MICROSCOPY

• Microscopy


  The technical field of using microscopes to view objects and areas that cannot be seen with the naked eye
• Microscopes


  • Help us see different microorganisms and cells, especially in different kinds of tissues, and to identify different kinds of bacteria
• Robert Hooke


  First scientist to observe microorganisms in the cork using a compound microscope in 1665
• Anton van Leeuwenhoek


  Uses knowledge to achieve a microscope with greater magnification, and was the first to make an observation of bacteria on water in 1674
• Bausch and Lomb


  Used brass to produce a more complex type of microscope and had mass production of microscopes in the 19th century
• Refractive index


  Measures the speed of light in a material, and is a structure that slows down the velocity of light
• Focal point


  Where the light rays are focused in a specific place
• Focal length


  The distance between the focal point and the center of the lens
• If we have a shorter focal point, we'll have greater magnification
• Light microscopy


  A type of compound microscope where an image is projected by the action of two or more series of lenses
• Types of light microscopes

  • Bright-field
  • Dark-field
  • Phase contrast
  • Fluorescence
• Bright field microscope


  Produces dark image against a brighter background, using ordinary light passing through the preparation
• Bright field microscope


  • Condenser collects and focuses a cone of light that illuminates the tissue slide
  • Objective lenses enlarge and project the illuminated image of the object towards the eyepiece, with different magnifications (4x, 10x, 40x)
• Microscope resolution


  The ability of a lens to separate or distinguish small objects that are close together
• Wavelength of light is the major factor in resolution, and is inversely proportional to resolution
• Maximum resolving power of light microscope is 0.2μm (micrometre)
• Working distance


  The distance between the front surface of the lens and the surface of the specimen
• Dark field microscope


  Produces bright image of the object against a dark background, used to observe living and unstained preparations
• Spirochetes


  • Treponema pallidum (syphilis)
  • Leptospira species (leptospirosis)
• Phase contrast microscope


  Enhances the contrast between intracellular structures having slight difference in refractive index, excellent for observing living cells
• Differential interference contrast microscope


  Creates images by detecting differences in refractive indices and thickness of different parts of specimen, excellent for observing living cells
• Fluorescence microscopy


  Exposes specimens to ultraviolet (UV)/ blue light, and specimens are stained with fluorochromes to show a bright image of the object resulting from the fluorescent light emitted by the specimen
• Electron microscopy


  Beams of electrons are used to produce images, with a wavelength approximately 1:2000 of the light microscope beam, increasing the resolution factor by 10:3
• Types of electron microscopes

  • Transmission electron microscopes
  • Scanning electron microscopes
• Transmission electron microscope


  Used to produce images, requiring the specimen to be cut very thin, chemically fixed and stained with electron dense material like uranyl acetate or lead citrate, with a resolution of 3nm and magnification up to 1 million times
• Scanning electron microscope


  Uses electrons reflected from the surface of a specimen to create a 3D image
• Confocal scanning laser microscope


  Uses a laser beam to illuminate spots on the specimen and creates a 3D image
• Scanning probe/scanning tunnelling microscope


  The up and down movement of a probe as it maintains current is detected and used to create an image of the surface of the specimen