1. Introduction to the Microscope
History
Types
Care
Parts & functions
Focusing

2. Question 1

3. Question 1: Bacteria

4. Question 2

5. Question 2: DNA

6. Question 3

7. Question 3: Breast Cancer Cell

8. Question 4

9. Question 4: Alga- red tides

10. Question 5

11. Question 5: Bed bug

12. Why? To see microscopic world
Learning Targets
Describe how a microscope works.
Calculate the total magnification of an image.
Compare the different types of microscopes.
Why? To see microscopic world

13. Microscope History
Circa 1000AD – The first vision aid was invented (inventor unknown) called a reading
stone. It was a glass sphere that magnified when laid on top of reading materials.

14. Microscope History
Circa 1284 –
Italian, Salvino D'Armate is credited with inventing the first wearable eye glasses.

15. Microscope History
1590 – Two Dutch eye glass makers, Zaccharias Janssen and son Hans Janssen experimented with multiple lenses placed in a tube. The Janssens observed that viewed objects in front of the tube appeared greatly enlarged, creating both the forerunner of the compound microscope and the telescope.

16. Microscope History
1665 – English physicist, Robert Hooke looked at a sliver of cork through a microscope lens and noticed some "pores" or "cells" in it.

17. Microscope History
1674 – Anton van Leeuwenhoek built a simple microscope with only one lens to examine blood, yeast, insects and many other tiny objects. Leeuwenhoek was the first person to describe bacteria, and he invented new methods for grinding and polishing microscope lenses that allowed for curvatures providing magnifications of up to 270 diameters, the best available lenses at that time.

18. Microscope History
18th century – Technical innovations improved microscopes, leading to microscopy becoming popular among scientists. Lenses combining two types of glass reduced the "chromatic effect" the disturbing halos resulting from differences in refraction of light.

19. Microscope History
1830 – Joseph Jackson Lister reduces spherical aberration or the "chromatic effect" by showing that several weak lenses used together at certain distances gave good magnification without blurring the image. This was the prototype for the compound microscope.

20. Microscope History
1872 – Ernst Abbe, then research director of the Zeiss Optical Works, wrote a mathematical formula called the "Abbe Sine Condition". His formula provided calculations that allowed for the maximum resolution in microscopes possible.

21. Microscope History
1903 – Richard Zsigmondy developed the ultramicroscope that could study objects below the wavelength of light. He won the Nobel Prize in Chemistry in 1925.

22. Microscope History
1932 – Frits Zernike invented the phase-contrast microscope that allowed for the study of colorless and transparent biological materials for which he won the Nobel Prize in Physics in 1953.

23. Microscope History
1931 – Ernst Ruska co-invented the electron microscope for which he won the Nobel Prize in Physics in An electron microscope depends on electrons rather than light to view an object, electrons are speeded up in a vacuum until their wavelength is extremely short, only one hundred-thousandth that of white light. Electron microscopes make

24. Microscope History
1931 – Ernst Ruska
it possible to view objects as small as the diameter of an atom.

25. Microscope History
1981 – Gerd Binnig and Heinrich Rohrer invented the scanning tunneling microscope that gives three-dimensional images of objects down to the atomic level. Binnig and Rohrer won the Nobel Prize in Physics in The powerful scanning tunneling microscope is the strongest microscope to date.

26. How does a microscope work?
Magnification
enlargement of an object
compare size of image to actual size of object
total magnification
ocular power x objective power = total magnification

27. Microscopes

28. Microscopes . . Poor Resolution = Blurry Image
Resolution – capacity to show 2 points that are close together as separate .
. .
10x
1000x
Poor Resolution = Blurry Image
Good Resolution = Clear Image

29. Resolution

30. How does a microscope work?
Parfocal
both low and high power objectives are adjusted to the same focus
easily switch between both objectives

31. What happens as magnification increases?
field of view decreases
brightness decreases
resolving power increases

32. Staining Coloring cell structure With dyes to reflect light
Certain cell parts absorb certain stains
Kills cells or disturbs contents
Vital stains-dyes that highlight structures in living cells

33. Microscope Care Always carry with 2 hands
Never touch the lenses with your fingers.
Only use lens paper for cleaning
Do not force knobs
Keep objects clear of desk and cords
When you are finished with your "scope", rotate the nosepiece so that it's on the low power objective, roll the stage down to lowest level, rubber band the cord, then replace the dust cover. .

34. Types of Microscopes
Compound Microscope
Dissection Microscope
Scanning Electron Microscope (SEM)
Transmission Electron Microscope (TEM)

35. What are the different types of microscopes?
Compound light microscope
Stereoscopic dissecting microscope
Electron microscope

36. Compound Microscope
Compound microscopes are light illuminated. The image seen with this type of microscope is two dimensional. This microscope is the most commonly used. You can view individual cells, even living ones. It has high magnification. However, it has a low resolution.

