Transmission electron microscopy is useful for studying subcellular compartment storage disorders at high resolution. It uses electrons rather than light to image structures, allowing visualization of organelles and other structures as small as 2 angstroms. Immunoelectron microscopy can identify the intracellular location of specific proteins. Studies of lysosomal storage disorders show accumulation of materials within lysosomes by electron microscopy. Mitochondrial disorders also show abnormal mitochondria by electron microscopy. New techniques like immunoelectron microscopy, 3D reconstruction, and nanotechnology may provide further insights into subcellular storage disorders.

1. "Transmission electron microscopy of subcellular
compartment storage disorders"
Dr. Consolato Sergi
Department of Laboratory Medicine and Pathology
University of Alberta, Edmonton, Canada

2. Outline
 Principles of transmission electron microscopy.
 Cell as subcompartments.
 Electron microscopy and light microscopy for storage
disorders.
 Immunoelectronmicroscopy, 3D reconstruction, and scanning
electron microscopy as well as new techniques
(nanotechnology?) for subcellular compartment storage disorders

3. Electron Microscopy at Glance
 It is a scientific instrument that use a beam of highly energetic
electrons to examine objects on a very fine scale.
 Wavelength of electron beam is much shorter than light,
resulting in much higher resolution.
 Two different types of EMs are:
 Transmission Electron Microscope (TEM): TEM allows one
the study of the inner structure and contours of objects
(tissues, cells, virusses)
 Scanning electron Microscope (SEM): SEM is applied to
visualize the surface of tissues, macromolecular aggregates
and materials.

4. Transmission Electron Microscope (TEM)
 Electrons scatter when they pass through thin sections of a
specimen.
 Transmitted electrons (those that do not scatter) are used to
produce image.
 Denser regions in specimen, scatter more electrons and appear
darker.

5. Transmission Electron Microscope (TEM)
Gun emits electrons
Electric field accelerates
Magnetic (and electric) field control
path of electrons
Electron wavelength @ 200KeV:
2x10-12 m
Resolution normally achievable @
200KeV: 2 x 10-10 m 2Å

6. Electron Microscope vs. Light Microscope
Electron Microscope
 High resolution, higher
magnification (up to 2 million times).
View the 3D external shape of an
object (SEM).
 2 different types of electron
microscopes: scanning electron
microscopes (SEM) and transmission
electron microscopes (TEM).
Light Microscope
 Useful magnification (only up to
1000-2000 times).
 3D external shape is not visible
by optical microscopy.
 2 types of microscopes: are
compound microscopes and stereo
microscopes (dissecting
microscopes).

7. 1- A stream of electrons is formed (by the electron source) and
accelerated toward the specimen using a positive electrical
potential.
2-This stream is confined and focused using metal apertures and
magnetic lenses into a thin, focused, momochromatic beam.
3-This beam is focused onto the sample using a magnetic lens.
4-Interaction occur inside the irradiated sample, affecting the
electron beam.
5-These interactions and effects are detected and transformed into
an image.
How Does Electron Microscope Work ?

8. http://middletownhighschool.wikispaces.com/Lysosomes+and+Peroxisomes
Lysosomes
 Cytoplasmic vacuole filled with
hydrolytic enzyme
 Heterophagy: Lysosomal digestion of
ingested material contatined in phagocytic
vacuoles.
 Autophagy: lysomal digestion of cell’s
own compenents
http://middletownhighschool.wikispaces.com/Lysosomes+and+Peroxisomes

9. Lysosome Diseases
Lysosomal storage disorders (LSD):
 Enzyme deficiencies that result in accumulation of
metabolites.
 > 50 different diseases, each characterized by
accumulation of specific substrate.
 Majority of LSD are AR inherited, with 3 exceptions: x-
linked disorders Fabry disease, Hunter syndrome
(mucopolysaccharidosis) and Danon disease.
 Incidence: 1 in in 1500 to 7000 live birth.

