Goals of Outline

• Overview of Fluorescent Microsphere Method
• Recent advances
• Other applications
  • Histology - microcirculation
  • Aerosols - regional ventilation
  • Cryomicrotome - spatial distributions
• Fluorescent Microsphere Resource Center

Brief tutorial on fluorescence

• Fluorescence occurs when a molecule absorbs energy in the form of light and then immediately releases this energy again in the form of light.
• Excitation wavelength - characteristic wavelength that molecule absorbs
• Emission wavelength - characteristic wavelength that molecule emits
• Stokes shift - difference between excitation and emission wavelength
• Quantum efficiency - ratio of emitted energy to absorbed energy
• Compounds can be separated on the basis of either their excitation or their emission spectra.

• Intensity of the emitted light, F, is described by the relationship

  F = ØI0(1-e-§bc)

  [1]

  where Ø is the quantum efficiency, I₀ is the incident radiant power, § is the molar absorptivity, b is the path length of the cell, and c is the molar concentration of the florescent dye . For dilute concentrations (§bc < 0.05), equation [1] reduces to the form:

  F = kØI0§bc

  [2]

  If Ø, I₀, § and b remain constant, the relationship between the fluorescent signal and dye concentration should be linear for dilute dye concentrations.

• sensitivities are equal
• fluorescent molecules are not hazardous
  • no disposal costs
• fluorescent molecules do not decay

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

History and evolution
1963 - All radioactive work to present
1981 - Gottlieb et al, Fluorescent microspheres
• Identified ischemic myocardium
1991 - Kowalik et al, Colored microspheres
• Absorption spectroscopy
1993 - Glenny et al, Fluorescent microspheres
• Emission spectroscopy
Physical properties of fluorescent microspheres
• Fluospheres™ (Molecular Probes and Triton Technologies)
  • Polystyrene
  • Diameter = 14.7 µm (scanning EM)
  • Diameter variability (CV) = 2.1% (Coulter)
  • Density = 1.02 g/ml (gradient centrifugation)
  • Fluorescence variability = 3.4 - 5.0% (FACS)
• 8 commercially available colors

Fluorescent Spectra

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

• Correlation between fluorescent and radiolabeled determined flow = 0.99

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

• physiology studies in field
• labs that are not cleared for radioactivity
• countries that do not allow radioactivity

Sources of error

• Methodolgic noise greater than radioactive microspheres
  • solvent volumes
  • filtering methods
  • signal variability (CV = 3%)
• Time requirements greater than radioactive microspheres
  • sample handling

Future Applications of Fluorescent Microspheres

• Histology
  • Fluorescent microspheres are easily visualized, allowing perfusion to be quantified at the microscopic level.
  • Provides ability to define source of blood when more than one circulation exists.
• Cryomicrotome
  • Currently in developmental stage
  • Automatically determines the spatial location of fluorescent microspheres in small organs
    • rats and rabbit lungs
    • canine hearts
  • Produces text file with X, Y, and Z locations of each microsphere
  • Able to differentiate four different colors

Aerosolized microspheres to measure regional ventilation
• 1 µm diameter fluorescent microspheres.
• Produced dry aerosol, administered to mechanically ventilated pigs.
• Pulmonary perfusion quantified using 15 µm diameter microspheres via central vein.
• Currently in press - H. T. Robertson et. al. [tomrobt@u.washington.edu]

Chronic studies
• initial studies suggest that radiolabels leach off of radioactive microspheres but fluorescent microspheres can be measured months after injection

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.