Study of Tissue Morphology –
Electron Microscopy
MMedSc Core Module
PATH6100
Laboratory Methods & Instrumentation
Learning Objectives
• To understand the working principles of
electron microscopes
• Common applications of ultrastructural
examination of tissue morphology in
medical science
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
Merits of EM
• high magnification at high resolution
• technique largely standardized
• some ultrastructural features are highly
specific for certain cell types or diseases
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
Joel 100SX
(TEM)
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.
Specimen-Beam Interaction
Auger
electron
Incident
electron beam
Backscattered
electrons
X-ray
Secondary
electrons
Light
Specimen
Absorbed electrons
Elastically
scattered
electrons
Unscattered
electrons
Elastically
scattered
electrons
Principles of TEM
• Image capture – on negative or by digital
camera
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
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
Semithin sections
stained with toluidine blue
Ultrathin
sections
on grid
Operation of TEM
•
•
•
•
Tedious operation
Time consuming
Works in the dark
Photography required
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)
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
Major Use of TEM in medicine
•
•
•
•
•
Renal diseases
Typing of tumors
Muscle diseases
Skin diseases
Miscellaneous diseases
HKU EM lab. in QMH (86-95)
14%
7%
Kidney
23%
Tumours
Muscle
56%
Others
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
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
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
Major Use of TEM in medicine
•
•
•
•
•
Renal diseases
Muscle diseases
Typing of tumors
Skin diseases
Miscellaneous diseases
More applications
• Immuno-electron microscopy
• Detection of viral particles – in feces, tissue
fluids and tissues
The Scanning Electron Microscope
Scan
generator
Signal
amplifier
To vacuum pump
Image
Waveform
monitor
Leica S360
Contrast Formation by Geometrical Configuration
Incident
Electron
Beam
Incident electron
beam
Contrast
Scanning EM
• Seldom used for diagnostic purposes
• Useful for research
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
X-Ray Emisssion
Incident
electron
X-ray photon
Ejected
orbital
electron
Excitation
Relaxation
X-ray microanalysis