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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
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
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
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