A Phosphorescence Lifetime Ocular Fluorometer

It is important that scientists study the production of a clear fluid called “aqueous humor” by the eye. About three to four drops of aqueous humor fill the space between the cornea and iris, called the “anterior chamber.” Aqueous humor is produced continuously by a specialized tissue behind the iris, called the “ciliary body.” It flows into the anterior chamber through the pupil and leaves the eye through a set of channels near the outer rim of the iris. Aqueous humor is produced fast enough that its entire volume in the anterior chamber is replaced once every hour. Its continuous flow brings nutrients to the lens of the eye and the cornea and washes away waste products. This helps maintain a healthy and clear cornea and lens, since these tissues do not have their own blood supply. In glaucoma the path that aqueous humor takes to leave the anterior chamber becomes partially obstructed and greater pressure is necessary to push aqueous humor out. The pressure throughout the eye gradually increases, and when pressure gets too high, the optic nerve becomes pinched where it leaves the eye. The increased pressure on the nerve causes a gradual blindness as the nerve fibers lose their capacity to carry information to the brain. One of the simplest treatments to lower intraocular pressure in eyes that have glaucoma is to administer eye drops that contain a medicine that reduces production of aqueous humor. Several medications for this purpose are now available and scientists continue to search for new medications that are more effective in reducing pressure without side effects. It is important in this kind of research to know how the ciliary body works to produce aqueous humor and how biochemical components of aqueous humor change as production rate of aqueous humor changes. Several techniques are now available to measure production rate of aqueous humor, its pH (acidity), and the amount of protein in the aqueous humor without disturbing the eye. Many of these measurements are made by applying a small amount of a fluorescent dye to the eye and allowing it to cross the cornea and mix with the aqueous humor. The fluorescence is then measured with a device called an “ocular fluorophotometer.” By measuring several properties of fluorescent light from the dye, we can determine these characteristics of the aqueous humor. Over the past year we have designed and constructed a new ocular fluorophotometer. This device consists of several lenses that focus blue or ultraviolet light into the eye, and several that collect the green or yellow fluorescent light. Light is carried to and from the lenses through glass optical fibers. The instrument also has the mechanical apparatus to allow us to position its focal point in the cornea or anterior chamber. The optical fibers can be connected to light sources and light detection modules that are designed to measure specific properties of the fluorescent light. In the pending grant period we plan to develop a new light source and light detection system that will be coupled to our new ocular fluorophotometer. This new system will allow us to measure another property of some dyes that can be applied to the eye, phosphorescence. Like fluorescence, phosphorescence is emitted by a dye when the dye is illuminated by light of an appropriate color. The difference is that phosphorescent light lasts much longer than fluorescent light after a brief flash of excitation light: phosphorescence is emitted over several thousandths of a second to several seconds (depending on the dye), while fluorescence is emitted in less than one millionth of a second. Although phosphorescence is usually less intense than fluorescence, the rate of its decrease after a flash is much easier to measure. The reason that phosphorescence is of interest to scientists who study glaucoma is that the intensity and duration of phosphorescence from most dyes is dependent on the biochemical components of the solution in which the dye is dissolved. For example, the concentration of dissolved oxygen strongly effects phosphorescence. As the concentration of oxygen increases, both the intensity and the duration of phosphorescence decrease. This property is called “quenching.” We hope to develop a technique that makes use of quenching of phosphorescence from certain dyes to measure the concentration of oxygen in the aqueous humor. The light source and light measurement module necessary to measure phosphorescence from the eye must be capable of producing an intense flash of light over a period of about one millionth of a second. This is similar to an electronic flash used on a camera. A special computer will be designed to control the flash and the measurement of the light from the eye over a precise time period (less than one one hundredth of a second) after each flash. When completed, the instrument will undergo many hours of testing on control solutions in a test tube before we attempt to make measurements in a living eye. If successful, this instrument and method will provide a new means of studying and understanding physiologic properties of aqueous humor.