ELECTRON MICROSCOPY FOR BIOLOGISTS

ELECTRON MICROSCOPY FOR BIOLOGISTS : 

ELECTRON MICROSCOPY FOR BIOLOGISTS INTRODUCTION Schematiska bilder är mestadels från www.matter.org.uk/tem

What is microscopy? : 

What is microscopy? Microscopy techniques - produce images at greater than life size - Magnification, M M = D/d (size of feature in the image and d the size of feature in object).

“The purpose of microscopy is insight, not images” - R.W. Hamming : 

“The purpose of microscopy is insight, not images” - R.W. Hamming Understanding of biological systems is derived from a knowledge of the spatial and temporal arrangement of structural entities ranging in size from organisms to macromolecules.

The scale of things : 

The scale of things

Proportions : 

Proportions

Detection : 

Detection light microscopy: photons interact with the specimen electron microscopy: electrons interact with the specimen: TEM and SEM scanning probe microscopy utilizes a variety of different interactions of a fine tip with the specimen. Atomic forces

Resolving power line : 

Resolving power line

Visualization : 

Visualization Obtaining an image. Record the images for analysis or for documentation purposes. Film recording is the only technique available on older equipment. Silver based photographic film, expensive, enables good resolution. Modern instruments use digital image capture. Transferred to a computer screen, stored or printed Digital images are readily accessible for subsequent computer analysis.

Analysis : 

Analysis Counting or measuring of objects in the images. May involve considerable computer processing of the images before the objects can be analysed. 3-D modelling

Physical limitations : 

Physical limitations In transmission electron microscopy electrons pass through thin specimens (50-1000 nm). BULK BEAM In scanning electron microscopy signals emitted from the surface of thick specimens. NARROW BEAM

why do we bother with clumsy electron-microscopy? : 

why do we bother with clumsy electron-microscopy? The resolution of a microscope = smallest distance between two points in the specimen > perceived as separate in the image. resolution is determined by the wavelength of the radiation used. resolving power of a particular system (Rayleigh criterion) DR = 0.61 l / N.A. where DR is the resolution l is the wavelength of the radiation used Numerical Aperture = sin of half the angle of the cone of radiation entering the lens Resolution gain by a shorter wavelength or collect more “data” from your specimen by having a larger numerical aperture.

Slide 12: 

visible wavelength 390nm ~ 750nm. Many cellular structures are much smaller than this. It is impossible to make lenses with a NA > 1.4 – limit of resolution due to aberrations is ~170nm. high absorbance for shorter wavelength radiation UV lenses of quartz or fluorite are expensive RADIO WAVES | MICROWAVES | INFRARED | VISIBLE | ULTRAVIOL.| X-RAYS | g -rays

electrons accelerated by 60 KVl = 0,003 nm : 

electrons accelerated by 60 KVl = 0,003 nm

TEM column : 

TEM column

The way forward is to use electrons : 

The way forward is to use electrons Short wavelength given by the de Broglie wavelength equation E = hc/l E = the kinetic energy of the particle,h = Planck’s constant, c = velocity of light in a vacuum l = the wavelenth of the particle. e- beams can readily be generated, using an electron gun. because of their charge they can be focused by electromagnetic lenses. Electrons can be generated The kinetic energy they acquire is given by E = Vq V = the accelerating voltage q = the charge on the electron

elektronkanon : 

elektronkanon

Elektronmikroskopi på web : 

Elektronmikroskopi på web http://www.matter.org.uk/tem/ http://nobelprize.org/physics/educational/microscopes/tem/index.html

Transmission electron microscopes comparable to light microscopes : 

Transmission electron microscopes comparable to light microscopes Electron beam path electron gun generates a beam condensor controls illumination objective lens magnifies image of the object Projector lenses, like an eyepiece, magnifies further

Constraints on the use of electrons: : 

Constraints on the use of electrons: Vacuum is essential: Moderate energy (60-200keV) electrons have a path length of a few mm in air Electron optical systems require a path ~1 meter The sample can’t be volatile or wet The absorbance of a material for electrons depends on its density. The practical thickness limit is < 500nm for 200kV electrons. Electrons are charged and interact strongly with matter; ionize. If electrons accumulate in the specimen it will repel the electrons in the beam. (worst in SEM) electrons falling on the specimen must be conducted to earth. C or Au sputtering Electron beam has considerable kinetic energy,  specimen temperature. Potentially resulting in damages.

Lens effect : 

Lens effect A strong magnetic field is generated by passing a current through a set of windings. This field acts as a convex lens, bringing off axis rays back to focus. Focal length can be altered by changing the strength of the current. The image is rotated, to a degree that depends on the strength of the lens.

Magnification : 

Magnification

Lens magnification : 

Lens magnification

Double condenser system : 

Double condenser system

1st condenser : 

1st condenser

2nd condenser : 

2nd condenser

Condenser aperture : 

Condenser aperture

Objective aperture : 

Objective aperture

Dark field and diffraction : 

Dark field and diffraction

Intermediate lens : 

Intermediate lens

Illumination adjustments : 

Illumination adjustments

Total magnification : 

Total magnification

Depth of field : 

Depth of field

Smaller aperture : 

Smaller aperture

Beam alignment : 

Beam alignment

Beam alignment : 

Beam alignment

Stigmators : 

Stigmators

Interaction with the specimen : 

Interaction with the specimen

Electrons interact : 

Electrons interact

The preparation of the material : 

The preparation of the material key importance that the preparation of the material be properly controlled. should be applied with understanding.

Specimen preparation : 

Specimen preparation