A Fluorescence Lifetime Ocular Fluorometer

The eye is composed of several tissues and fluids that must remain clear so that a sharp image can be formed on the retina. In the front section of the eye, a clear fluid called “aqueous humor” fills the space between the cornea and iris and helps maintain the clarity of the cornea and lens. A volume of about three to four drops of aqueous humor fill this space, called the “anterior chamber.” Aqueous humor is not stagnant, but is constantly being mixed in the anterior chamber by convection and eye movements. It is secreted by specialized tissue behind the iris called the “ciliary body,” flows through the pupil into the anterior chamber, and exits near the outer rim of the iris. Aqueous humor is secreted fast enough that the entire volume in the anterior chamber is replaced once an hour. The flow of aqueous humor through the anterior chamber brings nutrients to the cornea and lens and carries away waste products, much like blood does in vascularized tissue. This helps maintain a healthy and clear cornea and lens. In glaucoma, the path that aqueous humor takes to leave the anterior chamber becomes partially obstructed and a greater pressure is necessary to push aqueous humor out. As a result, the pressure throughout the eye gradually increases. When the pressure gets too high, the optic nerve becomes pinched where it enters the eye and a gradual blindness follows as nerve fibers lose their capacity to carry information to the brain. Ophthalmologists have several means to control intraocular pressure and stop or slow loss of the optic nerve. Sometimes the eye can be treated surgically or with a laser to provide an easier path for aqueous humor to leave the eye. One of the simplest treatments in most patients is to prescribe eye drops that decrease the rate that aqueous humor is produced. Scientists continue to search for new medicines to reduce intraocular pressure with few side effects. To better understand which medicines will work best it is important to understand the basic principles of how the ciliary body produces aqueous humor. One important aspect of this is to understand how flow of aqueous humor changes as its components change. The measurement of biochemical components of aqueous humor without disturbing the eye is a challenging problem that many scientists have been studying over the past several years. A good deal of progress has been made in measuring changes in characteristics of light emitted by fluorescent dyes in response to the composition of the fluid in which they are dissolved. If these dyes are added to the aqueous humor, by putting them in eyedrops, then we can study the composition of the aqueous humor by measuring their fluorescence. An instrument that measures fluorescence in the eye is called an “ocular fluorophotometer.” We have designed and built two types of ocular fluorophotometers in our laboratory to study aqueous humor in eyes of normal human subject and animals. One is used to measure a dye called fluorescein. We can calculate the production rate of aqueous humor by measuring how fast fluorescein leaves the eye after it is given as eye drops. The other fluorophotometer measures several other types of dyes and is used to study the acidity (pH}, protein content, and other biochemical properties of the aqueous humor. In the pending grant, we propose to build a third unique ocular fluorophotometer to make another type of measurement. This instrument will be designed to measure the level of oxygen in the aqueous humor. Oxygen is necessary to keep all tissues healthy. It is not known how oxygen in the aqueous humor changes under different conditions. Does oxygen content change when drugs are applied? Does it change as flow rate changes? What effect does oxygenation of the aqueous humor have on flow? Answers to these questions are important to understanding new approaches to treating glaucoma. The new instrument will measure a fluorescent dye called pyrenebutyric acid (PBA). When a bright flash of ultraviolet light is directed toward PBA, fluorescent light is emitted for a very brief but measurable time, about 0.1 millionth of a second. The duration of this fluorescence, or “fluorescence lifetime, 11 depends on the molecules in the aqueous humor that surround the PBA molecules. Since the fluorescence lifetime is very sensitive to oxygen, the oxygen content of the aqueous humor can easily be determined if fluorescence lifetime is measured. Several instruments are commercially available to measure fluorescence lifetime of solutions in a test tube, but making such a measurement in a living eye is much more difficult and we must build our own instrument to accomplish this task. The entire project is expected to take 2 to 3 years. During the first year we plan to design, build, and test the portion of the instrument that contains the lenses that will concentrate light on the eye and gather fluorescence from the eye. At a later time, a light source and light detector to measure fluorescence lifetime will be added and the instrument will be tested. Fluorescence lifetime of PBA has not been measured in the eye, although a related technique has been used to measure oxygen in suspensions of living cells. Although building such an instrument will be a challenge, many advances in technology have taken place over the past five years, and an instrument for measuring oxygen in the anterior chamber by measuring fluorescence lifetime is well within our capability. With such a device, we will learn more about the eye and how to approach and treat eye disorders like glaucoma.