Microscopy

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  • Microscopy
    • Light Microscope
      • Light microscopes are the most commonly used microscopes used to examine cells. This is because the equipment used is relatively inexpensive, requires minimal user training, and yields good results in terms of identifying cell structures and activity.
      • A number of lenses are used to produce an image. Image viewed directly through eyepieceLight passes from bulb under the stage, through condenser lense, then through specimen Beam of light then focused through objective lens, then through eyepiece lens Four different objective lenses (x4, x10, x40, x100) allow for specimen to be viewed at different magnifications Eyepiece lens magnifies image again, usually x10 Total magnification is given by multiplying the magnification of the 2 lenses Magnification possible with light microscope: up to 1500x ??
      • Magnification Up to 1500x in total
      • Resolution 200nm Limited by the wavelength of visible light
      • Specimen Preparation Staining Applying a coloured stain to the sample which binds to certain chemicals/structures, improving their visibility Acetic orcein stains DNA dark red Gentian violet stains bacterial cell walls Sectioning Specimen is embedded in wax to preserve structure of sample cell walls while cutting them into a thin slice
      • Advantages:  Wide range of specimens can be observed Specimens can be alive Specimens can be whole, or embedded in wax ten sectioned
      • Disadvantages:              Non-coloured specimens must be stained for specific organelles or molecules Relatively low resolution does not give detailed information
    • Electron Microscopes
      • Transmission Electron Microscope
        • A Transmission Electron Microscope (TEM) emits an electron beam through a very thin prepared sample. Electrons penetrate the denser parts of the sample with greater difficulty and this gives the contrast in the 2D image produced.
        • Magnification TEM: Up to x500, 000
      • Resolution 0.1nm 2000x times more than light microscope Produces detailed images
      • Specimen Preparation Specimen needs to be prepared correctly Fixed to make it firm Dehydrated and embedded in resin Stained using metal salts or metal particles Mounted on a copper grid Placed in a vacuum Staining Specimens are stained with metal salts or particles This causes electrons to scatter differently, giving contrast
      • Scanning Electron Microscope
        • A Scanning Electron Microscope (SEM) emits an electron beam directly onto a sample such that none of the electrons penetrate it. Instead, they ‘bounce off’ the sample and are received on a sensor, producing a 3D image.
          • Magnification: SEM: Up to x100, 000
      • Advantages: Produces detailed images of the structures inside cells SEM produces detailed 3D images showing contour of cells  
      • Disadvanatges: Electron beams deflected in air, so sample must be in a vacuum Samples must be dead Extremely expensive Large piece of equipment Use requires a high degree of skill and training
    • Magnification:  The degree to which the size of an image is larger than the object itself
      • Resolution:  The degree to which it is possible to distinguish between two points on an object that are very close together The higher the resolution, the greater the detail that you can see?

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