Scanning Electron MicroscopePollen

Chris Cardew 28th February 2016

Light Microscope

• A modern light microscope - 1000x .• The resolving power of the microscope limited

by the wavelength of the light used for illumination.

• Using light with a shorter wavelength--a small improvement.

• Using oil --- small improvement, but all together only brought the resolving power of the microscope to just under 100 nm.

Dr Ganga H

Dr Ganga H

Resolution of a microscope

• Wavelength of the illumination source ( λ )

• The numerical aperture of the lens (N.A.)

Limit of resolution = 0.61 λ/N.A.

• The maximum value of N.A. for a light microscope is approx. 1.4. therefore even the short blue light ( λ = 436 nm) of the visible spectrum will yield a resolution of only 190 nm.

• The wavelength of an electron beam is about 100,000 times less than that of visible light and hence the resolution of an electron microscope is far superior to that of the light microscope.

• NA is the light gathering ability and resolution of the acceptance cone of an objective

Dr Ganga H

Dr Ganga H

THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE

VacuumAir-filledInteriorMagnetsGlassLenses

High voltage (50kV) tungsten filament

Tungsten or quartz halogen lamp

Radiation source

X 500,000x1000 – x1500Maximum magnification

0.14nmFine detail

app. 200nm or 0.2micron

Maximum resolving power

Electronsapp. 4nm

Visible light390nm (red) – 760nm

Electromagnetic spectrum used

ELECTRON MICROSCOPELIGHT MICROSCOPEFEATURE

© 2007 Paul Billiet ODWS

Focus Lens is movable Rigidly fixed, adjust lens currents

THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE

Copper gridGlass slideSupport

Heavy metalsWater soluble dyesStains

Ultramicrotome Slices - 50nm

Parts of cells visible

Microtomeslices - 20 000nm

Whole cells visible

SectioningResinWaxEmbedding

Glutaraldehyde,OsO4 formaldehydeFixation

ELECTRON MICROSCOPELIGHT MICROSCOPEFEATURE

© 2007 Paul Billiet ODWS

Focussing screen Human eye (retina), photographic film

Fluorescent screen,photographic film

Scanning Electron Microscope

A scanning electron microscope (SEM) is a type of electron microscope that produces images of a sample by scanning it with a focused beam of electrons

‘The German scientists Max Knoll and Ernst Ruska realized that, since the wavelength (the distance between successive peaks or troughs of any wave) associated with electrons was much shorter than the wavelength of visible light, a microscope based on electron imaging should be able to pick out much finer detail than an optical microscope. This is because any tiny object or detail that has dimensions smaller than the wave falling on it will not influence or affect the wave’

Patricia HodgesPollen Drawings

Rex SawyerHoney Identification

Field Thistle pollen x400 using a light microscope

Field Thistle 2500 times

Intine

Field Thistle 2500 x 42.4um

Field Thistle x 2000

Intine

Field Thistle x 2000

Field Thistle 6000 x

Field Thistle x 8000

Field Thistle x 2500

Field Thistle x 2500

Field Thistle x 3000

Field Thistle x 3000

Field Thistle x 2500

Field Thistle x 2500

Field Thistle x 2000

Field Thistle x 2000

Field Thistle x 3000

Field Thistle x 2000

Field Thistle x 3000

Meadowsweet 4000 x enlargement

Meadowsweet 4000 x enlargement

Meadowsweet 6000 x enlargement.17.2um long

Meadowsweet x 4000

Corn Flower 3000x

Corn Flower 3000x

Corn Flower 5000x

Hogweed x 4000

Hogweed x 4000