MICROSCOPES

1
MICROSCOPES
2
PROPERTIES OF LIGHT

• ELECTROMAGNETIC SPECTRUM
• RADIO WAVES
• AS MUCH AS 2000 METERS LONG
• GAMMA RAYS
• AS SHORT AS 0.01 nm
• VISIBLE LIGHT
• NEAR MIDDLE
• BETWEEN 400-700 nm

3
WAVELENGTH

• DISTANCE BETWEEN TWO PEAKS
• 400-700 nm ARE VISIBLE
• FREQUENCY
• INTENSITY
• HEIGHT OF WAVE
• ALSO MAY BE DESCRIBED AS
• PHOTONS
• STREAM OF PARTICLES
• RAYS
• A NARROW BEAM

• SHORTER THE WAVELENGTH THE GREATER THE ENERGY
• VISIBLE LIGHT
• RED HAS LONGEST WAVELENGTH
• BLUE HAS SHORTEST
• GREEN BETWEEN
• WHITE LIGHT IS MIXTURE OF VARIOUS WAVELENGTHS

4
ACTIONS OF LIGHT

• REFLECTION
• BOUNCES BACK
• TRANSMISSION
• PASSES THROUGH
• ABSORPTION
• IF INTENSITY IS DIMINISHED
• HAS TRANSFERRED SOME OR ALL OF ITS ENERGY TO THE
  OBJECT

5
REFLECTION

• BOUNCES OFF OBJECT
• IF IT STRIKES STRAIGHT IT WILL BOUNCE STRAIGHT
  BACK
• IF IT STRIKES AT AN ANGLE IT WILL BOUNCE OFF AT
  ANGLE

6
TRANSMISSION

• RAYS PASS THROUGH PURE WATER OR CLEAR GLASS
  UNDIMINISHED
• IF NOT SOME LIGHT IS ABSORBED
• IF ALL WAVELENGTHS ARE ABSORBED EQUALLY THE
  INTENSITY OF THE LIGHT IS DIMINISHED BUT COLOR
  REMAINS THE SAME
• IF SOME WAVELENGTHS ARE ABSORBED MORE THAN OTHERS
  THE COLOR CHANGES

7
LIGHT MAY ALSO BE REFRACTED OR DIFFRACTED
8
REFRACTION

• RAY OF LIGHT MEETS A SUBSTANCE OF DIFFERENT
  DENSITY
• WHEN LIGHT ENTERS A SUBSTANCE OF GREATER DENSITY
  IT SLOWS DOWN
• EDGE THAT ENTERS FIRST SLOWS FIRST AND SECOND
  EDGE SLOWS LATER
• THE AMOUNT OF BENDING DEPENDS ON THE ANGLE AND
  HOW MUCH THE LIGHT IS SLOWED
• ALSO OCCURS WHEN LIGHT ENTERS A SUBSTANCE OF
  LESSER DENSITY
• OCCURS IN OPPOSITE DIRECTION

9
REFRACTIVE INDEX

• THE RATIO OF THE SPEED OF LIGHT TRAVELING IN A
  VACUUM TO ITS SPEED IN SOME PARTICULAR MATERIAL
• ESSENTIALLY A MEASURE OF THE ABILITY OF THE
  MEDIUM TO BEND LIGHT

10
THE REFRACTIVE INDEX IS WRITTEN ON THE SIDE OF
THE LENS
11
ABERRATIONS
12
SPHERICAL ABERRATIONS
13
CHROMATIC ABERRATION
14
CHROMATIC ABBERATIONS
15
DIFFRACTION
16
REAL IMAGE

• OBJECTIVE FORMS IN BODY OF THE TUBE

17
VIRTUAL IMAGE

• PROJECTED UP THROUGH BODY TO OCULAR
• IMAGE OF AN IMAGE
• RECEIVED BY EYE

18
MAGNIFICATION

• CONVEX LENSES MAGNIFY
• REFRACT LIGHT TO MEET AT A FOCAL POINT

19
RESOLUTION
20
RESOLUTION

• CAPACITY TO DISTINGUISH TWO ADJACENT OBJECTS OR
  POINTS FORM EACH OTHER
• HUMAN LENS CAN RESOLVE TWO OBJECTS (AT 10 INCHES)
  AS TWO POINTS AS LONG AS THEY ARE NOT CLOSER THAN
  0.2MM APART

