Estimated fluorescein elimination rate constant (k_d) following instillation of the probe drop.

<p>k_d was obtained as the slope of the fluorescence decay by fitting to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198831#pone.0198831.e004" target="_blank">Eq 4</a> according to ln F_dP (t) = ln F⁰dP—k_d t. The mean and SD values are 0.0142 and 0.0107 sec^-1, respectively (n = 49 eyes and 29 subjects).</p

Inter-subject variability of fluorescein clearance after the probe drop in two subjects.

<p>The excitation slit was focused on the tear film and the fluorescence <i>vs</i>. time profile was obtained for 10 min. F⁰_dP and k_d are the intercept and slope, respectively, of the fluorescence decay in the tear film as per the exponential decay curve (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198831#pone.0198831.e004" target="_blank">Eq 4</a>) using non-linear least squares. Half-lives t^d_1/2 indicated in the inset were calculated from k_d. <b>Panels A and B</b>: Data from a subject showing rapid clearance of fluorescein with half-lives of only 88 and 96 seconds in the left and right eyes, respectively. <b>Panels C and D</b>: Data from a different subject showing a relatively slower clearance with half-lives of 208 and 210 seconds in the left and right eyes, respectively.</p

Mathematical notations.

<p>Mathematical notations.</p

Schematic of the multi-drop protocol for the measurement of epithelial permeability.

<p>At t = 0, a 0.35% fluorescein drop (0.35 gm of fluorescein/100 mL PBS buffer) is instilled on the bulbar conjunctiva and the tear fluorescence is measured (shown by red unfilled circles). After clearance of the dye (usually < 15 min), two drops of 2% fluorescein (2 gm of fluorescein/100 mL PBS buffer) are instilled 10 min apart (T₁ and T₂). About fifteen minutes after the second drop, the ocular surface is washed with CMC solution (carboxymethyl cellulose solution; Blue arrow). Next, stromal fluorescence is measured 3–4 times at time T_s (usually within 5–10 min after T₃). AUC_dL1 and AUC_dL2, which are assumed to be equal, are estimated based on the area under the curve calculated for the 0.35% drop (AUC*). The tear fluorescence in response to the probe drop is fitted to a single-exponential decay to determine F⁰_dp and k_d. F⁰_dp is then used to estimate F⁰_dL1 and F⁰_dL2. k_d for the 2% drops is assumed to be the same as that for the 0.35% drop. Hence, the first 0.35% drop is referred to as the probe drop. The 2% drops have been employed to load the stroma with measurable levels of fluorescein so that noise-free measurements of the stromal accumulation can be obtained. Therefore, the 2% drops are referred to as the loading drops.</p

Corneal epithelial permeability to fluorescein in humans by a multi-drop method

<div><p>Purpose</p><p>The permeability of the corneal epithelium to fluorescein P_dc is an indicator of the health of the ocular surface. It can be measured in a clinical setting by determining the accumulation of fluorescein in the stroma following administration of the dye on the ocular surface. Here we demonstrate a new multi-drop method for the measurement of P_dc by a spot fluorometer.</p><p>Methods</p><p>Twenty-nine healthy participants were recruited for this study. First, a probe-drop of fluorescein (0.35%, 2 μL) was instilled on the conjunctiva. The clearance of the dye from the tears was immediately measured using the fluorometer. Following this, two loading drops (2%; 6 μL each) were administered 10 min apart. Fifteen minutes later, the ocular surface was washed and fluorescence from the stroma F_s was measured. Permeability was calculated using P_dc = (Q x F_s)/ (2 x AUC), where Q is the stromal thickness and AUC is the area under the fluorescence <i>vs</i>. time curve for the loading drops.</p><p>Results</p><p>After the probe drop, the tear fluorescence followed an exponential decay (elimination rate constant; k_d = 0.41 ± 0.28 per min; 49 eyes of 29 subjects), but the increase in F_s was negligible. However, after the loading drops, the measured F_s was ~ 20-fold higher than the autofluorescence and could be recorded at a high signal to noise ratio (SNR > 40). The intra-subject variability of k_d was insignificant. Since fluorescein undergoes concentration quenching at > 0.5%, the value of AUC for the loading drops was estimated by scaling the AUC of the probe drop. The calculated P_dc was 0.54 ± 0.54 nm/sec (n = 49). A Monte Carlo simulation of the model for the multi-drop protocol confirmed the robustness of the estimated P_dc.</p><p>Conclusions</p><p>The new multi-drop method can be used in place of the single-drop approach. It can overcome a lack of sensitivity in fluorometers of high axial resolution. The P_dc estimated by the multi-drop method is ~ 11-fold higher than previously reported but closer to the value reported for other drugs with equivalent octanol/water partition coefficient.</p></div

Calculated permeability of the corneal epithelium in healthy subjects.

