Shriners Research Center, Portland OR

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William Horton | Hans Peter Bächinger | Lynn Sakai | Peter Hurlin | Scott Stadler | Ronen Schweitzer | Douglas Keene
6th Pan Pacific Connective Tissue Symposium | Skeletal Growth Workshop

Keene Laboratory

Douglas R. Keene


Assistant Investigator
Image Center Director

Portland Shriners Research Center
Instructor of Molecular & Medical Genetics
Oregon Health & Science University

"Ultrastructural Abnormalities of Extracellular Matrix Proteins"

The EM Facility uses a variety of techniques to visualize the molecular components of bone, cartilage and skin at high magnification using the electron microscope. Using specialized techniques of immunocytochemistry, this laboratory continues to be vital in identifying the specific location of newly discovered molecules. This knowledge allows us to hypothesize about their functional role in overall tissue architecture and function. When we examine a diseased tissue in which this newly acquainted architecture is disturbed, the molecule becomes suspect and is therefore a candidate for study and evaluation.

Several connective tissue diseases including epidermolysis bullosa (a blistering disease) have been attributed to a defect in a single molecule using this methodology. This facility is also at the forefront of using specialized techniques to optimally preserve tissue structure for examination at high magnification. Standard techniques, which have been in use for many years, cause artificial redistribution of many tissue components, significantly altering the appearance of a tissue when viewed using the electron microscope. Using ultra-rapid freezing technology we provoke far less damage, permitting us to see far greater tissue detail. These new techniques will allow us to precisely determine the location of many small molecules, which would otherwise be moved out of place. We expect that accuracy of our understanding of tissue micro-architecture will significantly improve as a direct result of these studies.

Image Description
Upper left: Transmission electron micrograph of cartilage chondrocyte and matrix prepared using high pressure freezing technology, which best preserves cell and matrix components. (x 40,000)

Upper right: Negative stain preparation of homogenized skin demonstrating freshly isolated collagen fibrils and also a fibrillin-containing beaded microfibril. (x 50,000)

Lower left: Rotary shadowed image of a type IV collagen tetramer with monoclonal antibody bound to two of the four arms. (x 150,000)

Lower right: Transmission electron micrograph of human amnion showing the convoluted basement membrane zone which borders the epithelium (top) and connective tissue matrix (bottom). (x 10,000)

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