopy is an optical microscopy illumination technique in which small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast changes in the image. A phase contrast microscope does not require staining to view the slide. This microscope made it possible to study the cell cycle.
- 18. PHASE CONTRAST MICROSCOPY Living cells and most cell organelles are often difficult if not impossible to see by brightfield microscopy because they do not absorb, refract or diffract sufficient light to contrast with the surrounding medium. The phase contrast microscope was developed to improve contrast differences between cells/organelles and the surrounding medium, making it possible to see cells/organelles without staining.
- 19. PHASE CONTRAST MICROSCOPY The technique is based on the fact that cells differ in refractive index from their surroundings and thus bend some of the light rays passing through them. Light rays passing through a transparent specimen (most unstained cells are transparent) emerge as either direct rays unaffected by passage through the specimen (unaltered in intensity and phase) or diffracted rays bent as they pass through the specimen (altered intensity and/or phase). This effect is amplified by the phase annulus coupled with special phase rings in the objectives leading to a dark image on a light background
- 20. Phase Contrast Optical Microscopy This phase contrast microscope has two main parts Annular phase plate Annular diaphragm
- 21. Alternatives to optical microscopy electron microscope X-ray microscope Scanning Tunneling Microscope (STM) Atomic force microscope (AFM)
- 22. Alternatives to optical microscopy The use of electrons and x-rays in place of light allows much higher resolution - the wavelength of the radiation is shorter so the diffraction limit is lower. To make the short-wavelength probe non- destructive, the atomic beam imaging system (atomic nanoscope) is proposed STM and AFM are scanning probe techniques using a small probe which is scanned over the sample surface
- 23. Electron microscopy Electron Microscopes yield the following information: Topography (hardness, reflectivity...etc.) Morphology (strength, reactivity...etc.) Composition (melting point, reactivity, hardness...etc.) Crystallographic Information (conductivity, electrical properties, strength...etc.)
- 24. The basic steps involved in EMs A stream of electrons is (by the Electron Source) accelerated toward the specimen This stream is focused using metal apertures and magnetic lenses Interactions occur inside the irradiated sample, affecting the electron beam
- 25. Electron microscopy Transmission electron microscope Scanning electron microscope
- 26. Transmission electron microscopy Transmission electron microscopy (TEM) involves a high voltage electron beam emitted by an electron gun, usually fitted with a tungsten filament cathode as the electron source. The electron beam is focused by electrostatic and electromagnetic lenses, and transmitted through a specimen that is in part transparent to electrons and in part scatters them out of the beam. When it emerges from the specimen, the electron beam carries information about the structure of the specimen that is magnified by the objective lens system of the microscope. The spatial variation in this information (the "image") is recorded by projecting the magnified electron image onto a fluorescent viewing screen coated with a phosphor or scintillator material such as zinc sulfide.
- 27. Scanning Electron Microscope Unlike the TEM, where electrons of the high voltage beam form the image of the specimen, the Scanning Electron Microscope (SEM) produces images by detecting low energy secondary electrons which are emitted from the surface of the specimen due to excitation by the primary electron beam. In the SEM, the electron beam is rastered across the sample, with detectors building up an image by mapping the detected signals.
- 28. Applications of electron microscope Biology and life sciences Cryobiology Protein localization Electron tomography Cellular tomography Cryo-electron microscopy Toxicology viral load monitoring 3D tissue imaging Virology Vitrification
- 29. X-Ray Microscopy X-ray microscope uses electromagnetic radiation charge-coupled device (CCD) detector to detect X-rays This views biological samples in their natural state. X-rays cause fluorescence in most materials
- 30. Applications of Microscopy Forensic science Food product contaminant analysis Pharmaceutical applications Identification of cell types Pathology and histology