In Vivo Noncontact Imaging of Conjunctival Goblet Cells with Customized Widefield Fluorescence Microscopy

Abstract

Purpose: Conjunctival goblet cells (CGCs) play a crucial role in maintaining ocular surface health by producing mucins. However, assessing CGC changes in ocular diseases remains limited by invasive techniques and subjective evaluations. This study aims to develop a noncontact cellular resolution fluorescence microscopy for in vivo CGC imaging and investigate CGC dynamics in a dry eye disease (DED) mouse model.

Design: Experimental study.

Subjects: Freshly ex vivo porcine eyes, New Zealand white rabbits, and C57BL/6 mice.

Methods: Based on the intrinsic fluorescence properties of moxifloxacin, a high-resolution noncontact widefield fluorescence microscopy (WFFM) was customized with an all-in-focus algorithm to optimize in vivo CGC imaging over the curved conjunctival surface. A DED mouse model was established by topically applying 0.2% benzalkonium chloride (BAC) to the ocular surface daily for 7 days, followed by a 7-day recovery period without BAC. In vivo CGC alterations were assessed using WFFM on days 0, 3, 7, and 14. Additional assessments included the phenol red thread tear test, corneal sodium fluorescein staining, and periodic acid-Schiff (PAS) assay.

Main outcome measures: Conjunctival goblet cell density and area ratio.

Results: The WFFM system achieved a cellular resolution of 1 μm and a field of view of 1.4 mm × 1.4 mm. Imaging validation in mice and rabbits allowed for the distinguishing and quantitative assessment of individual CGCs or clusters on the curved conjunctival surface in vivo. Significant reductions in CGC density and area ratio on days 3 and 7 after BAC induction were observed in DED mouse in vivo with WFFM, with their values returning to the baseline 7 days after BAC removal, which was consistent with PAS staining results.

Conclusions: The customized WFFM enables in vivo cellular imaging of CGCs, offering a safe and accurate method for continuous monitoring of CGC pathophysiology in ocular surface diseases such as DED.

Financial disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Keywords: Cellular imaging; Conjunctival goblet cells; Dry eye disease; Noncontact imaging.

Figure 1

Widefield fluorescence microscopy system. A, Schematic of imaging system. B, Photo of the system. C, Imaging of a grid target board; the minimum grid size is 0.2 mm. D, Imaging of a USAF resolution target showing that element of 500 lp/mm was readable. BF = emission filter; BS = dichroic mirror; EF = excitation filter; ETL = electrically tunable lens; L1 = achromatic lens (f = 50 mm); L2 = achromatic lens (f = 30 mm); LED = light emitting diode; OL = objective lens; TL = tube lens (f = 100 mm).

Figure 2

Evaluation of focal plane change. A, Schematic of axial sweep acquisition of the focal plane. B, A photo of the DOF target. Detail of the DOF target, highlighting a detail of the imaged field of view from the 15 lp/mm section. C, Flow chart showing the image processing procedure. D–F, Images at 3 different focal plane depths on DOF target with 15 lp/mm category. G, The reconstructed all-in-focus image of DOF target merging 70 images around the estimated focal distance for each depth. DOF = depth of focus; ETL = electrically tunable lens; OL = objective lens.

Figure 3

Imaging of ex vivo CGCs. A, Mouse CGCs imaged by WFFM. B, Periodic acid–Schiff staining shows a distribution broadly similar to that of WFFM. C, Confocal imaging of mouse CGCs. D, Correlation analysis reveals strong positive correlation between WFFM and PAS histology images of ex vivo mouse GCCD (n = 10). E–G, Porcine CGCs with the same procedure. H, Ex vivo porcine GCD obtained by WFFM and PAS histology shows a strong positive correlation (n = 10). Black arrows are pointing to the same location. CGCs = conjunctival goblet cells; GCCD = goblet cell cluster density; GCD = CGC density; PAS = Periodic acid–Schiff; WFFM = widefield fluorescence microscopy.

Figure 4

In vivo imaging of CGCs. A–C, Representative images of the mouse palpebral CGCs at different focal planes. D, Corresponding all-in-focus image shows CGCs are aggregated in clusters. E–G, Representative images of the rabbit bulbar CGCs at different focal depths. H, All-in-focus image shows that most rabbit CGCs occur individually. CGCs = conjunctival goblet cells. Red colored squares are regions of interest for quantification.

Figure 5

Effect of BAC on ocular surface and tear volume. A–D, Corneal fluorescence staining images of BAC-treated mice on days 0, 3, 7, and 14. E, Fluorescence staining scores on days 3 and 7 are significantly higher than on day 0, no statistical difference between day 14 and day 0, and no significant change in tear volume at different time points (n = 6). ∗P < 0.05, ∗∗∗P < 0.001. BAC = benzalkonium chloride; ns = not significant.

Figure 6

CGC changes after BAC instillation. A–D, Representative images of CGCs on days 0, 3, 7, and 14. E and F, significant reduction in GCD and area ratio on days 3 and 7, returning to baseline levels by day 14 (n = 6). G–J, Representative periodic acid–Schiff staining images of CGCs on days 0, 3, 7, and 14. K, The number of CGCs showing a trend consistent with GCD and area ratio (n = 4). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. BAC = benzalkonium chloride; CGC = conjunctival goblet cell; GCD = goblet cell density; ns = not significant.