Bringing light into the chaos:
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
Part two: Bringing colour into
the light
Fluorescence microscopy and special
applications:
Widefield, Confocal microscopy, Multiphoton,
FRET, FLIM,FRAP, Photoactivation, TIRF
What is light?
Light is electromagnetic radiation.
What we usually describe as light is
only the visible spectrum of this
radiation with wavelengths between
400nm and 700nm.
The elementary particle that defines
light is the photon.
b)
a)
There are 3 basic dimensions of light
a) Intensity (amplitude) which is related to the perception of brightness
b) Frequency (wavelength), perceived as colour
c) Polarization (angle of vibration) which is not or weakly perceptible to humans
What is a microscope?
What is a microscope?
Theoretically a microscope is an array of two lenses.
Focal plane
Image plane
Image plane
Objective
lens
Tube lens
Eyepiece
lens
Modern microscope with ICS
(Infinity Colour corrected System)
Classic compound microscope
Your friend - the objective
Objectives can be classified into transmitted light and reflected-light (Epi) versions.
Flat-field correction and
aberration correction
Describe two main criteria for the quality of an objective:
Flatness of the intermediate image
Elimination of chromatic errors
Spherical aberration
Spherical aberration causes beams parallel to but away from
the lens axis to be focussed in a slightly different place than
beams close to the axis. This manifests itself as a blurring of
the image.
Flat-field correction and
aberration correction
Describe two main criteria for the quality of an objective:
Flatness of the intermediate image
Elimination of chromatic errors
Chromatic aberration
Chromatic aberration is caused by a lens having different
refractive indexes for different wavelengths. Since the focal length
of a lens is dependent on the refractive index, different wavelengths
will be focused on different positions in the focal plane.
Chromatic aberration is seen as fringes of colour around the image.
It can be minimised by using an achromatic doublet (= achromat) in which two materials
with differing dispersion are bonded together to form a single lens.
Objective types
elimination of chromatic errors
flatness of the intermediate image
• 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
magnification by the objective
x
the magnification by eyepiece
BUT maximum magnification does not mean maximum resolution!
What is resolution?
Resolution describes the minimal distance of two points that can be distinguished.
Picture taken from http://microscopy.fsu.edu/primer/anatomy/numaperture.html
What is the numerical aperture?
NA is an estimate of how much light from the sample is collected by the objective
α2
α1
Objective lens
Oil (n = 1.5)
Air (n = 1.0)
Coverslip (n = 1.5)
Glass slide (n = 1.5)
NA = n sin 
n = refractive index
α = angle of incident illumination
Numerical aperture, NOT
magnification determines resolution!
Resolution of 0.175µ Bead Pair
1.1
0.9
0.8
1.4 NA
0.7
0.6
0.5
0.4
0.7 NA
0.3
0.2
0.1
1.10
0.96
0.81
0.66
0.51
0.37
0.22
0.07
-0.07
-0.22
-0.37
-0.51
-0.66
-0.81
-0.96
0.0
-1.10
Normalized Intensity
1.0
Microns
Increasing NA
A lens with a larger NA will be able to
visualize finer details and will also collect
more light and give a brighter image than
a lens with lower NA.
Your tricky friend - the objective
How can we use the properties of light
to create contrast?
Which properties can be used?
Absorption
Scattering
Refraction
Phase
Polarization
Contrasting techniques
Brightfield
DIC
Phase contrast
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.
Cross section of sunflower root
(http://www.zum.de/Faecher/Materialien/beck/12/bs12-5.htm)
 all our microscopes can be used for brightfield
Piece of artificially grown skin
(www.igb.fhg.de/.../dt/PI_BioTechnica2001.dt.html )
Darkfield
Principle: The illuminating rays of light are directed through the sample from the side by putting a
dark disk into the condenser that hinders the main light beam to enter the objective. Only light that
is scattered by structures in the sample enters the objective.
Application: People use it a lot to look at Diatoms and other unstained/colourless specimens
Darkfield
Symbiotic Diatom colony
(www1.tip.nl/~t936927/making.html)
Brightfield
 we do not have microscopes set up for darkfield
Phase contrast in theory
Principle: Incident light [Io] is out of phase with transmitted light [I] as it was slowed down while
passing through different parts of the sample and when the phases of the light are synchronized
by an interference lens, a new image with greater contrast is seen.
Phase ring
I0
not aligned
aligned
Phase stops
I
 most of our microscopes are set up
for phase contrast
Phase contrast in practice
Application: Phase contrast is the most commonly used contrasting technique in
this institute. All tissue culture microscopes and the time-lapse microscopes are set
up for phase.
BUT: MOST OF YOU ARE USING IT IN THE WRONG WAY!! Because you
do not use the right phase stop with the corresponding objective!
brightfield
wrong phase stop
right phase stop
Polarization Contrast
Principle: Polarized light is used for illumination. Only when the vibration direction of the polarized
light is altered by a sample placed into the light path, light can pass through the analyzer. The
sample appears light against a black background. A lambda plate can be used to convert this
contrast into colours.
Application: Polarization contrast is used
to look at materials with birefringent
properties, in which the refractive index
depends on the vibration direction of the
incident light, e.g. crystals or polymers.
Analyzer
Lambda
plate
Brightfield
Polarization Polarization
contrast
contrast with
Lambda plate
Polarizer
 we do not have microscopes set up for polarization contrast
D(ifferential) I(nterference) C(ontrast)
Principle: Also known as Nomarski microscopy. Uses polarized light for illumination.
Synchronizing of the different phases of incident and transmitted light is done by a set of prisms
and filters introduced into the light path.
Δ
~Δn/Δx
Δ>0
A
B
ΔX
ΔX
C
n1
n2
n3
X
ΔX
 most of our microscopes are set up for DIC
Contrasting techniques - a summary
• Brightfield -absorption
Light is transmitted through the sample. Only useful for specimens that can be contrasted via dyes. Very little contrast in unstained
specimens.
• 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.
• Phase Contrast- phase interference
Incident light [Io] is out of phase with transmitted light [I] and when the phases of the light are synchronized by an interference lens, a new
image with greater contrast is seen
• Polarization Contrast -polarization
Uses polarized light for illumination. Only when the vibration direction of the polarized light is altered by a sample placed into the light
path, light can pass through the analyzer. The sample appears light against a black background.
• Differential Interference Contrast (DIC) –
polarization + phase interference
Also known as Nomarski microscopy. Synchronizing of the different phases of incident and transmitted light is done by a set of special
condenser lens mounted below the stage of a microscope
• Fluorescence Contrast (->Ireen)
Final words from your friend - the objective
10x
Coverslip-types:
1:
0.13 - 0.17 mm
1.5:
0.16 - 0.19 mm
2.0:
0.19 - 0.23 mm
20x
40x
60x
Objective
Useful links
• Margaret and Tom (ext. 6872)!!!
• Zeiss – Microscopy from the very beginning
http://www.zeiss.de/C1256B5E0047FF3F?Open
• Molecular Expressions homepage
http://micro.magnet.fsu.edu/index.html
• Wikipedia
http://en.wikipedia.org/wiki/Main_Page