<p>The values of P_dc (as calculated by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198831#pone.0198831.e024" target="_blank">Eq 22</a>) ranged from 0.07 nm/sec to 2.59 nm/sec. The mean and SD are 0.54 and 0.54 nm/sec, respectively (n = 49 eyes and 29 subjects; median = 0.32 nm/sec).</p

Effect of instilled volume on fluorescein clearance (k_d) in four different subjects.

<p>The data also shows the intra-subject variability of fluorescein clearance in repeat trials. <b>Panels A-D</b> show k_d estimated from independent probe drops of fluorescein (0.35%) at 2 or 6 μL each dropped in a single subject ~15 min apart. Paired t-tests show lack of any significant difference between the means of k_d measured at the two instilled volumes.</p

Estimated tear fluorescence at time t = 0 after instillation of the probe drop (F⁰_dP,).

<p>The Y intercept (tear fluorescence at t = 0, F⁰_dP) of the fluorescence <i>vs</i>. time plot was obtained by fitting data to the exponential decay as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198831#pone.0198831.e004" target="_blank">Eq 4</a>: ln F_dP (t) = ln F⁰dP—k_d t. The mean and SD values are 206.51 and 113.89 mV, respectively (n = 49 eyes and 29 subjects).</p

Effect of the axial resolution on the measured fluorescence.

<p>The depth of the focal diamond is 2δ. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198831#pone.0198831.g002" target="_blank">Fig 2</a>, we note that δ is 140 μm. <b>Panel A</b>: Focal diamond (RED lines) across the tear film with fluorescein. Note that the tear film does not span the entire depth of the focal diamond. In the figure, F^A_d and C_F refer to the measured tear fluorescence and concentration of fluorescein in the tear film, respectively. <b>Panel B</b>: Tear film is thicker compared to that in Panel A so that F^B_d >F^A_d. <b>Panel C</b>: Focal diamond is positioned in the stroma. In this case, the measured fluorescence F^C_S would be proportional to C_F but independent of the stromal thickness, which is greater than 2δ. <b>Panel D</b>: Estimation of ICF. The intersection of the tear film with the focal diamond is a parallelepiped of volume given by t_d x 2W x H, where t_d is the thickness of the tear film, 2W is the maximum width of the focal diamond, and H is the thickness of the excitation slit beam. The volume of the focal diamond is given by 2δ x W x H, where 2δ is the axial resolution of the instrument (2δ ~ 280 μm). Hence, the ratio of the volume of the focal diamond to that of volume of intersection between tear film and focal diamond (defined as ICF, instrument correction factor) is given by (2δ x W x H) / (t_d x 2W x H). Hence, ICF would be δ/t_d, which is equal to 47 assuming a tear film thickness of ~ 3 μm [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198831#pone.0198831.ref038" target="_blank">38</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198831#pone.0198831.ref041" target="_blank">41</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198831#pone.0198831.ref043" target="_blank">43</a>]. This estimation assumes that the fluorescence in the stroma for a given concentration of fluorescein is not different from an equivalent amount of fluorescein in water. More specifically, fluorescein is assumed unbound in the stroma.</p

Linearity, depth resolution, and concentration quenching.

<p>Three solutions of fluorescein, prepared with PBS at 1, 10, and 50 μM (corresponding to 0.038%, 0.38%, and 1.88% (w/v), respectively) were contained in T-25 flasks. Depth-resolved fluorescence measurements were then obtained by positioning the flask along with the optical axis of the fluorometer. The angle between the excitation and the emission arms was held at 45°. Concentrations of fluorescein in % (provided in parenthesis) are in w/v basis. <b>Panel A</b>: Typical fluorescence <i>vs</i>. depth profile obtained with 1 μM solution. Note that the fluorescence of the solution remains constant over the depth of the scan. The fluorescence change from background to the new plateau occurs over a transition depth ~ 280 μm (inset on the right). This is a measure of the axial resolution of the instrument, which can also be specified as full-width at half maximum (FWHM; 140 <i>μ</i>m). <b>Panel B</b>: Fluorescence <i>vs</i>. depth profiles obtained with fluorescein at 1, 10, and 50 μM solutions. Unlike the fluorescence profile for 1 μM solution (thin line), the fluorescence for 50 μM (thick line) decreases with increasing depth, indicating concentration quenching. The fluorescence <i>vs</i>. depth profile for the 10 μM solution (0.38%) also shows concentration quenching, but it is marginal. Hence, the concentration of fluorescein in the probe drop was kept below 0.38% in all our experiments. The inset in Panel B summarizes the concentration quenching at different depths for the three different solutions.</p