Bringing light into the chaos: A general introduction to optics and light microscopy Juliana Schwarz 19/03/07.

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