microscopy

• Microbiology


  Study of organisms and agents that are too small to be seen by the naked eye

  
• Organisms


  • Bacteria
  • Fungi
  • Protozoa
  • Algae
• Agents


  • Viruses
• Microscope


  Came from the Greek words mikros (small) and skopein (to watch/see)
• Anton Van Leeuwenhoek was the first to observe microorganisms (i.e. bacteria)
• Parts of the Microscope


  • #1 Draw Tube – Reflects light up to the viewer's eye
  • #2 Revolving nosepiece or turret – Allows for quick change of objectives
  • #3 Low Power Objective – The first lens you use when doing proper microscope work. Usually 10 X
  • #4 High Power Objective – The second lens you use when doing proper microscope work. Usually 40 X
  • #5 Oil immersion Objective – The highest magnification used. Usually 100 X. NEVER use the course adjustment when using this lens.
  • #6 Stage Clips – Use to keep the slide in place.
  • #7 Iris/Diaphragm – Use to vary the amount of light passing through the slide.
  • #8 Light Source – Sends light up through the diaphragm and through the slide for viewing
  • #9 Eye Piece—The part you look at with your eye. Usually 10 X magnification.
  • #10 Arm – Used to safely transport microscope
  • #11 Stage – Slides are placed on this
  • #12 Coarse Adjustment Knob – Used to make large changes in focus. NOTE Never use this when viewing on high power
  • #13 Fine Adjustment Knob – Used to small adjustments of focus
  • #14 Base – Used to safely transport the microscope

  
• Bright-Field Microscope


  • Produces a dark image against a brighter background
  • Has several objective lenses
  • Parfocal microscopes remain in focus when objectives are changed
  • Total magnification is the product of the magnifications of the ocular lens and the objective lens

  
• Path of light through the microscope


  1. Light travels from the illuminator and enters the condenser

  2. Light then enters the condenser which focuses light onto the specimen slide situated on the stage

  3. Light passes through the specimen on the slide

  4. Next light enters the objective lens. [If using the 100x objective lens, then light enters immersion oil first then the 100x lens.]

  5. Light then reaches the prism in the eyepiece, which shifts light path onto the ocular lens. Then light reaches your eye.

  
• Magnification (M)


  • M = IS/AS (Image Size/Actual Size)
  • Linear M: Eyepiece - 10X, LPO - 10X, HPO - 40X, OIO - 100X
  • Total M: (Linear M of eyepiece)(Linear of objective), e.g. Total M of OIO = (10X)(100X) = 1,000X
• Important Features of Objectives


  • Focal Point (F)
  • Focal Length: LPO = 16mm, HPO = 4 mm, OIO = 1.8 MM
  • Resolving Power/Resolution (R): d = 0.5λ / nsinϴ
  • Numerical Aperture (NA): LPO = 0.25, HPO = 0.65, OIO = 1.25
• Relationship of M and FL


  The closer the object to the focal point is, the higher is the magnification rate. If the object is placed 2X of the focal length, then the object and the real image will have the same size.
• Relationship of d and R


  d = limit of resolution, R = resolving power
• Numerical Aperture (NA)


  • Measure of the resolving power of an objective
  • Higher NA means better light-gathering properties and resolution, but shorter working distance
• Magnification increase leads to decrease in light admitted
• Higher working distance means greater distance between the front surface of lens and surface of cover glass or specimen