The letter responds to the comments from P. Tyagi in the same issue.

This study provides the first direct evidence of water transport through the urothelium, traditionally 1 considered impermeable. Using Optical Coherence Tomography (OCT), we observe that the urothelium absorbs and 2 expels water under varing concentrations of NaCl, challenging long-held views about its impermeability. The discovery 3 that osmotic stress can induce urothelial damage has significant implications for bladder disorders like interstitial cystitis 4 and overactive bladder, where urothelial integrity ### Competing Interest Statement The authors have declared no competing interest.

A multimodal imaging system combining Optical Coherence Tomography and Confocal laser scanning microscopy can provide high-resolution depth-resolved structural imaging and non-invasive cellular tracking for zebrafish imaging. This study applies three genetically modified zebrafish lines to target glial cells, tumors, and macrophages to explore functional applications. It uses an 850nm near-infrared SLED for OCT and a 480nm blue emission laser for confocal imaging, both sharing the same sample arm that enables simultaneous scanning. Achieving a 250kHz acquisition rate, this tandem system offers OCT B-Scan and confocal image display in real time. The platform can aid advanced cellular activity monitoring, surgery, and in vivo biological mechanism analysis, including myopia, inflammation, and tumor progression.

Significance:We provide the first direct evidence of the urothelial response to water transport through the urothelium, traditionally considered impermeable. Using optical coherence tomography (OCT), we observe that the urothelium absorbs and expels water under varying concentrations of NaCl, challenging long-held views about its impermeability. The discovery that osmotic stress can induce urothelial damage has implications for bladder disorders such as interstitial cystitis and overactive bladder, where urothelial integrity is compromised. Aim:Traditionally considered impermeable, the urothelium has recently been implicated in water transport due to the presence of aquaporins. Despite this, direct evidence of the urothelial response to water movement through the urothelium remains elusive. We aim to provide such evidence by examining urothelial responses to NaCl solutions using OCT. Approach:Fresh porcine bladder samples were subjected to OCT imaging to observe urothelial responses under varying osmolarity conditions, using NaCl solutions ranging from 0.31 to 2.07    Osm / L . Urothelial optical pathlength thickness was measured pre-NaCl and post-NaCl application. In addition, histological and scanning electron microscopy (SEM) analyses were conducted to assess cellular integrity and damage. Results:OCT imaging revealed a significant increase in urothelial optical pathlength thickness following deionized water application, indicative of water absorption. Conversely, exposure to higher osmolarity NaCl solutions resulted in urothelial shrinkage, suggesting water efflux. Histological analysis demonstrated intact cellular structures at lower osmolarities ( 0.31    Osm / L ) but significant cellular disruption at higher concentrations ( ≥ 1.03    Osm / L ). SEM analysis corroborated these findings, showing progressive damage to umbrella cells with increasing osmolarity. Conclusions:We provide evidence that the urothelium is a dynamic barrier capable of water transport, influenced by osmotic gradients. The observed osmotic-induced urothelial damage may have important implications for the pathophysiology of conditions such as interstitial cystitis and overactive bladder, offering insights into potential diagnostic and therapeutic strategies. These findings warrant further investigation using human tissue.

Previous studies indicated that macrophages play a role during lens regeneration in newts, but their function has not been tested experimentally. Here we generated a transgenic newt reporter line in which macrophages can be visualized in vivo. Using this new tool, we analyzed the location of macrophages during lens regeneration. We uncovered early gene expression changes using bulk RNAseq in two newt species, Notophthalmus viridescens and Pleurodeles waltl. Next, we used clodronate liposomes to deplete macrophages, which inhibited lens regeneration in both newt species. Macrophage depletion induced the formation of scar-like tissue, an increased and sustained inflammatory response, an early decrease in iris pigment epithelial cell (iPEC) proliferation and a late increase in apoptosis. Some of these phenotypes persisted for at least 100 days and could be rescued by exogenous FGF2. Re-injury alleviated the effects of macrophage depletion and re-started the regeneration process. Together, our findings highlight the importance of macrophages in facilitating a pro-regenerative environment in the newt eye, helping to resolve fibrosis, modulating the overall inflammatory landscape and maintaining the proper balance of early proliferation and late apoptosis.

