Fig. 11. Measuring aqueous flow with fluorophotometry: an example. Fluorescein is introduced into the eye using topical instillations of the dye on the evening before testing. By the next morning, a uniform corneal stromal distribution of fluorescein exists, which is released almost exclusively through the endothelium into the anterior chamber. Fluorescein-stained aqueous humor is mixed with unstained newly formed aqueous humor and removed from the anterior chamber by the usual channels of outflow. Although many factors must be considered and controlled, the essential measurements are corneal stromal concentration (using a small sample area carefully separated from the aqueous humor, as shown) and aqueous humor concentration (measured by moving the sampling area into the anterior chamber while avoiding the region of newly formed aqueous humor at the pupil). Brubaker has provided typical data from a normal subject. (Brubaker RF: Clinical evaluation of the circulation of aqueous humor" in Duanes.) Measurements of fluorescein concentration in the right eye at 0800 hours were cornea, 806 ng/mL; anterior chamber, 94.3 ng/mL. An hour later the readings were cornea, 598 ng/mL; anterior chamber, 79.5 ng/mL. To determine the mass of fluorescein in each compartment, the volume of each compartment is determined using measurements of corneal thickness (in this case, 0.49 mm), corneal diameter (13.5 mm), and anterior chamber depth (2.8 mm). The volume of the cornea is estimated using the formula for the volume of a cylinder: In this case: The volume of the anterior chamber is estimated using the formula for a segment of a sphere: where h = axial depth of the anterior chamber and y = anterior chamber diameter. In this case: The total mass of fluorescein in the cornea and the anterior segment can then be calculated: The substraction of 1.5 ng/ml corrects for corneal autofluorescence in this patient. So, at 0800 hrs, By similar means, the mass at 0900 hours is calculated to be 60.6 ng, so the loss of fluorescein over the 1-hour period = 18 ng. As a first approximation, it is assumed that this 18 ng/60 minutes loss occurs completely from aqueous flow. Calculating average aqueous concentration during the hour: So, 1000 μL of aqueous humor “carries” 86.9 ng of fluorescein. We can then derive the flow rate necessary to remove this mass of fluorescein: In fact, because a small amount (5% to 10%) of the fluorescein leaves the anterior segment by diffusion into the iris, the true aqueous flow rate is correspondingly less. In practice, the test is more complicated than this example suggests. For one thing, the readings are usually repeated over several hours, and the necessary measurements and calculations are often more complex than this example. More detailed discussions are available.