1. Study of Tissue Morphology – Electron Microscopy
MMedSc Core Module
PATH6100
Laboratory Methods & Instrumentation

2. Learning Objectives
To understand the working principles of electron microscopes
Common applications of ultrastructural examination of tissue morphology in medical science

3. Introduction to Electron Microscopy
Ultrastructural study = Electron microscopy
Two types of EM: Transmission (TEM) and Scanning (SEM)
Resolution of TEM : best at about 0.2 nm (nanometer = 10^-9 m), which is about 1000x better than ordinary light microscope
TEM is far more useful for medical investigations than SEM

4. Merits of EM high magnification at high resolution
technique largely standardized
some ultrastructural features are highly specific for certain cell types or diseases

5. Drawbacks of EM equipment expensive
procedures time consuming (staff costly)
small samples lead to possible sampling error and misinterpretation
optimum tissue preservation requires special fixative and processing
much experience is needed for interpreting the results

6. Joel 100SX
(TEM)

8. Principles of TEM
Illumination - Source is a beam of high velocity electrons accelerated under vacuum, focused by condenser lens (electromagnetic bending of electron beam) onto specimen.
Image formation - Loss and scattering of electrons by individual parts of the specimen. Emergent electron beam is focused by objective lens. Final image forms on a fluorescent screen for viewing.

9. Specimen-Beam Interaction
Incident electron beam
Auger electron
Backscattered electrons
X-ray
Secondary electrons
Light
Specimen
Absorbed electrons
Elastically scattered electrons
Elastically scattered electrons
Unscattered electrons

10. Principles of TEM
Image capture – on negative or by digital camera

11. Fixation of tissues for EM
Must be prompt
Cut to 1-2 mm cubes
Use sharp razor blade, avoid crushing
2.5% glutaraldehyde for 4 to 12 hours
Postfixation in 1% osmium tetroxide

12. Tissue preparation for TEM
Dehydration in alcohol
Embedding in resin
Semithin sections cut at 0.5 micron thick, stained with toluidine blue
Selection of sample blocks
Ultrathin sections at 0.1 micron thick, stained with lead citrate and uranium acetate

13. Semithin sections
stained with toluidine blue

14. Ultrathin
sections
on grid

15. Operation of TEM Tedious operation Time consuming Works in the dark
Photography required

16. Practical Hints
Take photos at the appropriate magnifications depending on target of interest). e.g.
3,000x for immune deposits in glomeruli,
8,000x or higher for measuring glomerular basement membrane thickness (350nm)
100,000x for amyloid fibers (10 nm)

17. Practical Hints Use negative films (?)
Take photos at LOWER magnifications
Fluorescent screen offers inferior image and there is a tendency to examine at higher magnifications
When prints are made, negative images can be easily enlarged several times with good results

18. Major Use of TEM in medicine
Renal diseases
Typing of tumors
Muscle diseases
Skin diseases
Miscellaneous diseases

19. HKU EM lab. in QMH (86-95)

20. EM for renal diseases
Is the most frequent application of EM in diagnostic pathology
A review of cases handled by EM laboratory in a recent 10 year period showed that 56% were renal tissues

21. EM for renal diseases confirmation of immune complex deposition
precise location of immune deposits
detection of other deposits: light chain deposition, cryoglobulin, amyloidosis, non-amyloid fibrils
structural changes in glomerular basement membrane: thinning, thickening, splitting, collagen fibrils …
inclusion bodies in cytoplasm

22. Examples of differential diagnosis of tumors
carcinoma, melanoma, sarcoma
adenocarcinoma, mesothelioma
thymoma, thymic carcinoid, lymphoma, seminoma
small round cell tumors: Ewing’s sarcoma, embryonal rhabdomyosarcoma, lymphoma, neuroectodermal tumors
spindle cell soft tissue tumors
endocrine and non-endocrine tumors

23. Major Use of TEM in medicine
Renal diseases
Muscle diseases
Typing of tumors
Skin diseases
Miscellaneous diseases

24. More applications Immuno-electron microscopy
Detection of viral particles – in feces, tissue fluids and tissues

25. The Scanning Electron Microscope
Image
Scan
generator
Waveform
monitor
Signal
amplifier
To vacuum pump

26. Leica S360

27. Contrast Formation by Geometrical Configuration
Incident Electron Beam
Incident electron beam
Contrast

28. Scanning EM
Seldom used for diagnostic purposes
Useful for research

29. X-ray microanalysis
For detection of elements – usually on crystalline structures
When high velocity electrons strike an atom, X-ray of characteristic wave lengths specific to the atomic number of the atom is emitted

30. X-Ray Emisssion Excitation Relaxation X-ray photon Incident electron
Ejected
orbital
electron
Excitation
Relaxation

31. X-ray microanalysis

32. EM for typing of tumors
Most useful when employed in conjunction with other investigations: light microscopy, immunohistochemistry and molecular techniques
To resolve a definite differential diagnosis between a few entities formulated at the light microscopic level