37. Compound Microscope Images
Paulownia Wood c.s. 200x
Frog’s blood
1,000x

38. Microscope Parts Ocular lens Body Tube Revolving Nosepiece Arm
Objective Lens
Stage
Stage Clips
Coarse adjustment knob
Diaphragm
Fine adjustment knob
Light
Base

39. magnifies; where you look through to see the image of your specimen.
ocular lens
Ocular lens
magnifies; where you look through to see the image of your specimen.
They are usually 10X or 15X power.  Our microscopes have an ocular lens power of 10x.

40. supports the tube and connects it to the base
arm
supports the tube and connects it to the base
arm

41. the flat platform where you place your slides
stage
the flat platform where you place your slides
stage

42. coarse adjustment knob
moves stage (or body tube) up and down
coarse adjustment knob

43. after using the coarse adjustment knob
fine adjustment knob
small, round knob on the side of the microscope used to fine-tune the focus of your specimen
after using the coarse adjustment knob
fine adjustment knob

44. the bottom of the microscope, used for support
base
the bottom of the microscope, used for support
base

45. connects the eyepiece to the objective lenses
body tube
body tube
connects the eyepiece to the objective lenses

46. the part that holds two or more objective lenses
revolving nosepiece
the part that holds two or more objective lenses
and can be rotated to
easily change power
revolving nosepiece

47. Adds to the magnification
objective lenses
Adds to the magnification
Usually you will find 3 or 4 objective lenses on a microscope.  They almost
always consist of 4X, 10X, 40X and 100X powers.  When coupled with a 10X (most common)
objective lens

48. objective lenses
eyepiece lens, we get total magnifications of 40X (4X times 10X), 100X , 400X and 1000X.
The shortest
lens is the lowest power, the longest one is the lens with the greatest power.  Lenses are color coded.
objective lenses

49. push in (spring loaded) thereby protecting the lens and the slide.
objective lenses
The high power objective lenses are retractable (i.e. 40XR).  This means that if they hit a slide, the end of the lens will
push in (spring loaded) thereby protecting the lens and the slide.
objective lenses

50. Images reproduced from: http://micro.magnet.fsu.edu/
12:18 AM

51. Objectives PLAN-APO-40X 1.30 N.A. 160/0.22  - Infinity corrected
Flat field Apochromat Magnification Numerical Tube Coverglass
Factor
Aperture Length Thickness
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52. “Half” of a Zeiss Microscope objective
Photo taken in
Zeiss Museum
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53. Objectives This defines a “resel” or “resolution element”
Limit for smallest resolvable distance d between 2 points is (Rayleigh criterion):
d =
1.22

This defines a “resel” or “resolution element”
Thus high NUMERICAL APERTURE is critical for high magnification
In a medium of refractive index n the wavelength gets shorter:n
12:18 AM

54. Numerical Aperture
Resolving power is directly related to numerical aperture.
The higher the NA the greater the resolution
Resolving power:
The ability of an objective to resolve two distinct lines very close together
NA = n sin u
(n=the lowest refractive index between the object and first objective element) (hopefully 1)
u is 1/2 the angular aperture of the objective
12:18 AM

55. Refractive Index Matched RI Mis-Matched RI Water Objective n = 1.52
Oil
Air
n = 1.0
n=1.52
n = 1.52
Coverslip
n=1.33
n=1.52
Specimen
Water
Matched RI
Mis-Matched RI
12:18 AM

56. moves it left and right, the other moves it up and down.
stage clips
Stage clips hold the slides in place.  If your microscope has a mechanical stage, you will be able to move the slide around by turning two knobs.  One
moves it left and right, the other moves it up and down.
stage clips

57. controls the amount of light going through the specimen
diaphragm
controls the amount of light going through the specimen
Many microscopes have a rotating disk under the stage.  This diaphragm has different sized holes and is used to vary the intensity and size of the cone of light
diaphragm

58. you desire and the particular objective lens in use.
diaphragm
that is projected upward into the slide.  There is no set rule regarding which setting to use for a particular power. Rather, the setting is a function of the transparency of the specimen, the degree of contrast
you desire and the particular objective lens in use.
diaphragm

59. light
makes the specimen
easier to see
light

60. Using the Microscope
The proper way to focus a microscope is to start with the lowest power objective lens first and while looking from the side, crank the lens down as close to the specimen as possible without touching it.  Now, look through the eyepiece lens and focus upward only until the image is sharp.  If you can't get it in focus, repeat the process again.  

61. Using the Microscope
Once the image is sharp with the low power lens, you should be able to simply click in the next power lens and do minor adjustments with the focus knob.  If your microscope has a fine focus adjustment, turning it a bit should be all that's necessary.   Continue with subsequent objective lenses and fine focus each time. 

62. Using High Power
Rotate to 40x objective, locate desired portion of specimen in the center of the field. Refocus very carefully so that the specimen is focused as sharply as
possible. (Do not
alter focus for the
Following steps )

63. Using High Power
Partially rotate so that 40x and 100x objectives straddle the specimen.

64. Using High Power
Place a small drop of oil on the slide in the center of the lighted area. (Take care not to dribble on the stage.)
Put the small drop
of oil directly over
the area of the
specimen to be
Examined.

