Development of moxifloxacin-based fluorescence microscopies for medical applications

Abstract

In life sciences and medicine, optical microscopy is an essential tool for examining living tissues at a cellular level. Optical microscopes have evolved over time to meet the needs of various experiments and samples. High-magnification microscopes that utilize reflection, absorption, and fluorescence techniques are commonly used for examination, surgical guidance, and lesion localization. Confocal microscopy, a key example of a biomedically applicable optical microscope, uses pinholes to achieve high resolution and optical sectioning, allowing for the acquisition of high-contrast images through reflectance and fluorescence. This technique offers advantages in faster and more sensitive diagnosis through cellular-level examination. However, confocal microscopy has limitations, such as a restricted field of view (FOV) at high magnification, speed limitations due to the point- scanning method, and challenges with using fluorescent materials in living organisms. Recently, it was discovered that the ophthalmic antibiotic moxifloxacin has properties that allow for high-contrast cellular imaging through cellular fluorescence labeling. Moxifloxacin non-specifically stains general cells and specifically stains some secretory cells. Despite the ability to diagnose tumors with a high moxifloxacin fluorescence signal in resected human skin cancer and brain tumor tissues, confocal microscopy has only been performed on ex-vivo resected tissues due to limited imaging speed for in-vivo imaging. Therefore, the goal of this study is to develop a new optical system for in-vivo medical applications based on the non-specific and specific cell staining properties of moxifloxacin. In this study, two new optical systems were developed based on the characteristics of tissue for in-vivo imaging. The first system was designed for ocular surface imaging, combining wide-field fluorescence microscopy with extended depth of focus technology to adjust focus according to depth. The second system is a handheld microscope that integrates moxifloxacin-based fluorescent staining with existing high-speed 3D technology. It addresses the inflexibility of current systems by incorporating a novel image delivery method. First, I developed an ocular surface examination system that can be used for ocular surface diagnosis such as dry eye syndrome using the secretory cell-specific target staining characteristics of moxifloxacin. I was the first to discover that moxifloxacin specifically stains goblet cells (GCs) of the eye and colon, among secretory cells. In the conjunctiva of the eye, GCs are cells that secrete tear mucus and play an important role in homeostasis. Because GCs are distributed only on the conjunctiva surface, GC imaging techniques using moxifloxacin can operate without optical sectioning capabilities. Consequently, I successfully imaged in-vivo rat conjunctival goblet cells under anesthesia using a general wide-field fluorescence microscope (WFFM). Typical high-magnification microscope configurations can improve imaging speed, but there is a trade-off between the high resolution at which cells can be imaged and the depth at which they can be imaged. To increase the depth of focus without the loss of resolution, I developed an ocular surface GC diagnosis system using extended-depth-of-focus (EDOF) technology. Moxifloxacin-based EODF microscopy has been developed with axial swept wide-field fluorescence microscopy (MBAS-WFFM), which images all depths of focus and combines focal regions, and an electro-tunable lens (ETL) based EDOF system that improves the speed of MBAS-WFFM. The system was verified in mouse and rabbit models and applied to human conjunctiva. By adding an automatic GC analysis algorithm based on a Unet, an artificial intelligence model widely used in the biomedical field, I have completed the ocular conjunctiva diagnostic device by enabling high-speed GC density and morphology analysis. Next, I developed a tumor-guided imaging system utilizing the non-specific staining properties of moxifloxacin. In tumor examination, it is crucial to remove all tumors while preserving as much normal tissue as possible, necessitating a high-resolution 3D imaging method. To address this limitation, I developed a dual-channel confocal microscope (DC- CM) capable of simultaneously imaging 5-ALA fluorescence, which is used clinically, and moxifloxacin to assess the brain tumor-guiding performance of moxifloxacin. This approach demonstrated increased sensitivity by providing precise information. To overcome the speed limitations of traditional confocal microscopes, I introduced light sheet microscopy. Scanning oblique plane microscopy (OPM), which uses oblique illumination and oblique surface scanning with a single objective lens, was developed as a moxifloxacin-based fluorescence imaging system. Moxifloxacin-based sOPM demonstrated a tenfold increase in throughput in both resected human normal and tumor brain tissues, while delivering the same level of detailed information as confocal microscopy. Despite the high throughput of moxifloxcain-based sOPM, sOPM has limitations in bioapplication due to the large volume and inflexibility of the system due to the image relay method that reconstructs the tilted image of the existing OPM. Finnally to deal with this problem, I introduced a new image relay method based on image fiber bundles to this system and miniaturized it into a handheld form. Hand held sOPM was verified for performance in excised ex-vivo mouse tissue, and was then shown to have potential for in vivo use by tracking corneal damage recovery in mice in vivo. In this study, two moxifloxacin-based fluorescence microscopes (MBFM) were developed for medical applications such as ocular surface examination and tumor guidance. High contrast visualization of GC with moxifloxacin labeling was found for the first time. Based on this, the first EDOF microscope capable of non-contact imaging was developed and used to examine ocular conjunctival GCs. An automatic AI algorithm was developed to analyze GCs, enabling rapid density and morphology inspection. Sufficient verification has been achieved in in-vivo rabbits, and clinical trials are underway with next-generation EDOF sytem. And MBFM was developed for brain tumor delineation based on non-specific cell labeling of moxifloxacin. The effectiveness of brain tumor guide using MBFM was verified by simultaneous imaging with 5-ALA fluorescence used in existing clinical trials. Unlike 5- ALA, which can only identify the presence or absence of a tumor, MBFM images round cells at high density to detect tumors. MBFM overcomes the speed limitations of existing confocal microscopes with light sheet technology and was introduced into sOPM. Although sOPM showed a throughput that is more than 10 times faster than confocal microscopy, its large volume and lack of flexibility limit its bioapplications. Ultimately, to solve this problem, Hand held sOPM, a new image relay method using flexible fiber bundles, was developed. This system was validated through in vivo mouse cornea imaging.