Development of High-performance Microscopy for Noninvasive Examination of Conjunctival Goblet Cells

Abstract

Conjunctival goblet cells (CGCs) are specialized epithelial cells that secrete mucins to form the mucous layer of the protective tear film. The mucus layer provides both lubrication and a physical barrier against pathogens. In addition to producing mucins, CGCs also secrete several antimicrobial substances, which help to directly neutralize potential pathogens. CGCs can interact with and modulate the immune system in various ways. They are involved in the recruitment of immune cells to the site of infection, and they can also secrete factors to regulate immune responses for ocular surface homeostasis. Recent research suggested that CGCs might also play a role in antigen presentation, which is an essential part of the adaptive immune response. This would indicate that CGCs are not just passive barriers to infection, but active participants in the immune response. Previously, the status of CGCs was examined by using impression cytology (IC). IC removes cells on the surface by impressing with a filter paper and CGCs were visualized after dehydration and Periodic acid–Schiff (PAS) staining. Decrease of CGC density was reported in various ocular surface diseases including dry eye disease. Although CGCs would be an important biomarker for the diagnosis of ocular surface diseases, current CGC examination methods have limitations. IC is mildly invasive, lacks standardization, and takes a long time for processing and examination. Reflectance confocal microscopy (RCM) can visualize CGCs non-invasively by scanning a laser focus and collecting reflection through a pin hole. However, RCM suffers limitations of low and non-specific reflection contrasts, small imaging field-of-views (FOVs) of a few hundred micrometers. Recently, our laboratory had established that moxifloxacin antibiotic ophthalmic solution has bright intrinsic single-photon and multi-photon fluorescence, good tissue penetration, and high intracellular concentration. We also discovered that moxifloxacin specifically labels CGCs and successfully imaged CGCs in high contrasts by using both 3D fluorescence microscopies and wide-field fluorescence microscopy. However, when it comes to in-vivo CGC imaging, high-speed CGC imaging on the surface of conjunctiva was challenging. This was due to the shallow depth-of-field (DOF) of conventional microscopy when imaging the curved and tilted conjunctiva. Thus, I started my research aimed at developing high-performance fluorescence microscopy with extended DOF (EDOF), and further applied it to humans. Various techniques for achieving EDOF imaging were explored, and I chose a method using an actuator to achieve an EDOF. Initially, an EDOF was realized by directly moving the objective lens, and in-vivo mouse model imaging was demonstrated. Subsequently, a high-speed EDOF was implemented using a high-stroke deformable mirror (DM), and in-vivo rabbit model imaging was demonstrated. Ultimately, for human application, a high-speed surface tracking technology was developed and combined with EDOF imaging. CGC imaging in human subjects was demonstrated. At the beginning of the study, EDOF was realized by moving the objective lens axially and sweeping the focal plane. Thirty images are taken while sweeping the focal plane 1 mm and the acquired images are locally focused at different locations within same FOV. These multiple images were processed to generate single all-in-focus image. Using this moxifloxacin-based axially swept wide-field fluorescence microscopy (MBAS-WFFM), I successfully imaged CGCs in the mouse conjunctiva in an in-vivo mouse experiment. Through a longitudinal study of chemically damaged mouse conjunctiva, I was able to observe both the decrease and recovery in CGCs. While MBAS-WFFM succeeded in imaging CGCs in the curved conjunctiva, its effectiveness was limited to small animals like mice having small eyes and was affected by breathing motions, mainly due to slow imaging speed. Therefore, I developed a high-speed EDOF microscopy that is not affected by substantial breathing motion. This moxifloxacin-based extended depth-of-field (MB-EDOF) microscopy adapted a deformable mirror (DM) for the high-speed axial sweeping of focal plane during single image frame acquisition. The raw images acquired by MB-EDOF microscopy had both in-focus and out-of-focus information. Out-of-focus information was removed by deconvolution using axially accumulated PSF. High-speed MB-EDOF microscopy using a high-stroke DM was developed for noninvasive and real-time CGC examination. The system could run at the imaging speed of 15 fps, which was more than 10× faster than the previous system and had an extended DOF of 800 μm, which was approximately 25 times of the standard DOF. The performance of the new system was verified by CGC imaging of both mouse and rabbit models. Real-time breathing-motion-insensitive imaging and mosaic imaging were demonstrated. This animal experiment was conducted in collaboration with team of Prof. Chang Ho Yoon from Seoul National University College of Medicine and with team of Prof. Hong Kyun Kim from School of Medicine, Kyungpook National University. While this system could image one FOV in a very short time, it was still time-consuming to image larger areas requiring lateral translation due to the refocusing in the changed height of the conjunctiva surface beyond the EDOF. For human applications, CGC imaging would be better completed within a short time (e.g., 10 s) to avoid subject discomfort. Large-area imaging, also, would be preferred owing to the high spatial variation of CGC density in the conjunctiva. Therefore, high-speed moxifloxacin-based fluorescence microscopy with surface tracking is required for robust large-area imaging in human subjects. Herein, I developed high-speed EDOF wide-field microscopy with surface tracking for noninvasive real-time CGC imaging in human subjects. EDOF imaging was achieved by axially sweeping the focal plane with an electrically tunable lens with an EDOF of 800 μm and an imaging speed of >10 frames per second. A novel long-range surface tracking based on oblique plane imaging was developed for rapid large-area imaging with lateral translation. EDOF microscopy with surface tracking enabled to capture 5 × 2 patch images in human volunteer subjects within approximately 10 seconds. Large-area CGC imaging and subsequent CGC density analysis were demonstrated. Further clinical studies using EDOF microscopy with surface tracking will be conducted to determine the optimal location and imaging area for the robust and reliable information of CGCs and their reference values. This new imaging system could rapidly examine CGCs in humans and this information may be valuable for precision diagnosis and optimal treatment of ocular surface diseases.