Chapter 3

Fluorophotometry for Pharmacokinetic Assessment

Bernard E. McCarey

Abstract  The corneal epithelium provides a semi-impermeable barrier between the eye and the environment. With continuous intercellular tight junctions, the corneal epithelium exerts a high resistance to passage of ions. Fluorescein staining of the corneal epithelium and stroma has been a subjective measure of the quality of the epithelial barrier. Maurice (1963) was an early pioneer in devising a slit lamp-based instrument to quantify the fluorescein in the cornea of human subjects. Cunha-Vaz (Br J Ophthalmol 59:649-656, 1975) expanded the technology to perform multiple measures of fluorescein concentration as the instrument’s focal plane moved through the eye. Since these early instruments, several commercial fluorophotometers have become available with application to drug delivery of fluorescent tracers.

3.1  Commercial Fluorophotometer

A commercially available fluorophotometer, the Fluorotron Master, OcuMetrics, Mountain View, CA, uses a scanning slit to measure fluorescence at various depths within the eye from the tear film to the retina (Fig. 3.1) The fluorescence values are recorded from the retina to the cornea in 148 spatial steps of 0.25 mm within the optical focal range of 38 mm. A self-calibration is performed against an internal target before each scan. The scan is completed in 15 s. The focal diamond for sampling the ocular tissue fluorescence has dimensions of 100 mm height by 1.9 mm width.

B.E. McCarey (*)

Emory University School of Medicine, Eye Center, 1365B Clifton Road, Suite B2600, Atlanta, GA 30322, USA

e-mail: ophtbmc@emory.edu

AAPS Advances in the Pharmaceutical Sciences Series 2, DOI 10.1007/978-1-4419-9920-7_3, © American Association of Pharmaceutical Scientists, 2011

Fig. 3.1  The focal diamond (Df) is the overlapping volume from the converging blue light beam and the detector green light beam (Gray et al. 1985)

In practice, the sample window is sensitive to fluorescence over approximately­ a 1 mm depth spatial distance (Fig. 3.1). An anterior segment adaptor lens can be installed on the fluorophotometer to reduce the spatial steps to 0.125 mm for an ­optical range of 18.5 mm. The fluorescence values of the ocular structures are scanned in the reverse order from that of the standard objective lens, i.e., from the cornea to the retina. The adaptor doubles the standard angle between the excitation and emission pathways to 28°. This increases the resolution while providing a sufficient range to record the fluorescence from the tear/cornea, aqueous humor, crystalline lens, and anterior vitreous. The focal diamond for the anterior segment lens has a height of 50 mm and width of 950 mm. The instrument can measure the natural fluorescence of the ocular tissue to a sensitivity of 0.1 ng/mL sodium fluorescein and has a linear response up to 2,000 ng/mL (OcuMetrics 1995).

Since the instrument has a 1-mm-depth spatial distance, a spatial resolution correction is necessary to adjust the fluorescence of thinner structures such as the 0.5 mm cornea or the choroidal–retinal structure of the eye. As the focal diamond travels through the cornea, the fluorophotometer will record a bell-shaped fluorescence curve (Fig. 3.2). This principle is further illustrated in Fig. 3.3. As the focal diamond advances through the ocular structures, a symmetrical curve of the tissue fluorescence will be documented (Fig. 3.4).

Fig. 3.4  The fluorophotometer excitation and emission beam are focused through the ocular structures­ . As the beam is advanced through the eye, the fluorescence values are used to plot the ocular fluorescence profile of the eye

3.2  Normal Human Subject and Rabbit Ocular Fluorescence

The autofluorescence of the crystalline lens increases with age (Chang and Hu 1993; Chang et al. 1995). Figure 3.5 is an autofluorescence scan of a 57-year-old male with the crystalline lens fluorescence of 300 ng/mL and the tear/cornea peak at 10 ng/mL. Figure 3.5a was captured with the anterior chamber lens which provides a greater resolution to separate the tear/cornea peak from the lens peaks. Figure 3.5b was captured with the standard objective lens. In contrast, the fluorescent profile for the young rabbit (<6 months old) illustrated a different tear/cornea to lens ratio (Fig. 3.6). The rabbit crystalline lens fluorescence is 2 ng/mL and the tear/cornea peak at 3.5 ng/mL.

Fig. 3.5  The ocular scan with the anterior chamber lens plots the natural fluorescence through the anterior segment of the eye of a 57-year-old male. (a) Captured with the anterior chamber objective lens. (b) Captured with the standard objective lens