10. Lysosomal storage disorders (LSD) cont’
 Tay-Sachs disease:
 Neurological genetic disorder that effects lipid storage.
 Due to a mutation in the HEX A gene found on
chromosome 15, which plays a role to catalyze the
breakdown of gangliosides (GM2).

11. Lysosomal storage disorders (LSD) cont’
Sialidosis:
Sialidosis (NEU1, 6p21.3) is a severe
inherited disorder that affects many
organs and tissues.
 This disorder is divided into two types,
which are distinguished by the age at
which symptoms appear and the severity
of signs and symptoms.
o Type I (cherry-red spot myclonus
syndrome)
o Type II (mucolipidosis I).

13. Spleen, Immunohistochemistry (anti-CD68)
l placenta showing a foam cell alteration (arrow) of the cytoplasm of the
trophoblastic cells overlying the villous Stroma, m placental trophoblast with
an intense staining reaction (dark) for acid phosphatase, n faint staining in an
age-related placenta control

14. Transmission electron microscopy of the placenta showing
storage of membrane bound material in the placental trophoblast
(arrow) overlying the villous stroma (asterisk; ×1100)

15. Alpha-1 antitrypsin (AAT)
deficiency (SERPINA 1, 14q32.1) is
a clinico-pathologic condition in
which the body does not make
enough of a protein, called protease
inhibitor that protects the lungs and
liver from damage, such as
pulmonary emphysema and liver
cirrhosis.
AAT

16. Liver, Hematoxylin-Eosin staining

17. Liver, Transmission EM
Liver, Transmission EM

18.  An intracellular organelle.
 There are 100 to 1000s of mitochondria/cell.
 All mitochondria come from the mother.
 Mitochondria have their own DNA.
 Found in all cell types, except the RBC.
 Major functions of mitochondria:
 Makes energy in the form of ATP.
 Programmed cell death (apoptosis).
Mitochondria

19. Organ Systems Possible Problems
Brain
developmental delays, mental retardation, dementia, seizures, neuro-
psychiatric disturbances, atypical cerebral palsy, migraines,
strokes
Nerves
weakness (which may be intermittent), neuropathic pain, absent
reflexes, dysautonomia, gastrointestinal problems (ge reflux,
dysmotility, diarrhea, irritable bowel syndrome, constipation,
pseudo-obstruction), fainting, absent or excessive sweating
resulting in temperature regulation problems
Muscles weakness, hypotonia, cramping, muscle pain
Kidneys
renal tubular acidosis or wasting resulting in loss of protein,
magnesium, phosphorous, calcium and other electrolytes.
Heart cardiac conduction defects (heart blocks), cardiomyopathy
Liver hypoglycemia (low blood sugar), liver failure
Eyes visual loss and blindness
Ears hearing loss and deafness
Pancreas and Other
Glands
diabetes and exocrine pancreatic failure (inability to make digestive
enzymes), parathyroid failure (low calcium)
Systemic
failure to gain weight, short stature, fatigue, respiratory problems
including intermittent air hunger, vomitting
Problems That May Be Associated with Mitochondrial Cytopathies

21. 1a-d: In mitochondrial myopathies the ultrastructural features may
range from large, subsarcolemmal accumulations of mitochondria
to myofibers containing disordered myofibrils, degenerating
mitochondria and vacuoles

22. Immunoelectron-microscopic and storage disease
You can say immunoelectron microscopy uses antibodies to detect
the intracellular location of structures of particular proteins at high
resolution by electron microscope

23. Detection by immunoelectron microscopy of GL-3 in cutaneous cell
components of a patient with Fabry disease. (A) Positive signals are shown in
the lysosome of pericytes. The limiting membrane of the lysosome is clearly
demonstrated in the sample processed without deosminization. Arrowheads
indicate the limiting membrane of the lysosome (B–F) Cytoplasmic lysosomal
deposits are strongly labelled with 10-nm streptavidin–colloidal gold conjugate
in endothelial cells

24. Nanotechnology and storage disease

25. Thank you