21
RESOLVING POWER
 

22
NUMERICAL APERATURE

• NA
• LENS SIZE AND USE OF IMMERSION OIL
• R wavelength 2NA

23
REFRACTIVE INDEX AND THE OIL IMMERSION LENS

• ERNST ABBE DEVELOPED OIL IMMERSION LENS TO
  CORRECT ABERRATIONS

24
CONTRAST
25
COMPOUND MICROSCOPES

• LIGHT MICROSCOPE
• PARTS

26
TOTAL MAGNIFICATION

• OCULAR X OBJECTIVE

27
MEASURING OBJECTS SEEN IN THE LIGHT MICROSCOPE
28

• known distance
  between
• One division two lines on stage
  micrometeron ocular Number of
  divisions on ocularmicrometer
  micrometer in mm

29
EXAMPLE

• number of
  calibration
• Length of ocular divisions X factor
  for one
• Organism occupied
  ocular division
• 5 X 1.54 microns 7.70
  microns

30
METRIC UNITS OF LENGTH

• METER 39.37 inch
• DECIMETER 3.94 inch
• CENTIMETER 0.39 inch
• MILLIMETER
• MICROMETER
• NANOMETER

31
WORKING DISTANCE
32
PARFOCAL

• FOCUS AT SCANNING OBJECTIVE
• MOVE INCREMENTALLY UP IN POWER WITH ONLY FINE
  ADJUSTMENTS

33
FIELD OF VIEW
34
SLIDE PREPARATIONS
35
WET MOUNTS

• SIMPLE WET MOUNT
• HANGING DROP SLIDE

36
MAKING A BACTERIAL SMEAR
37
USING THE OIL IMMERSION LENS
38
FOCUS CAREFULLY WITH THE 40X OBJECTIVE
39
ROTATE LENS HALFWAY
40
APPLY A DROP OF OIL TO SLIDE
41
ROTATE 100X LENS OVER SLIDE
42
THREE IMPORTANT RULES

• 1. Never use an oil immersion lens without the
  oil.
• 2. Never get oil on any other lens.
• 3. Clean up all oil when finished.

43
RETURNING THE MICROSCOPE TO STORAGE

• 1 Lower stage fully  2 Return the 4x objective
  to the viewing position.  3 Reduce rheostat to
  lowest setting, turn off power switch  4 Wrap
  cord snugly, no sharp bends, tuck in plug. 
• Loosely drape over oculars.
• DO NOT WRAP AROUND CONDENSER!! 5 Replace
  dust cover, return to storage, arm towards you. 
  6 Report any problem with the instrument
  immediately.

44
STAINS

• VITAL STAINS
• FIXED SLIDES

45
TYPES OF STAINS

• BASIC DYES
• ACIDIC DYES
• MORDANTS

46
TYPES OF STAINS

• SIMPLE STAINS
• DIFFERENTIAL STAINS
• SPECIAL STAINS

47
SIMPLE STAINS
48
DIFFERENTIAL STAINS

• GRAM STAIN
• ACID FAST STAIN

49
GRAM STAIN
50
ACID FAST STAIN
51
SPECIAL STAINS

• SPORE STAINS
• FLAGELLAR STAINS
• NEGATIVE STAINS

52
SPORE STAINS
53
FLAGELLAR STAINS
54
NEGATIVE STAIN
55
CAPSULAR STAIN
56
OTHER TYPES OF MICROSCOPES

• PHASE CONTRAST
• DARK FIELD
• FLUORESCENCE
• DIFFERENTIAL INTERFERENCE CONTRAST MICROSCOPE
• CONFOCAL
• ELECTRON MICROSCOPE

57
DARK FIELD
58
PHASE CONTRAST
59
DIFFERENTIAL INTERFERENCE CONTRAST
60
IMMUNOFLOURESCENCE
61
CONFOCAL
62
ATOMIC FORCE
63
TRANSMISSION ELECTRON MICROSCOPE
64
SCANNING ELECTRON MICROGRAPH