MICROPARA MICROSCOPY

• Light is electromagnetic radiation
• Microscope images

  • Photographs of cells taken using a microscope, called micrographs
  • Fibroblast grown in culture
  • Piece of artificially grown skin
  • Cross section of sunflower root
• Numerical aperture


  An estimate of how much light from the sample is collected by the objective
• A lens with a larger numerical aperture


  Will visualize finer details and collect more light, giving a brighter image than a lens with lower numerical aperture
• The higher the numerical aperture of a lens


  The better the resolution of a specimen that can be obtained with that lens
• Basic dimensions of light

  • Intensity (amplitude) related to the perception of brightness
  • Frequency (wavelength) perceived as colour
  • Polarization (angle of vibration) not or weakly perceptible to humans
• Resolution

  The minimal distance of two points that can be distinguished
• Types of Light Microscopes

  • Bright field Light Microscope
  • Phase Contrast Light Microscope
  • Dark-Field Light Microscope
  • Fluorescence Light Microscope
• Magnification


  A measure of how much larger a microscope causes an object to appear
• Light Microscopy uses visible light and glass lenses
• History of microscopes

  Starting with use of a simple lens in ancient times, to the first compound microscope around 1590, and up to the microscopes used in 7th grade life science, the microscope has allowed scientists to make discoveries about the “invisible world”
• A microscope is an array of two lenses
• Photon


  The elementary particle that defines light
• Principle of Light Microscopy

  When a ray of light passes from one medium to another, it bends by phenomena called refraction. The bending of light slows the speed and is determined by the refractive index of the medium
• The resolution of a microscope or lens is the smallest distance by which two points can be separated and still be distinguished as separate objects
• The microscope has become one of the most recognizable symbols of science
• Darkfield microscopy is used to look at Diatoms and other unstained/colourless specimens
• The smaller the resolution value, the higher the resolving power of the microscope and the better the clarity and detail of the image
• Brightfield microscopy is only useful for specimens that can be contrasted via dyes
• Numerical aperture, NOT magnification, determines resolution
• Maximum magnification does not mean maximum resolution
• Fluorescence microscopy is used in the visualization of bacterial agents and to identify specific antibodies produced against bacterial antigens
• Thank you!
• Phase contrast is the most commonly used contrasting technique in Light Microscopy
• Contrasting techniques in Light Microscopy

  • Brightfield
  • Darkfield
  • Phase contrast
  • DIC