Beyond Microspheres -- New Applications For The Imaging Cryomicrotome

Clyde H. Barlow, Jon Ewen, Diane Lee, Stacey Binnie, Katherine Barlow,
Torleiv Flatebo-Ringer, Jesse Thompson and Jeffrey J. Kelly

Barlow Scientific, Inc., Olympia, WA  98502   USA

C. H. Barlow, J. Ewen, D. Lee, S. Binnie, K. Barlow, T. Flatebo-Ringer, J. Thompson and J.J. Kelly  "Beyond microspheres -- new applications for the imaging cryomicrotome".  Fourth International Conference on Fluorescent Microsphere Methods.  Academical Medical Center, Division of Medical Physics, Amsterdam, the Netherlands.  October, 1999.  Digital fluorescence imaging coupled with serial sectioning of frozen tissues produces maps of the three dimensional location of fluorescent microspheres used to evaluate regional blood flow. The technology to obtain regional flow maps in this manner has many applications for morphometric analysis of tissues and regional flow measurements using indicators other than microspheres.
Microsphere methods provide an elegant method for recording the history of regional flow at selected times. Microsphere methods also have drawbacks. Flow measurements in a tissue region require approximately 400 microspheres for a 95% confidence level and the use of large numbers of microspheres to measure flows in small volumes can alter regional flow by diffuse ischemia. To overcome these problems we are developing a "molecular microsphere" method using fluorescent dyes that stain tissue in proportion to flow. Dye fluorescence is correlated with simultaneously injected fluorescent microsphere deposition by serial imaging with the imaging cryomicrotome.
Morphometry of vascular trees can also be determined. Fluorescently labeled Batson's #17 casting medium (Polysciences, Inc.) is infused into the aortic root of rat hearts. After the polymer cures, hearts are frozen and analyzed in the imaging cryomicrotome in the same manner as for fluorescent microsphere analysis. The coronary artery tree is filled to a level that is dependent on the viscosity of the filling medium. Arterial trees are currently being imaged to the 10-20 micrometer diameter level. Combining vascular tree mapping with fluorescent microsphere based regional flow imaging should allow distributions of vascular networks and regional flow to be measured in the same organ.

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Last modified: 01/08/2000