Microscopy

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Microscopy

• Lab Three

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Compoun
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• Care and handling
• Always carry with TWO HANDS, one on the arm and
  one supporting the base.
• Never hold the microscope sideways or upside
  down.
• Check the oculars for fingerprints or other
  smudges. If necessary, use LENS PAPER only.
  Fog first.

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Specimen control - hold and manipulate the
specimen

• stage - where the specimen rests
• clips - used to hold the specimen still on the
  stage
• stage controls - device that allows you to move
  the specimen in controlled, small increments
  along the x and y axes (useful for scanning a
  slide)

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Illumination - shed light on the specimen

• lamp - produces the light
• rheostat - alters the current applied to the lamp
  to control the intensity of the light produced

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Illumination continued

• condenser - lens system that aligns and focuses
  the light from the lamp onto the specimen
• Iris diaphragm- 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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• Molecular Expressions Microscopy Primer Anatomy
  of the Microscope - Köhler Illumination -
  Condenser Aperture Diaphragms Interactive Java
  Tutorial

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Lenses - form the image

• objective lens - gathers light from the specimen
• ocular- transmits and magnifies the image from
  the objective lens to your eye
• turret - rotating mount that holds many objective
  lenses
• tube - holds the eyepiece at the proper distance
  from the objective lens and blocks out stray
  light

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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

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Total Magnification

• Total magnification is the product of the ocular
  (10x) and the objective magnification
• Pg. 41

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Letter e slide
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• 1. Begin with low power objective in place and
  the STAGE ALL THE WAY UP.
• 2. Focus by lowering the stage.
• Low power first, then switch to higher power
  scopes should be nearly parfocal.
• On high power, use FINE FOCUS ONLY. You can run
  the objective into the slide.
• Adjust interocular width and diopter for your
  eyes until you see only one circle.

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Field of view vs. Depth of Field

• Field of view- circular area you see when you
  look through the ocular

• Depth of field-the amount of the field of view
  that is in sharp focus

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Crossed Threads Slide
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Brightness - How light or dark is the image?

• Brightness can be changed by adjusting the
  rheostat and adjusting the condenser and
  diaphragm/pinhole apertures.

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Resolution

• The minimum distance between two points at which
  they can still be distinguished as separate
  points
• - more magnification doesnt help an object be
  seen more clearly once resolution is at it
  maximum

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Contrast

• What is the difference in lighting between areas
  of the specimen?
• Contrast is related to the illumination system
  and can be adjusted by changing the intensity of
  the light and the diaphragm aperture.

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Contrast

• The image on the left has more appropriate
  contrast than the image on the right

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3 types of illumination for a compound microscope
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Bright Field Microscopy Transmitted Light
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Darkfield Illumination

• Scattered light

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Preparing a wet mount slide

• Place the specimen on the slide
• Rest the coverslip at an angle over the specimen
• Allow the coverslip to drop rapidly, hopefully
  avoiding air bubbles

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Get a Stage Micrometer Slide
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Pg.50
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Compound light microscope Bright field
(transmitted light)Dark field (scattered
light) Dissecting microscope Reflected
lightProvides a 3D image Electron
microscope Transmission (TEM)Scanning (SEM)
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Look at specimens under Dissecting Microscope
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Electron microscopes use electrons instead of
light. Magnets focus and scan a beam of
electrons. Transmission electron microscopes
(TEM) - beam passes through the subject
Scanning electron microscopes (SEM) - beam is
reflected from surface of object
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Scanning Electron Micrograph of Bacteria (X14,000)
Bacteria
Courtesy of Dr. Michael Craig (BMS Dept./SMSU)
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Scanning Electron Micrograph of a Butterfly Wing
Scale (X10,000)
Wing Scale
Courtesy of Dr. Michael Craig (BMS Dept/SMSU)
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Scanning Electron Micrograph of a Hens Egg Shell
(X1,800)
Egg Shell
Courtesy of Dr. Michael Craig (BMS Dept./SMSU)
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Scanning Electron Micrograph of a Drosophila
Female (X130)
Drosophila Female
Courtesy of Dr. Michael Craig (BMS Dept./SMSU)
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When you are finished A. Remove any slide from
the stage and make sure that the stage is
clean. B. Turn the light off. C. Put the low
power objective in place. D. Turn the stage all
the way up. E. Adjust the stage control so that
it is centered E. Replace the dust cover.F. Put
Away
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Assignment

• Page 50
• A Fill out table in class
• B answer questions 1-13 (use manual, text, and
  web resources to answer)
• C use web resources to research a different
  type of microscope than discussed in lab today
  and write a 1 paragraph summary of how it is used