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A general introduction to
optics and light microscopy
Juliana Schwarz
19/03/07
Overview
Part one:
The root of all evil
Basic terms and applications
in light microscopy
b) a)
Image plane
• CP-Achromat
Good colour correction – exactly for two wavelengths. Field flatness in the image center,
refocusing also covers the peripheral areas. For fields of view up to dia. 18 mm. Versions
for phase contrast.
• Achroplan
Improved Achromat objectives with good image flatness for fields of view with dia. 20 or
even 23 mm. Achroplan for transmitted light and Achroplan Ph for phase contrast.
• Plan-Neofluar
Excellent colour correction for at least three wavelengths. Field flattening for the field of
view with dia. 25 mm. Highly transmitting for UV excitation at 365 nm in fluorescence. All
methods possible, special high-quality variants are available for Pol and DIC.
• Plan-Apochromat
Perfect colour rendition (correction for four wavelengths!). Flawless image flatness for
fields of view with dia. 25 mm. Highest numerical apertures for a resolving power at the
very limits of the physically possible.
Still your friend - the objective
What is magnification?
Magnification is defined by the
α2
α1
Objective lens
Oil (n = 1.5)
Air (n = 1.0)
Coverslip (n = 1.5)
Glass slide (n = 1.5)
1.1
1.0
0.9
Normalized Intensity
0.8 1.4 NA
0.7
0.6
0.5
0.4
0.7 NA
0.3
0.2
0.1
0.0
0.07
0.22
0.37
0.51
0.66
0.81
0.96
1.10
-1.10
-0.96
-0.81
-0.66
-0.51
-0.37
-0.22
-0.07
Microns
Increasing NA
Absorption
Scattering
Refraction
Phase
Polarization
Contrasting techniques
DIC Darkfield
Taken from: http://fig.cox.miami.edu/~cmallery/150/Fallsyll.htm
Contrasting techniques
• Brightfield
• Darkfield
• Phase Contrast
• Polarization Contrast
• Differential Interference Contrast (DIC)
• Fluorescence Contrast (Ireen)
Brightfield
Principle: Light is transmitted through the sample and absorbed by it.
Application: Only useful for specimens that can be contrasted via dyes. Very little contrast in
unstained specimens. With a bright background, the human eye requires local intensity fluctuations
of at least 10 to 20% to be able to recognize objects.
Application: People use it a lot to look at Diatoms and other unstained/colourless specimens
Darkfield
Phase ring
Phase stops
BUT: MOST OF YOU ARE USING IT IN THE WRONG WAY!! Because you
do not use the right phase stop with the corresponding objective!
Analyzer
Lambda
plate
Δ
~Δn/Δx
Δ>0
C n1
A B n2
n3
X
ΔX ΔX ΔX
• Darkfield -scattering
The illuminating rays of light are directed from the side so that only scattered light enters the microscope lenses, consequently the cell
appears as an illuminated object against the view.
10x
20x
Coverslip-types: 40x
60x
1: 0.13 - 0.17 mm
1.5: 0.16 - 0.19 mm
2.0: 0.19 - 0.23 mm
Objective
Useful links
• Margaret and Tom (ext. 6872)!!!
• Wikipedia
http://en.wikipedia.org/wiki/Main_Page