Newts have a remarkable ability to regrow organs, providing key insights into the mechanisms of tissue regeneration. Lens regeneration in newts only initiates from the dorsal iris, and the underlying mechanism remains unknown. Understanding this process is critical for triggering non-competent adult tissues to support somatic cellular plasticity. Studies have focused on the difference in morphological and genetic variations between dorsal and ventral iris during the lens regeneration process using endpoint ex vivo tissue imaging and analysis, however, lens regeneration is a dynamic process. Therefore, this study aims to characterize the lens regeneration process in vivo with OCT and OCTA to non-invasively visualize the morphological variation of the anterior chamber and vasculature injury responses in the iris. Our results demonstrate that OCT is an ideal platformfor in vivo monitoring lens regeneration in newts and provides a dynamic insight into this process.

Lens regeneration in the adult newt illustrates a unique example of naturally occurring cell transdifferentiation. During this process, iris pigmented epithelial cells (iPECs) reprogram into a lens, a tissue that is derived from a different embryonic source. Several methodologies both in vivo and in culture have been utilized over the years to observe this phenomenon. Most recently, Optical Coherence Tomography (OCT) has been identified as an effective tool to study the lens regeneration process in continuity through noninvasive, real-time imaging of the same animal. Described in this chapter are three different methodologies that can be used to observe the newt lens regeneration process both in vivo and ex vivo.

OCTSharp is a versatile open-source OCT software that aims to support real-time OCT imaging development in research labs. For high-speed imaging, OCT demands high-performance real-time data processing and visualization. However, hardware control and sophisticated memory management prevent agile prototyping. The proposed OCTSharp allows users to tailor OCT for specific purposes in a short development cycle. This software is based on managed C# environments with accessible hardware control, minimal memory management, and GPU support. We demonstrate that OCT software based on C# is highly efficient and can satisfy most research-level OCT applications.

To image the underlying structures of a scattering medium, raster scanning imaging technologies capture least scattered photons (LSPs) and reject multiple scattered photons (MSPs) in backscattered photons. However, MSPs can still squeeze into the images, resulting in limited imaging depth, degraded contrast, and significantly reduced lateral resolution. Great efforts have been made to understand how MSPs affect imaging performance through modeling, but how the backscattered photons are distributed in a scattering medium during imaging remains a conceived picture base on simulations. Here, we demonstrate a method of reconstructing BSPP using beam-offset optical coherence tomography (OCT), where OCT images are acquired at offset positions from the illumination beam. By separating LSPs and MSPs, we can quantify imaging depth, contrast, and lateral resolution and access the depth-resolved modulated transfer function (MTF). This approach presents great opportunities for better retrieving tissue optical properties, correctly interpreting images, or directly using MTF as the feedback for adaptive optical imaging.

Optical coherence tomography (OCT) demands massive data processing and real-time displaying during high-speed imaging. Current OCT imaging software is predominantly based on C++, aiming to maximize performance through low-level hardware management. However, the steep learning curve of C++ hinders agile prototyping, particularly for research purposes. Moreover, manual memory management poses challenges for novice developers and may lead to potential security issues. To address these limitations, OCTSharp is developed as an open-source OCT software based on the memory-safe language C#. Within the managed C# environment, OCTSharp offers synchronized hardware control, minimal memory management, and GPU-based parallel processing. The software has been thoroughly tested and proven capable of supporting real-time image acquisition, processing, and visualization with spectral-domain OCT systems equipped with the latest advanced hardware. With these enhancements, OCTSharp is positioned to serve as an open-source platform tailored for various applications.

BACKGROUND:Aging and regeneration are heavily linked processes. While it is generally accepted that regenerative capacity declines with age, some vertebrates, such as newts, can bypass the deleterious effects of aging and successfully regenerate a lens throughout their lifetime.RESULTS:Here, we used Spectral-Domain Optical Coherence Tomography (SD-OCT) to monitor the lens regeneration process of larvae, juvenile, and adult newts. While all three life stages were able to regenerate a lens through transdifferentiation of the dorsal iris pigment epithelial cells (iPECs), an age-related change in the kinetics of the regeneration process was observed. Consistent with these findings, iPECs from older animals exhibited a delay in cell cycle re-entry. Furthermore, it was observed that clearance of the extracellular matrix (ECM) was delayed in older organisms.CONCLUSIONS:Collectively, our results suggest that although lens regeneration capacity does not decline throughout the lifespan of newts, the intrinsic and extrinsic cellular changes associated with aging alter the kinetics of this process. By understanding how these changes affect lens regeneration in newts, we can gain important insights for restoring the age-related regeneration decline observed in most vertebrates.

