Microscopy

• Microscope:

  • Came from the Latin word “microspium”
  • Greek words “mikros” that means small; “skopein” which means to look at
  • It is a laboratory instrument used to examine objects that are too small to be seen by the naked eye
• Microscopy is the science of investigating small objects and structures using a microscope
• Units of Measurement:

  • 1 meter (m)
  • 10 = 101 = decimeter (dm)
  • 100 = 102 = centimeter (cm)
  • 1000 = 103 = millimeter (mm)
  • 10-6 = micrometer (um) = 0.000001m or micron (unit used for bacteria)
  • 10-9 = nanometer (nm) = 0.000000001m (unit used for viruses)
  • 10-10 = angstrom = 0.0000000001m or 0.1nm
• Light Microscopy refers to the use of any kind of microscope that uses visible light to make specimens observable
• Compound Light Microscope:

  • Contains more than one magnifying lens: objective lens and ocular lens
  • Maximum magnifying power of 1000x
  • Parfocal: specimen remain in focus
• Total Magnification:

  • Calculated by multiplying the magnifying power of the objective lens by the magnifying power of the ocular lens
  • Ocular Lens x Objective Lens = Total Magnification
• Working Distance: the distance between the front surface of the lens and surface of the cover glass or specimen
• Microscope parts:

  • Ocular Lens (eyepiece): remagnifies the image formed by the objective lens
• • Base: serves as a support for microscopes
• • 1000 AD - Reading stone is a sphere of glass that can be placed on top of text to magnify it.
• • 1021 AD - Book of Optics by Ibn al-Haytham (also known as the Father of Optics). Ibn al-Haytham is a mathematician, astronomer, and a scientist. He completely transformed how light and vision were understood. 
• • 13th Century - The first wearable eyeglasses called spectacle was created by Salvino D’Armati. These eyeglasses were made of wood.
• • 1590 - Hans Lippershey and his son Zaccharias Hanssen, were experimenting with a variety of lenses. They discovered the first early compound microscope. They utilized two lenses to magnify an object with higher magnification.
• 1608 - First Telescope created by Hans Lippershey in Poland.
• • 1609 - Galileo Galilei created the first compound microscope using convex and concave lenses. He was the first one to use a compound microscope. 
• 1625 - Giovanni Faber responsible for coining the term microscope.
• • 1665 - Robert Hooke built a compound microscope. Hooke then wrote a book called “Micrographia”. He used his compound microscope to observe thin slices of cork. Hooke was also the first one to see cells by the use of a microscope.
• 1676 - Anton Van Leeuwenhoek is the first person to report the microorganisms that he called animalcules. Leeuwenhoek used the microscope to examine blood, yeast, and insects. He also invented a microscope with 50-300x magnification. Due to his contribution to microscopy, he is recognized as the Father of Microscope.
• • Body Tube - transmits the image from the objective lens to the ocular lens.
• Arm - it supports the body tube and connects it to the base
• Objective Lenses - primary lenses that magnify the specimen.
• • Revolving Nosepiece - the part of the microscope that holds two or more objective lenses and can be rotated to easily change power. 
• Stage - it holds the microscope slide in position.
• Stage Clip - it holds the slides in place.
• Condenser - focuses light through specimen.
• Diaphragm - it controls the amount of light entering the condenser.
• Illuminator - a steady light source (110 volts) used in place of a mirror.
• Coarse Focusing Knob - a rapid control which allows for quick focusing by moving the objective lens or stage up and down. It is used for initial focusing.
• Fine Adjustment Knob - a slow but precise control used to fine focus the image when viewing at the higher magnification.
• Base - it serves as a support for microscopes.
• Bright-Field Microscopy:

  • Most widely-used type of light microscope
  • Specimen produces an image darker than the surrounding brightly illuminated field
  • Can be used for both live, unstained material and preserved-stained material
• Dark Field Microscopy:

  • Specimen produces a light on dark background
  • Uses a darkfield condenser with an opaque disc to block light, allowing only direct light to enter the objective lens
  • Used to identify bacteria like Treponema pallidum, the causative agent of syphilis
  • Best for observing live, unstained specimens
• Phase-Contrast Microscopy:

  • Targets the internal structure of the cell
  • Uses a special condenser (annular diaphragm) and objective lenses to accentuate small differences in the refractive index of various structures within the organism
  • Specimen is contrasted against a gray background
  • Used for live specimens; excellent for internal cellular detail
• Differential Interference (Nomarski) Microscopy:

  • Uses prisms to add contrasting colors
  • Provides brightly colored, highly contrasting, three-dimensional images of live specimens
• Fluorescence Microscopy:

  • Uses UV radiation source and filters to protect the viewer’s eye
  • Takes advantage of fluorescence where molecules emit light after being triggered
  • Specimens stained with fluorescent dyes or combined with fluorescent antibodies emit visible light
  • Used usually for immunology; excellent diagnostic tool
• Confocal Microscope:

  • Scans specimens stained with fluorescent dyes using a laser beam
  • Multiple images are combined into a three-dimensional image by a computer
  • Can view unstained specimens using light reflected from specimens
• Two-Photon Microscopy:

  • Uses long wavelength (red) light
  • Allows imaging of living cells in tissues up to 1mm deep
  • Tracks the activity of cells in real time
• Super-Resolution Light Microscopy:

  • Uses two laser beams to scan cells one nanometer at a time, providing a resolution of less than 0.1mm
• Scanning Acoustic Microscopy:

  • Interprets the action of a sound wave sent through a specimen
  • Powerful and non-distractive
  • Used to study living cells attached to other surfaces, such as cancer cells, artery plaque, and bacterial biofilms