Interpretation of SEM images-iolo-printout images.pdfRomanesque Brocolli/Cauliflower (or Romanesco)...

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1 “One Sees Only What One Knows” SEM Image Interpretation Iolo ap Gwynn The University of Wales Bioimaging Laboratory Aberystwyth Wales [email protected] RMS/ESB Workshop Sorrento 2005 SEM Imaging • Information required ? – Signal types – Resolution ? • Specimen preparation – Preservation ? – Dehydration ? – Coating • Microscope settings • Interpretation – Analysis Potential SEM Information • If used incorrectly – Very little – Waste of time and effort • If used to its potential – Much – Dependent upon • Specimen preparation • Imaging conditions • Interpretation and analysis Quotation “Man sieht nur was man weiss” (One sees only what one knows) Goethe What is this? Romanesque Brocolli/Cauliflower (or Romanesco) Purpose of SEM Studies Reveal topographical surface detail SE – low voltage BSE – all voltages Detect sub-surface information BSE – optimise voltage Detect compositional differences BSE – Atomic number contrast XRA – element specific

Transcript of Interpretation of SEM images-iolo-printout images.pdfRomanesque Brocolli/Cauliflower (or Romanesco)...

Page 1: Interpretation of SEM images-iolo-printout images.pdfRomanesque Brocolli/Cauliflower (or Romanesco) Purpose of SEM Studies ... • Research background ... SEM Workshop ap Gwynn & Richards

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“One Sees Only What One Knows”SEM Image Interpretation

Iolo ap GwynnThe University of Wales Bioimaging Laboratory

AberystwythWales

[email protected]

RMS/ESB Workshop Sorrento 2005

SEM Imaging• Information required ?

– Signal types– Resolution ?

• Specimen preparation– Preservation ?– Dehydration ?– Coating

• Microscope settings• Interpretation

– Analysis

Potential SEM Information• If used incorrectly

– Very little– Waste of time and effort

• If used to its potential– Much– Dependent upon

• Specimen preparation• Imaging conditions

• Interpretation and analysis

Quotation

“Man sieht nur was man weiss”(One sees only what one knows)

Goethe

What is this?

Romanesque Brocolli/Cauliflower (or Romanesco)

Purpose of SEM Studies

Reveal topographical surface detailSE – low voltageBSE – all voltages

Detect sub-surface informationBSE – optimise voltage

Detect compositional differencesBSE – Atomic number contrastXRA – element specific

Page 2: Interpretation of SEM images-iolo-printout images.pdfRomanesque Brocolli/Cauliflower (or Romanesco) Purpose of SEM Studies ... • Research background ... SEM Workshop ap Gwynn & Richards

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Logical Analysis of Micrographs

CONSOLIDATED VIEW

MICROGRAPH

INTERPRETATION

A W Robards 1984

INFORMATION RETRIEVAL

Structural

CytochemicalNumerical

Instrumental effects Interactions Specimen preparation

Correlative information Supplementary considerations

Reasons for Lack of Positive Contrast e.g. BSE imaging

• Microscope settings?• Fixing/staining agent(s) has not

penetrated• Fixing/staining agent(s) has not reacted• Coating unsuitable• … etc.

Which is the correct image ?

Calcified/soft Cartilage Interface

Secondary Electron Images

Backscattered Electron Images

10 µm

8 kV 16 kV

0.5kV 1 kV 2 kV

4 kV

SE-upper detectorCalcified articular cartilage

4nm Pt/Pd

Surface of Glass Fibre Different Signal Comparison

1 kV SE4nm Pt/Pd

3 kV BSE4nm Pt/Pd

30 kV BSE4nm Pt/Pd + 60nm C

Page 3: Interpretation of SEM images-iolo-printout images.pdfRomanesque Brocolli/Cauliflower (or Romanesco) Purpose of SEM Studies ... • Research background ... SEM Workshop ap Gwynn & Richards

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Cultured fibroblastOsmium stained

BSE images

Images courtesy of R.G.Richards

8kV

15kV

BSE

Images courtesy of R.G.Richards

Image AnalysisQuantitative dataGrey level values (256 / pixel) Measurements

length, breadth, areas etc Counting

labels

Basic Principles of Image Analysis

Areas of interest definedexpressed as binary (0 & 1) image

Measurements on binary image.

The result of ‘thresholding’

The result of ‘processing’

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SomeMeasurements

Possible

1. Count the No of objects2. Area3. Length4. Breadth5. Perimeter

Others:6. Equivalent circle7. Ferets8. … and more

PC_Image

SelectMeasurements

Articular cartilagefracture propagation

= Undulation of matrix

3D Rendering by software(Alicona: Mex)

Combining Microscopies

Images courtesy of DO Meredith, AO

Take Home Messages

• Do microscopy – Carefully– Not a means to ‘decorate’ publications

• Research background– Carefully (broad sources)

• Ask a microscopist!– Before you start (potential information)– To interpret results

SEM Workshopap Gwynn & Richards 2005

ESB Sorrento


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