Bringing light into the chaos: A general introduction to optics and light microscopy Juliana Schwarz...
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Transcript of Bringing light into the chaos: A general introduction to optics and light microscopy Juliana Schwarz...
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 lightFluorescence 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.
There are 3 basic dimensions of lighta) Intensity (amplitude) which is related to the perception of brightnessb) Frequency (wavelength), perceived as colourc) Polarization (angle of vibration) which is not or weakly perceptible to humans
a)b)
What is a microscope?
What is a microscope?
Theoretically a microscope is an array of two lenses.
Objective lens
Tube lens Eyepiece lens
Focal plane Image plane
Classic compound microscope
Modern microscope with ICS (Infinity Colour corrected System)
Image plane
Your friend - the objectiveObjectives can be classified into transmitted light and reflected-light (Epi) versions.
Flat-field correction and aberration correction
Flatness of the intermediate image
Elimination of chromatic errors
Describe two main criteria for the quality of an objective:
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
Flatness of the intermediate image
Elimination of chromatic errors
Describe two main criteria for the quality of an objective:
Chromatic aberration
Chromatic aberration is caused by a lens having differentrefractive indexes for different wavelengths. Since the focal length of a lens is dependent on the refractive index, different wavelengthswill 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
• 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.
elimination of chromatic errors
flatness of the intermediate image
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
NA = n sin
What is the numerical aperture?NA is an estimate of how much light from the sample is collected by the objective
α1α2
Coverslip (n = 1.5)
Glass slide (n = 1.5)
Oil (n = 1.5)Air (n = 1.0)
n = refractive index
α = angle of incident illumination
Objective lens
Numerical aperture, NOT magnification determines resolution!
Resolution of 0.175µ Bead Pair
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
-1.1
0
-0.9
6
-0.8
1
-0.6
6
-0.5
1
-0.3
7
-0.2
2
-0.0
7
0.0
7
0.2
2
0.3
7
0.5
1
0.6
6
0.8
1
0.9
6
1.1
0
Microns
No
rmalized
In
ten
sit
y
1.4 NA
0.7 NA
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
Darkfield
Phase contrast
DICTaken 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
Piece of artificially grown skin (www.igb.fhg.de/.../dt/PI_BioTechnica2001.dt.html )
Cross section of sunflower root(http://www.zum.de/Faecher/Materialien/beck/12/bs12-5.htm)
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.
all our microscopes can be used for brightfield
DarkfieldPrinciple: 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
Brightfield
Darkfield
we do not have microscopes set up for darkfield
Symbiotic Diatom colony(www1.tip.nl/~t936927/making.html)
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.
I
I0
most of our microscopes are set up for phase contrast
Phase ring
alignednot aligned
Phase stops
Phase contrast in practiceApplication: 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!
wrong phase stopbrightfield right phase stop
Polarization ContrastPrinciple: 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.
we do not have microscopes set up for polarization contrast
Lambda plate
Polarizer
Analyzer
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.
Brightfield Polarization contrast
Polarization contrast with Lambda plate
D(ifferential) I(nterference) C(ontrast)
most of our microscopes are set up for DIC
A BC
Δ > 0
ΔX ΔX ΔX
n1
n2n3
Δ ~Δn/Δx
X
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.
Contrasting techniques - a summary
• Brightfield -absorptionLight is transmitted through the sample. Only useful for specimens that can be contrasted via dyes. Very little contrast in unstained specimens.
• Darkfield -scatteringThe 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 interferenceIncident 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 -polarizationUses 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 interferenceAlso 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
Coverslip-types:1: 0.13 - 0.17 mm1.5: 0.16 - 0.19 mm2.0: 0.19 - 0.23 mm Objective
10x20x
40x60x
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
• Wikipediahttp://en.wikipedia.org/wiki/Main_Page