A Simplified Method For Counting Fluorescent Microspheres

Jon Ewen, Clyde H. Barlow, Katherine Kelly and Jeffrey J. Kelly

Barlow Scientific, Inc., Olympia, WA  98502   USA

J. Ewen, C. H. Barlow, K. Kelly and J.J. Kelly  "A simplified method for counting fluorescent microspheres".  Fourth International Conference on Fluorescent Microsphere Methods.  Academical Medical Center, Division of Medical Physics, Amsterdam, the Netherlands.  October, 1999.  Fluorescent microsphere methods provide an elegant method for recording the history of regional flow in tissues at selected times. One feature of the fluorescent microsphere method that limits its wide use is the labor intensive analysis procedure. Currently used methods, of which we are aware, require each volume element (sample) to undergo digestion, isolation, and extraction followed by fluorescence analysis to estimate the numbers of microspheres present. We are currently developing an analysis method that uses no reagents and that should have an analysis time of about a minute per sample.
To measure regional flow, standard methods are used to inject fluorescent microspheres and to dissect tissues into individual samples. Each resulting tissue sample is placed between two sheets of plastic film and is compressed into a thin layer using a pneumatic press. The thinned sample is placed on a motorized translation stage for imaging. Fluorescence images are obtained using a xenon arc or tungsten halogen lamp excitation, and Sony XC-77 CCD camera for image acquisition. Interference filters in separate filter wheels provide excitation and emission wavelengths allowing images of microspheres containing different fluorochromes to be collected separately. The area of the thinned samples cannot be recorded in a single image due to resolution limitations. Therefore, the sample is translated beneath the camera and multiple images merged for microsphere counting. In the thinned sample, 15-micrometer diameter fluorescent microspheres can be clearly imaged and counted, providing an accurate determination of numbers of microspheres in each sample.

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Last modified: 01/08/2000