Robb Glenny, M.D.
Fluorescent Microsphere Resource Center
December 18, 1996

Fluorescent Microsphere Methods


Goals of Outline

Brief tutorial on fluorescence

Basic Fluorimetry

Fluorescence vs. radioactivity

Advantages of fluorescence vs. absorption spectroscopy

  1. two vs. one wavelength used to isolate color
  2. emitted light read at right angles to the exciting light, minimizing background signal
  3. fluorescent methods have a greater range of linearity

    Because of these differences, the sensitivity of fluorescence is approximately 1000 times greater than absorption spectrophotometric methods.

Disadvantages of fluorescence

  1. sensitivity of the signal to pH
  2. sensitivity of the signal to temperature

Fluorescent Microspheres

Fluorescent Dyes

Fluorescent Spectra

Fluorescent Spectra

Validation vs. Radiolabeled Microspheres (Glenny, Bernard, et al. 1993)

Fluorescent Microsphere Methods 1
Fluorescent Microsphere Methods 2

Correlation between fluorescent and radiolabeled

Greatest advantage of fluorescent microspheres is that they can be used in studies where radioactivity is not permitted.

Sources of error

Many of the same potential errors as radiolabeled microspheres with some additional ones.
Potential ErrorSolution
Signal quenchingdilute sample, less spheres
Loss of microspheres when isolatingmeticulous technique, centrifugation filters, single tube processing
Low signal:noise ratioincrease number of microspheres injected or sample size
Inaccurate solvent volumesaccurate pipettes, larger volumes, robotics
Dye stabilitycheck in solvent
Background signalchoose appropriate colors

Future Applications of Fluorescent Microspheres


Aerosolized microspheres results


Glenny, R. W., S. Bernard, and M. Brinkley. (1993). "Validation of fluorescent-labled microspheres for measurement of regional organ perfusion." J. Appl. Physiol. 74(5): 2585-2597.

Last modified: 2010-02-04 17:50:31 PST

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