Optical Microscope

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Optical Microscope Microscopy Workshop Florida
High Tech Corridor techPATHUniversity of South
Florida Dr. Luisa Amelia Dempere DirectorMajor
Analytical Instrumentation CenterUniversity of
Florida
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• Outline
• What is a Microscope?
• Relationship between the Microscope and the human
  eye
• History of Optical Microscope
• How the Optical Microscope Works?
• Important terms Parts Components Lenses Obse
  rvation modes
• Image Quality

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• What is a Microscope?
• Microscopes are instruments designed to produce
  magnified visual or photographic images of small
  objects. The microscope must accomplish three
  tasks
• produce a magnified image of the specimen
• separate the details in the image
• render the details visible to the human eye or
  camera

A light microscope is also known as an optical
microscope
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Relationship between the Microscope and the human
eye
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Relationship between the Microscope and the human
eye
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History of Optical Microscope
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History of Optical Microscope
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History of
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History of Optical Microscope
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History of Optical Microscope
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History of Optical Microscope
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History of Optical Microscope
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How the Microscope works? Important Terms

• Depth of field - vertical distance, from above to
  below the focal plane, that yields an acceptable
  image
• Field of view - area of the specimen that can be
  seen through the microscope with a given
  objective lens
• Focal length - distance required for a lens to
  bring the light to a focus (usually measured in
  microns)
• Focal point/focus - point at which the light from
  a lens comes together
• Magnification - product of the magnifying powers
  of the objective and eyepiece lenses
• Numerical aperture - measure of the
  light-collecting ability of the lens
• Resolution - the closest two objects can be
  before they're no longer detected as separate
  objects (usually measured in nanometers)

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Depth of Field
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Focal Plane
Depth of Field
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Depth of Field
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Resolution is the ability to tell two points
apart as separate points. If the resolving power
of your lens is 2um that means two points that
are 2um apart can be seen as separate points. If
they are closer together than that, they will
blend together into one point. Optical
microscopes are used daily in our lives for
example eyeglasses and a simple magnifying glass.
To increase the magnification of a microscope the
number of lenses must be increased. Although
sometimes the image becomes unclear that's when
the microscope's resolving power decreases. The
resolving power is the microscope's ability to
produce a clear image. In the 1870s, a man named
Ernst Abbe explained why the resolution of a
microscope is limited. He said that since the
microscope uses visible light and visible light
has a set range of wavelengths. The microscope
can't produce the image of an object that is
smaller than the length of the light wave. The
value for the resolution of a light microscope
has been constant at 200 nm (2,000 angstroms).
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• Parts and Components
• Specimen control - hold and manipulate the
  specimen
• Stage - where the specimen rests
• Clips - used to hold the specimen still on the
  stage
• Micromanipulator - device that allows you to move
  the specimen in controlled, small increments
  along the x and y axes
• Illumination - shed light on the specimen
• Lamp - produces the light (Typically, lamps are
  tungsten-filament light bulbs. For specialized
  applications, mercury or xenon lamps may be used
  to produce ultraviolet light.
• Rheostat - alters the current applied to the lamp
  to control the intensity of the light produced
• Condenser - lens system that aligns and focuses
  the light from the lamp onto the specimen
• Diaphragms or Pinhole Apertures - placed in the
  light path to alter the amount of light that
  reaches the condenser (for enhancing contrast in
  the image)

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• Lenses - form the image
• objective lens - gathers light from the specimen
• eyepiece - transmits and magnifies the image from
  the objective lens to your eye
• nosepiece - rotating mount that holds many
  objective lenses
• tube - holds the eyepiece at the proper distance
  from the objective lens and blocks out stray
  light
• Focus - position the objective lens at the proper
  distance from the specimen
• coarse-focus knob - used to bring the object into
  the focal plane of the objective lens
• fine-focus knob - used to make fine adjustments
  to focus the image
• Support and alignment
• arm - curved portion that holds all of the
  optical parts at a fixed distance and aligns them
  
• base - supports the weight of all of the
  microscope parts
• The tube is connected to the arm of the
  microscope by way of a rack and pinion gear. This
  system allows you to focus the image when
  changing lenses or observers and to move the
  lenses away from the stage when changing
  specimens.

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Parts and Components
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Lenses
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Lenses
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Aberrations
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Observation Modes

• Brightfield - This is the basic microscope
  configuration This technique has very little
  contrast.
• Darkfield - This configuration enhances contrast.
• Rheinberg illumination - This set-up is similar
  to darkfield, but uses a series of filters to
  produce an "optical staining" of the specimen.

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Observation Modes

• Phase contrast - In a phase-contrast microscope,
  the annular rings in the objective lens and the
  condenser separate the light. The light that
  passes through the central part of the light path
  is recombined with the light that travels around
  the periphery of the specimen. The interference
  produced by these two paths produces images in
  which the dense structures appear darker than the
  background.

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• Differential interference contrast (DIC) - DIC
  uses polarizing filters and prisms to separate
  and recombine the light paths, giving a 3-D
  appearance to the specimen (DIC is also called
  Nomarski after the man who invented it).
• Hoffman modulation contrast this contrast is
  similar to DIC except that it uses plates with
  small slits in both the axis and the off-axis of
  the light path to produce two sets of light waves
  passing through the specimen.
• Polarization - The polarized-light microscope
  uses two polarizers, one on either side of the
  specimen, positioned perpendicular to each other
  so that only light that passes through the
  specimen reaches the eyepiece. Light is polarized
  in one plane as it passes through the first
  filter and reaches the specimen.
  Regularly-spaced, patterned or crystalline
  portions of the specimen rotate the light that
  passes through. Some of this rotated light passes
  through the second polarizing filter, so these
  regularly spaced areas show up bright against a
  black background.
• Fluorescence - This type of microscope uses
  high-energy, short-wavelength light (usually
  ultraviolet) to excite electrons within certain
  molecules inside a specimen, causing those
  electrons to shift to higher orbits. When they
  fall back to their original energy levels, they
  emit lower-energy, longer-wavelength light
  (usually in the visible spectrum), which forms
  the image

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• Image Quality
• When you look at a specimen using a microscope,
  the quality of the image you see is assessed by
  the following
• Brightness - How light or dark is the image?
• Focus - Is the image blurry or well-defined?
• Resolution - How close can two points in the
  image be before they are no longer seen as two
  separate points?
• Contrast - What is the difference in lighting
  between adjacent areas of the specimen?

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Brightness
Focus
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Resolution
Contrast
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