Raster scanning imaging technologies capture least scattered photons (LSPs) and reject multiple scattered photons (MSPs) in backscattered photons to image the underlying structures of a scattering medium. However, MSPs can still squeeze into the images, resulting in limited imaging depth, degraded contrast, and significantly reduced lateral resolution. Great efforts have been made to understand how MSPs affect imaging performance through modeling, but the techniques for visualizing the backscattered photon profile (BSPP) in scattering media during imaging are unavailable. Here, a method of reconstructing BSPP is demonstrated using beam-offset optical coherence tomography (OCT), in which OCT images are acquired at offset positions from the illumination beam. The separation of LSPs and MSPs based on the BSPP enables quantification of imaging depth, contrast, and lateral resolution, as well as access to the depth-resolved modulated transfer function (MTF). This approach presents great opportunities for better retrieving tissue optical properties, correctly interpreting images, or directly using MTF as the feedback for adaptive optical imaging.

Background Aging and regeneration are heavily linked processes. While it is generally accepted that regenerative capacity declines with age, some vertebrates, such as newts, can bypass the deleterious effects of aging and successfully regenerate a lens throughout their lifetime. Results Here, we used Optical Coherence Tomography (OCT) to monitor the lens regeneration process of larvae, juvenile, and adult newts. While all three life stages were able to regenerate a lens through transdifferentiation of the dorsal iris pigment epithelial cells (iPECs), an age-related decline in the kinetics of the regeneration process was observed. Consistent with these findings, iPECs from older animals exhibited a delay in cell cycle re-entry. Furthermore, it was observed that clearance of the extracellular matrix (ECM) was delayed in older organisms. Conclusions Collectively, our results suggest that although lens regeneration capacity does not decline throughout the lifespan of newts, the intrinsic and extrinsic cellular changes caused by aging alter the kinetics of this process. By understanding how aging affects lens regeneration in newts, we can gain important insights for restoring the age-related regeneration decline observed in most vertebrates.

Water transport through the urothelium was directly visualized under OCT. Water transport can significantly compress the urothelium and even lead to the damage of the urothelium, possible etiologies of overactive bladder and bladder pain syndrome.

Purpose:To establish optical coherence tomography (OCT) as an in vivo imaging modality for investigating the process of newt lens regeneration.Methods:Spectral-domain OCT was employed for in vivo imaging of the newt lens regeneration process. A total of 37 newts were lentectomized and followed by OCT imaging over the course of 60 to 80 days. Histological images were obtained at several time points to compare with the corresponding OCT images. Volume measurements were also acquired.Results:OCT can identify the key features observed in corresponding histological images based on the scattering differences from various eye tissues, such as the cornea, intact and regenerated lens, and the iris. Lens volume measurements from three-dimensional OCT images showed that the regenerating lens size increased linearly until 60 days post-lentectomy.Conclusions:Using OCT imaging, we were able to track the entire process of newt lens regeneration in vivo for the first time. Three-dimensional OCT images allowed us to volumetrically quantify and visualize the dynamic spatial relationships between tissues during the regeneration process. Our results establish OCT as an in vivo imaging modality to track/analyze the entire lens regeneration process from the same animal.Translational Relevance:Lens regeneration in newts represents a unique example of vertebrate tissue plasticity. Investigating the cellular and morphological events that govern this extraordinary process in vivo will advance our understanding and shed light on developing new therapies to treat blinding disorders in higher vertebrates.

Intracellular motion (IM) is originated from the motion of molecules and organelles in the cytoplasm of eukaryotic cells. IM is essential for the proper functioning of cells. Instead of tracking specific molecules, coherent gated methods measure the speckle variation induced by IM. Initially, holographic optical coherence imaging was used to image the IM of tumor spheroids and drugs' responses1,2 . Later on, with optical coherence tomography (OCT), IM has been used as an endogenous contrast to reveal the cellular and subcellular structures with freshly excised tissue. The uniqueness of the coherent gated method is that it can detect IM at different depths without requiring fluorescence tagging. Therefore, the imaged objects can stay at a more natural status.

Newts have the exceptional capability of regenerating the lens through their lifetime. The transparency of the anterior chamber makes OCT an idea imaging technology to track the entire process of the lens regeneration in vivo without interruptions. We demonstrated, for the first time, that OCT can capture not only essential morphological changes similar to the changes observed in histology but some fine structures, like zonular fibers, which are not visible in histology. Our initial results warrant the future research of tailoring OCT for dynamically imaging the lens regeneration in newts.