Electron Optics - PowerPoint Presentation

Slide1

Electron Optics Basic Introduction

Bob Ashley

6-14-2013Slide2

Overview

Why electrons?

Wavelength and visible light

Effects of diffraction and resolution

Lens design

Defects and distortions

MagnificationSlide3
Slide4

Electron Duality

Electrons have a particle and wave nature

Wave and particle nature

SourceSlide5

Why Electrons?

Wavelength (λ)

Measurement of sinusoidal

wave distance peak to peak

Visible light small segment

Electrons wavelength dependent

on velocity

200kV scope 1.2 x 10

-3

nm

http://reich-chemistry.wikispaces.com/Fall.2008.MMA.Boyle.TimelineSlide6

The Wave

Radiate from source in widening circles

Diffraction phenomenon

Wave strike edge and bend

Interference of waves

http://

images.tutorvista.comSlide7

Phase

Difference between two waves having the same electrical degrees or time and having the same frequency are in phase.

Can interfere with other waves.

Constructive interference

Additive property of two waves

Destructive interference

Cancelling waves

Scattering

Wave deviation from trajectory

with resultant wave phase difference

Coherent

Constant phase difference

Not necessary to be in phase

IncoherentMultiple phases combine and cancel

Illustration of phase shift. The horizontal axis represents an angle (phase) that is increasing with time.

Wikipedia

Waves combine (constructive, coherent)

Waves combine 180° out of phase (destructive, coherent)

=

=

=

Waves that combine with varying phases nearly cancel out (incoherent addition)

www.scribd.com

/doc/27753743/Coherence-Incoherence-And-Light-ScatteringSlide8

Diffraction

Waves interfere with the initial wave front

Appear to have a series of bright parallel bands or fringes

Fresnel Fringes

freh-nell

Resolution is degraded

Edges fuzzy rather than distinctSlide9

Airy Discs

The airy discs are the ringed patterns of Fresnel fringes

When they overlap more difficult to discern two points as independent and thus resolution is poorer

Airy disc radius is the measurement of

resolution

Point Spread Function

Figure: Bizzola Electron Microscopy 1999

http://greenfluorescentblog.files.wordpress.com/Slide10

Some Math

The math behind resolution (radius of airy disc)

λ=

wavelength, n= refractive index (what medium the wave is passing through glass etc.), α= aperture angle of lens

0.612λ

r = ______________

n (sinα

)Slide11

Resolving Power

Light microscope

r = 172 nm

Electron Microscope

r = .003 nm theoretical

r = .27 nm point to point in JEOL 2100 scopeWhy?Slide12

The Holy Trinity

Resolution, Magnification, and Contrast

Tradeoffs

None can be fully actualizedSlide13

The Holy Trinity

Resolution

The ability to distinguish two closely placed entities that otherwise might appear as one

Adobe.com

Resolution, Magnification, and ContrastSlide14

The Holy Trinity

Magnification

T

he measure of the increase in diameter of a structure from it’s original size

Resolution, Magnification, and ContrastSlide15

Holy Trinity

Contrast

The ability to distinguish differences in intensity values between bright and dark areas.

Resolution, Magnification, and ContrastSlide16

Contrast

Two types in electron microscopy

Amplitude contrast (scattering contrast)

Subtractive effect where various shades are evident by loss of electrons

Main source of most electron microscope contrast (except cryo)

Phase Contrast (interference contrast)

Interference of diffracted waves cause intensity differences due to loss of energy and the corresponding shorter focal pointsAppear as bright ring or halo around the edge of an object

Fresnel

‘freh-nell

’ fringeSlide17

Lenses and Magnification

Double convex converging lens

Same optical properties of light microscopes and electron microscopes

Refraction

www.passmyexams.co.uk

Image formation in a lens

Same optical properties of light microscopes and electron microscopes

Bizzola Electron Microscopy 1999Slide18

Electromagnetic Lenses

Electrons move in helical pattern

Very influenced by magnetic fields

Mass is small and require “mean free path”- high vacuum

Bizzola Electron Microscopy 1999Slide19

Resolution Limiting Phenomena

Electromagnetic lens

d

efects

Spherical aberration

Chromatic aberrationAstigmatism

Beam coherenceSource of electron beam

WikipediaSlide20

Spherical Aberration

Due to geometry of electromagnetic lenses such that rays passing through the periphery of the lens are refracted more that rays passing along the axis

Circle of minimum confusion

Corrected in EM with apertures

to eliminate some of the peripheral rays

but results in decrease aperture angle and

therefore resolution

This is C

s

programs for image processing

2.0 mm in 2100, constant

http://electron6.phys.utk.edu/

Bizzola Electron Microscopy 1999Slide21

Chromatic Aberration

Distortion in lens in which there is a failure to focus different wavelength rays to converge on same point.

In light it’s the different color wavelengths

In electrons shorter wavelength electrons are more energetic and have a longer focal length than longer wavelength electrons.

Results in enlargement of focal point similar to Airy disc

Minimized by ensuring stable voltage of source

Good vacuumThinner specimens

Electrons transmitted through specimen will

change their energy and wavelengthSlide22

Astigmatism

Radial blur results when a lens field is not symmetrical in strength but stronger in one plane and weaker in another

Only part of image will be in focus at a given time

Point would appear elliptical rather than spherical

Corrected

by

Properly centered aperturesStigmators of condenser and objective lens

Nature Protocols 3, - 977 - 990 (2008)Slide23

Magnification in the Transmission Electron Microscope

Three magnify lenses in the electron microscope

Objective

Intermediate

Projector

image distance

Mag

=

___________________

object distance

Magnification is product of the individual magnifying powers of each lens M

T = MO

x MI x MP Light microscope 1,000x

EM 1,000,000x

Useful

magnification = resolution of eye (CCD) / resolution of lens systemSlide24

The TEM…To Be Continued

Bizzola Electron Microscopy 1999Slide25

Susan Hafenstein

Pixel sizeSlide26

Calculating your Pixel size

Knowing the size of each pixel in the digital image

Used to produce a magnification bar and Measure objects

For 3D cryoEM is is needed when determining the CTF and calculating the reconstructionSlide27

Information is imbedded in the ccd

(in DM3 format – accessible by Digital Micrograph program)

OR

available in posted table on “Microscope magnifications and pixel sizes”

OR

You can calculate from the known magnification used to record the imageSlide28

Film

There are 25,400 microns/inch.

25,400 microns/inch divided by dpi = scan step size

The Nikon Super Coolscan 8000ED scans at 4000dpi

25,400 microns/inch divided by 8000 = 6.35 micron

6.35 microns = 63,500 angstrom

Divide 63,500 by the Magnification of microscope to get the pixel size

63,500

---------- = 1.08 Angstrom / pixel

59,000Slide29

Calculation of Pixel Size From a CCD Image

You have to know the actual pixel size of the CCD cameras and the magnification of your image.

As an example: 15 microns = 150,000 Å..

camera pixel size (in Å)

------------------------------

= the pixel size at the specimen magnification

level Slide30

How big a box?

Diameter

Of object

20 %

Box size

(don

’

t forget to ‘feather’)

Note: Or + 50%, or X2, or X3Slide31

Example:

Picornavirus = 300Å

+ 60 Å

box = 360Å

if your pixel size is 2.14Å/pixel

you should select 168 pixel diameter for your box sizeSlide32

Workshop:Same data --- different programs

Same program --- different data

Beginners + intermediate + experienced