Human Biology Student Outline – Light Microscopy
Light Microscopy: History, Usage, and Utility
http://www.micrographia.com/tutoria/micbasic/micbpt01/micb0100.htm
http://www.biologie.uni-hamburg.de/b-online/e03/03.htm
http://www.micro.magnet.fsu.edu/primer/anatomy/numaperture.html
1.
Introduction –
2.
History A.
Robert Hooke
B.
3.
Anton van Leeuwenhoek
Microscopy
A.
Anatomy of the Light Microscope –
i.
Light Microscope (Compound Microscope)
a.
Stage
•
Mechanical Stage
b.
Illumination
c.
Condenser
•
Iris Diaphragm
d.
Body Tube
e.
Oculars
f.
Objectives
•
Objective
Revolving Nosepiece
Objective
Magnificaiton
Page 1
Ocular
Magnificaiton
Total
Magnificaiton
Human Biology Student Outline – Light Microscopy
Scanning
Low Power
High Power
Oil Immersion
g.
4x
10 x
45x
97x
Focus
•
Coarse Adjustment
•
Fine Adjustment
*
B.
10x
10x
10x
10x
Parfocalization
The Nature of Light
i.
Speed
Page 2
40x
100x
450x
970x
Human Biology Student Outline – Light Microscopy
ii.
Refraction
Indices of Refraction
Vacuum
1.000
Air
1.000277
Water
1.333
Flint Glass
1.57 – 1.75
n = index of refraction;
C = speed of light in vacuum;
V = speed of light in a particular material
http://hyperphysics.phyastr.gsu.edu/hbase/geoopt/refr.html#c1?n1=3&n2=4&th1=23&th2=34
Page 3
Human Biology Student Outline – Light Microscopy
C.
Fundamentals of Optics
i.
Focal Point
ii.
Anti-point
iii.
Image
Page 4
Human Biology Student Outline – Light Microscopy
b.
•
Resolving Power (RP)
Angular Aperture
Page 5
Human Biology Student Outline – Light Microscopy
•
“Airy Disks”
http://www.micro.magnet.fsu.edu/primer/anatomy/numapertu
re.html
Page 6
Human Biology Student Outline – Light Microscopy
u = 1/2 angular aperture
N.A. = sin u
Therefore the NA range will be roughly 0 to 1
sin 0 = 0
sin 90 = 1
Effect of sin on angle
Magnification
NA
Relative
Page 7
Human Biology Student Outline – Light Microscopy
1x
10x
40x
100x (oil)
(Plan Achromat)
Resolution
0.025
0.25
0.65
1.25
Poorest
Best
N.A. = n sine u
Snell’s Law seen above
Refractive Index of Air = 1
Refractive Index of Oil = 1.515
Refractive Index of Water = 1.33
Refractive Index of Glycerin = 1.47
Refractive Index of Glass = 1. 515
N.A. = n sine u
Page 8
Human Biology Student Outline – Light Microscopy
Resolution or Resolving Power

= wavelength of light
RP =

2 x NA
Page 9
Human Biology Student Outline – Light Microscopy
Example: For white light using a 10x objective
White Light (average wavelength = ~550 nm)
RP = 550nm = 550 = 1100 nm or 1.1 m
2 x 0.25 0.50
Blue Light
RP = 450nm = 450
2 x 0.25 0.50
= 900 nm or 0.9 m
Red Light
RP = 650nm = 650
2 x 0.25 0.50
= 1300 nm or 1.3 m
Resolution using a lens with fixed numerical aperture of 0.95 at
varying wavelengths
Wavelenght () of Light (nm)
Color
360
Deep Violet
400
Violet
450
Blue
500
Blue-Green
550
Green-Yellow
600
Orange
650
Red
700
Deep Red
Resolution (µm)
0.19
0.21
0.24
0.26
0.29
0.32
0.34
0.37
Resolution can also be increased by the following:
Page 10
Human Biology Student Outline – Light Microscopy
1.
2.
3.
4.
Blue filter, will allow only the shorter wavelengths of blue
to hit the organism
Condenser, kept at its highest position for maximum
amount of light to enter
Diaphragm should not be stopped, but opened
Immersion Oil, only when the 100x is used.
Example (100x oil immersion): (understand that the
resolving power has been increased due to a higher
refractive index)
With white light
RP = 550nm = 550
2 x 1.25 2.50
= 220 nm or 0.22 m
Slide in a blue filter and turn up the light intensity
RP = 450nm = 450
2 x 1.25 2.50
= 180 nm or 0.18 m
Page 11