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 Incident electron beam Auger electron Backscattered electrons X-ray Secondary electrons Light Specimen Absorbed electrons Elastically scattered electrons Elastically scattered electrons Unscattered 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)

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 Image Scan generator Waveform monitor Signal amplifier To vacuum pump

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 Excitation Relaxation X-ray photon Incident electron Ejected orbital electron Excitation Relaxation

X-ray microanalysis

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