Microscopical images cannot be interpreted intuitively on the basis of previous nonmicroscopical

Transcription

1 2 3 Limitations and Potential of the Scanning Microscope (SEM) Iolo ap Gwynn The University of Wales Bioimaging Laboratory Aberystwyth Wales iag@aber.ac.uk RMS/ESB Workshop Sorrento 2005 Microscopical images cannot be interpreted intuitively on the basis of previous nonmicroscopical experience A W Robards Past President Royal Microscopical Society Physical Resolution Limits Unaided eye ~ 0.1mm Light microscope ~ 0.2µm Scanning EM ~ 1nm Transmission EM ~ 0.1nm Object Sizes? Small organisms < 1mm Cells < 20µm Mitochondria: ~ 1µm Membranes ~ 10nm Microfilaments ~ 4-7nm Microtubules ~ 24nm Bacteria < 1µm Viruses < 100nm Macromolecules < 10nm Atoms < 1nm SEM 4 gun Condenser lens(es) Objective aperture Scan coils Objective lens(es) Signal Specimen Scanning Microscope 5 Detector Scan Generator Scanning Microscope Amplifier CRT or computer 6 1

2 Magnification 7 Traditional View of SEM 8 Coating: Pt/Pd + C 9 Ls Specimen raster nl* *Number of lines (nl) same for both scans Imaging raster Eye of Housefly 10kV Magnificat ion = Li Ls nl* 100nm Li Pollen Grains Bacteria & Phage SEM Imaging Information required? Signal types Resolution? Specimen preparation Preservation? Dehydration? Coating Microscope settings Interpretation Analysis 10 SEM Imaging Information required? Signal types Resolution? Specimen preparation Preservation? Dehydration? Coating Microscope settings Interpretation Analysis 11 - specimen interaction Rutherford scattering Elastic scattering Backscattered electron M L K N Characteristic X rays Secondary s :few ev :beam electron Inelastic scattering 12 2

3 Light Signals from specimen X-rays probe Secondary electrons (SE) Backscattered electrons (BSE) Auger electrons 13 Bulk samples High density beam beam Up to 10 µm Typical 5-10µm Reactive Volume (R) low Z Emissions Auger electrons Secondary electrons Backscattered electrons Characteristic X-rays 15 Absorbed electrons Low density Continuum X-rays Transmitted electrons Reactive volume (R) of electron-specimen interaction Fluorescent X-rays Purpose of SEM Studies 16 Calcified/soft Cartilage Interface 17 Beam-Specimen Interaction Chamber E-T SE Detector 18 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 Secondary Images Which is the correct image? Backscattered Images SE 3 SE 2 BSE 2 SE 1 BSE 3 BSE 1 3

4 19 20 Effective Probe Diameter Any Signal SE Emission From a Surface Too small noisy image Too large poor definition Effective Probe Diameter Dependent upon: ¾Beam energy ¾Signal collected ¾Lens settings ¾Energy spread ¾Specimen density ¾Coating material ¾Coating thickness 21 Primary beam Secondary e- - few ev Backscattered e- - beam energy Effective probe diameter Coating (Au, Pd, Pt, Cr etc.) Optimal Several microns Energy dependent Specimen Raster lines 22 1 kv 0.5kV Calcified articular cartilage SE-upper detector 10 µm 2 kv (C,H,O etc.) 23 Surface of Glass Fibre 24 Different Signal Comparison AccVoltage effect Note working distance 4 kv 8 kv 16 kv 1 kv SE 3 kv BSE 30 kv BSE + 60nm C 4

5 Depth nm Round fibroblast - adhering filopodia 25 Maximum depth of BSE emission from LR White resin vs. Beam Energy 26 Cultured fibroblast Osmium stained 27 BSE images 8kV kev 15kV Images courtesy of Llinos Harris BSE 28 Immunogold imaging Vinculin in focal adhesions 29 TEM by SEM (BSE signal) 30 5

6 Variants on the SEM Theme Potential SEM Information Field Emission Scanning EM (FEGSEM) Schottky type (heated) Cold type highest resolution Cryo SEM Variable (Low) Pressure SEM Environmental SEM Environmental FEGSEM (Schottky gun) Probe Micro Analyser (X-rays) 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 SEM Workshop ap Gwynn & Richards 2005 ESB Sorrento 6