Open-top Bessel beam two-photon light sheet microscopy for three-dimensional pathology.

Nondestructive pathology based on three-dimensional (3D) optical microscopy holds promise as a complement to traditional destructive hematoxylin and eosin (H&E) stained slide-based pathology by providing cellular information in high throughput manner. However, conventional techniques provided superficial information only due to shallow imaging depths. Herein, we developed open-top two-photon light sheet microscopy (OT-TP-LSM) for intraoperative 3D pathology. An extended depth of field two-photon excitation light sheet was generated by scanning a nondiffractive Bessel beam, and selective planar imaging was conducted with cameras at 400 frames/s max during the lateral translation of tissue specimens. Intrinsic second harmonic generation was collected for additional extracellular matrix (ECM) visualization. OT-TP-LSM was tested in various human cancer specimens including skin, pancreas, and prostate. High imaging depths were achieved owing to long excitation wavelengths and long wavelength fluorophores. 3D visualization of both cells and ECM enhanced the ability of cancer detection. Furthermore, an unsupervised deep learning network was employed for the style transfer of OT-TP-LSM images to virtual H&E images. The virtual H&E images exhibited comparable histological characteristics to real ones. OT-TP-LSM may have the potential for histopathological examination in surgical and biopsy applications by rapidly providing 3D information.

Noninvasive Imaging of Conjunctival Goblet Cells as a Method for Diagnosing Dry Eye Disease in an Experimental Mouse Model.

Purpose: The purpose of this study was to evaluate a noninvasive conjunctival goblet cell (GC) imaging method for assessing dry eye disease (DED) in an experimental mouse model. Methods: Moxifloxacin-based fluorescence microscopy (MBFM) was used to examine GCs noninvasively in 56 mice. Forty-two (42) DED-induced mice were divided into 2 groups and treated topically for 14 days with cyclosporine (CsA) or normal saline (NS). In vivo MBFM imaging and clinical DED evaluations were performed and goblet cell density (GCD) and goblet cell area (GCA) were obtained and compared with histological GCD using periodic acid-Schiff (PAS) staining. Correlation and receiver operating characteristic (ROC) analyses showed MBFM's high diagnostic value. Results: The GCD and GCA of the DED mice obtained from in vivo MBFM imaging were highly correlated with clinical DED parameters and GCD obtained from PAS histology. The therapeutic effect of CsA, as observed by in vivo MBFM, was significant with respect to that of NS treatment. The ROC curves derived from in vivo MBFM showed high diagnostic value in assessing DED. Conclusions: The proposed noninvasive method has high diagnostic value in assessing the severity of DED and the effect of treatment for this disease. Translational Relevance: A noninvasive imaging method using moxifloxacin-based fluorescence microscopy was evaluated for assessing DED in an experimental mouse model. The method showed high diagnostic value in assessing the severity of DED and the effect of treatment, bridging the gap between basic research and clinical treatment. The study provides a promising tool for diagnosing and monitoring DED.

Deep learning framework for automated goblet cell density analysis in in-vivo rabbit conjunctiva.

Goblet cells (GCs) in the conjunctiva are specialized epithelial cells secreting mucins for the mucus layer of protective tear film and playing immune tolerance functions for ocular surface health. Because GC loss is observed in various ocular surface diseases, GC examination is important for precision diagnosis. Moxifloxacin-based fluorescence microscopy (MBFM) was recently developed for non-invasive high-contrast GC visualization. MBFM showed promise for GC examination by high-speed large-area imaging and a robust analysis method is needed to provide GC information. In this study, we developed a deep learning framework for GC image analysis, named dual-channel attention U-Net (DCAU-Net). Dual-channel convolution was used both to extract the overall image texture and to acquire the GC morphological characteristics. A global channel attention module was adopted by combining attention algorithms and channel-wise pooling. DCAU-Net showed 93.1% GC segmentation accuracy and 94.3% GC density estimation accuracy. Further application to both normal and ocular surface damage rabbit models revealed the spatial variations of both GC density and size in normal rabbits and the decreases of both GC density and size in damage rabbit models during recovery after acute damage. The GC analysis results were consistent with histology. Together with the non-invasive high-contrast imaging method, DCAU-Net would provide GC information for the diagnosis of ocular surface diseases.

High-resolution open-top axially swept light sheet microscopy.

BACKGROUND: Open-top light-sheet microscopy (OT-LSM) is a specialized microscopic technique for the high-throughput cellular imaging of optically cleared, large-sized specimens, such as the brain. Despite the development of various OT-LSM techniques, achieving submicron resolution in all dimensions remains. RESULTS: We developed a high-resolution open-top axially swept LSM (HR-OTAS-LSM) for high-throughput and high-resolution imaging in all dimensions. High axial and lateral resolutions were achieved by using an aberration-corrected axially swept excitation light sheet in the illumination arm and a high numerical aperture (NA) immersion objective lens in the imaging arm, respectively. The high-resolution, high-throughput visualization of neuronal networks in mouse brain and retina specimens validated the performance of HR-OTAS-LSM. CONCLUSIONS: The proposed HR-OTAS-LSM method represents a significant advancement in the high-resolution mapping of cellular networks in biological systems such as the brain and retina.

