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+Peroxi somes 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

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 SystemsPossible 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 Musclesweakness, hypotonia, cramping, muscle pain Kidneys renal tubular acidosis or wasting resulting in loss of protein, magnesium, phosphorous, calcium and other electrolytes. Heartcardiac conduction defects (heart blocks), cardiomyopathy Liverhypoglycemia (low blood sugar), liver failure Eyesvisual loss and blindness Earshearing 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