65. Using High Power
Rotate so that the 100x oil immersion objective touches the oil and clicks into place.

66. Using High Power
Focus only with fine focus. Hopefully, the specimen will come into focus easily. Do not change focus dramatically.

67. Using High Power
Clean up!: When you have finished for the day, wipe the 100x oil immersion objective carefully with lens paper to remove all oil. Wipe oil from the slide thoroughly with a Kimwipe. Cleanse stage should any oil have spilled on it. Recap the immersion oil container securely, replace in drawer.

68. Compound Light Microscope

69. Compound Light Microscope
has 2 or more lenses (eyepiece and objective)
uses transmitted light (light passes through object)
must use thin specimen so light can pass thru
can use live specimens
usually doesn’t exceed total mag. of 2000x
Our scopes in class are capable of 400x
only see specimen in 2D

70. Compound Microscope Images
Human Hair (x 400)
Mite
Paramecium

71. Compound Light Microscope
Structures & Functions

72. Compound Light Microscope
Eyepiece/Ocular – The lens through which the scientist looks
Body Tube – Connects eyepiece to microscope
Revolving Nosepiece – Holds 3-4 objectives (magnifying lens), turns for objective selection.

73. Compound Light Microscope
Scanning Objective - Used for locating objects & scanning the slide quickly (Red Line- 4X)
Lowest power objective
Low Power Objective – Lens that allows you to find & center the object on a slide. Yellow line around the objective (10x)
High Power Objective - Lens that zooms in for closer viewing (40X)
Blue line around high power

74. Compound Light Microscope
Stage – platform upon which the slide rests
Mechanical Stage – movable clips that hold & move the slide
Iris Diaphragm – transparent lens through which light travels. Size and brightness can be adjusted.
Lamp – light source needed for viewing the specimen

75. bsapp.com

76. Matching Procedure Fire bullets from a suspected weapon
With the aid of a comparison microscope, compare these “test fires” to the suspected bullets
Striations must be identical for a positive match
bsapp.com

77. Comparison Microscope
Two scopes- One Field
bsapp.com

78. bsapp.com

79. bsapp.com

80. Dissection Microscope
A dissection microscope is light illuminated. The image that appears is three dimensional. It is used for dissection
to get a better look at the larger specimen. You cannot see individual cells because it has a low magnification.
(also called stereo microscope)

81. Sunflower with moth pupa in the stem
Dissection Microscope Images
Head of a moth pupa
60x
Sunflower with moth pupa in the stem
10x

82. Stereoscopic Dissecting Microscope
uses reflected light to produce image
range of magnification
10x-60x

83. Images from a Stereoscope
Penny – Abe’s face
Penny – back
Beetle

84. Stereo Dissecting Microscope
2 eyepieces to produce 3-D image
Uses reflected light to illuminate surface of specimen
Used on large objects which light cannot pass through
Magnifies object 5x – 60x

85. Scanning Electron Microscope - SEM
SEM use electron illumination. The image is seen in 3-D. It has high magnification and high resolution. The
specimen is coated in gold and the electrons bounce off to give you and exterior view of the specimen. The pictures are in black and white.

86. Scanning Electron Microscope Images
pigeon blood
cockroach antenna

87. Transmission Electron Microscope - TEM
TEM is electron illuminated. This gives a 2-D view. Thin slices of specimen are obtained. The electron beams pass
through this. It has high magnification and high resolution.

88. Transmission Electron Microscope Images
bacillus bacteria
dividing
mitochondrion

89. Electron Microscope 2 Types Scanning electron microscope (SEM)
Transmission electron microscope (TEM)

90. Electron Microscope Discovered in Germany in 1930’s and 1940’s
Uses beam of electrons to see image NOT light
Image is produced on a T.V. monitor in black & white (no light)
Much higher resolution
Limitations
Can’t view living things due to vacuum in interior
Very expensive
Very big, must have own foundation

91. Transmission electron microscope (TEM)
Electrons go through very thin slice of specimen – detailed image on T.V. screen
200,000x (can be increased to 1,000,000x)

92. Images from a TEM
Bacteria
E.coli bacteria dividing
Leaf

93. Scanning electron microscope (SEM)
Beam of electrons across a whole specimen (sprayed with fine metal coating)
3 dimensional view of surface features on T.V. screen
100,000x

94. Scanning Electron Microscope
uses reflected electrons to produce image

95. Images from a SEM Dentist Drill (x 50) Hypodermic Needle (x 100)
Mosquito (x 100)
Toilet
Paper
(x 500)

96. Phase Contrast: living cells
Blood
Cheek cell

97. WHICH SCOPE WOULD BE BEST TO USE TO LOOK AT:
A pollen grain
Mold on a piece of bread
Bacteria
Nucleus of a cell
Grasshopper