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SEM Imaging Potential SEM Information

Information required ?
Signal types If used incorrectly
One Sees Only What One Knows Resolution ? Very little
SEM Image Interpretation Specimen preparation Waste of time and effort
If used to its potential
Preservation ?
Iolo ap Gwynn
Much
The University of Wales Bioimaging Laboratory
Dehydration ? Dependent upon
Aberystwyth Coating Specimen preparation
Wales
iag@aber.ac.uk Microscope settings Imaging conditions

Interpretation Interpretation and analysis


Analysis
RMS/ESB Workshop Sorrento 2005

Quotation What is this? Purpose of SEM Studies

Reveal topographical surface detail


Man sieht nur was man weiss SE low voltage
(One sees only what one knows) BSE all voltages
Detect sub-surface information
Goethe BSE optimise voltage
Detect compositional differences
BSE Atomic number contrast
XRA element specific
Romanesque Brocolli/Cauliflower (or Romanesco)

1
Logical Analysis Calcified/soft Cartilage Interface
Reasons for Lack of Positive
of Micrographs Contrast e.g. BSE imaging
MICROGRAPH

INTERPRETATION
Microscope settings? Secondary Electron Images
Fixing/staining agent(s) has not
Instrumental effects Interactions Specimen preparation
penetrated
Which is the correct image ?
CONSOLIDATED VIEW
Fixing/staining agent(s) has not reacted
Correlative information Supplementary considerations
Coating unsuitable
INFORMATION RETRIEVAL
etc.
Structural Backscattered Electron Images
Numerical
Cytochemical
A W Robards 1984

0.5kV 1 kV 2 kV Surface of Glass Fibre Different Signal Comparison

Calcified articular cartilage 10 m SE-upper detector

4 kV 8 kV 16 kV

1 kV SE 3 kV BSE 30 kV BSE
4nm Pt/Pd 4nm Pt/Pd 4nm Pt/Pd
4nm Pt/Pd + 60nm C

2
BSE
Cultured fibroblast Image Analysis
Osmium stained
Quantitative data
BSE images Grey level values (256 / pixel)
Measurements
8kV length, breadth, areas etc
Counting
labels
15kV
Images courtesy of R.G.Richards
Images courtesy of R.G.Richards

Basic Principles of The result of The result of


Image Analysis thresholding processing

Areas of interest defined


expressed as binary (0 & 1) image
Measurements on binary image.

3
3D Rendering by software
Some 1. Count the No of objects
PC_Image (Alicona: Mex)
2. Area
Measurements 3. Length
Possible 4. Breadth
5. Perimeter
Select
Measurements
Others: Articular cartilage
fracture propagation
6. Equivalent circle = Undulation of matrix

7. Ferets
8. and more

Combining Microscopies Take Home Messages


Do microscopy SEM Workshop
Carefully ap Gwynn & Richards 2005
ESB Sorrento
Not a means to decorate publications
Research background
Carefully (broad sources)
Ask a microscopist!
Before you start (potential information)
To interpret results

Images courtesy of DO Meredith, AO