Feasibility of moxifloxacin and proflavine dual fluorescence imaging for detecting gastrointestinal neoplastic lesions: A prospective study.

OBJECTIVES: High-contrast and high-resolution imaging techniques would enable real-time sensitive detection of the gastrointestinal lesions. This study aimed to investigate the feasibility of novel dual fluorescence imaging using moxifloxacin and proflavine in the detection of neoplastic lesions of the human gastrointestinal tract. METHODS: Patients with the colonic and gastric neoplastic lesions were prospectively enrolled. The lesions were biopsied with forceps or endoscopically resected. Dual fluorescence imaging was performed by using custom axially swept wide-field fluorescence microscopy after topical moxifloxacin and proflavine instillation. Imaging results were compared with both confocal imaging with cell labeling and conventional histological examination. RESULTS: Ten colonic samples (one normal mucosa, nine adenomas) from eight patients and six gastric samples (one normal mucosa, five adenomas) from four patients were evaluated. Dual fluorescence imaging visualized detail cellular structures. Regular glandular structures with polarized cell arrangement were observed in normal mucosa. Goblet cells were preserved in normal colonic mucosa. Irregular glandular structures with scanty cytoplasm and dispersed elongated nuclei were observed in adenomas. Goblet cells were scarce or lost in the colonic lesions. Similarity analysis between moxifloxacin and proflavine imaging showed relatively high correlation values in adenoma compared with those in normal mucosa. Dual fluorescence imaging showed good detection accuracies of 82.3% and 86.0% in the colonic and the gastric lesions, respectively. CONCLUSIONS: High-contrast and high-resolution dual fluorescence imaging was feasible for obtaining detail histopathological information in the gastrointestinal neoplastic lesions. Further studies are needed to develop dual fluorescence imaging as an in vivo real-time visual diagnostic method.

Computational conjugate adaptive optics microscopy for longitudinal through-skull imaging of cortical myelin.

Myelination processes are closely related to higher brain functions such as learning and memory. While their longitudinal observation has been crucial to understanding myelin-related physiology and various brain disorders, skull opening or thinning has been required to secure clear optical access. Here we present a high-speed reflection matrix microscope using a light source with a wavelength of 1.3 µm to reduce tissue scattering and aberration. Furthermore, we develop a computational conjugate adaptive optics algorithm designed for the recorded reflection matrix to optimally compensate for the skull aberrations. These developments allow us to realize label-free longitudinal imaging of cortical myelin through an intact mouse skull. The myelination processes of the same mice were observed from 3 to 10 postnatal weeks to the depth of cortical layer 4 with a spatial resolution of 0.79 µm. Our system will expedite the investigations on the role of myelination in learning, memory, and brain disorders.

Longitudinal monitoring of pancreatic islet damage in streptozotocin-treated mice with optical coherence microscopy.

Pancreatic islets regulate glucose homeostasis in the body, and their dysfunction is closely related to diabetes. Islet transplantation into the anterior chamber of the eye (ACE) was recently developed for both in vivo islet study and diabetes treatment. Optical coherence microscopy (OCM) was previously used to monitor ACE transplanted islets in non-obese diabetic (NOD) mice for detecting autoimmune attack. In this study, OCM was applied to streptozotocin (STZ)-induced diabetic mouse models for the early detection of islet damage. A custom extended-focus OCM (xfOCM) was used to image islet grafts in the ACE longitudinally during STZ-induced beta cell destruction together with conventional bright-field (BF) imaging and invasive glucose level measurement. xfOCM detected local structural changes and vascular degradation during the islet damage which was confirmed by confocal imaging of extracted islet grafts. xfOCM detection of islet damage was more sensitive than BF imaging and glucose measurement. Longitudinal xfOCM images of islet grafts were quantitatively analyzed. All these results showed that xfOCM could be used as a non-invasive and sensitive monitoring method for the early detection of deficient islet grafts in the ACE with potential applications to human subjects.

High-contrast visualization of human skin cancers with combined reflectance confocal and moxifloxacin-based two-photon microscopy: An ex vivo study.

BACKGROUND AND OBJECTIVES: Precise determination of cancer margin during skin cancer surgery is crucial for complete resection and further clinical prognosis. Although reflection confocal microscopy (RCM) has been used for perioperative guiding, its reflection contrast has limitations in detecting cancer cells in the dermis. We previously developed combined reflection confocal (RC) and moxifloxacin-based two-photon (MB-TP) microscopy for sensitive cancer detection by using multiple contrast mechanisms. In this study, the performance of combined microscopy was characterized in various skin cancer specimens and compared with standard methods. MATERIALS AND METHODS: Seven human skin specimens in total including two normal ones, three basal cell carcinomas (BCCs), and two squamous cell carcinomas (SCCs) were collected and imaged in fresh condition. Moxifloxacin ophthalmic solution was topically instilled for cell labeling for 3-5 minutes, then mosaic imaging with the combined microscopy was conducted. The imaged specimens were imaged again after exogenous nuclear labeling for comparison and then processed for standard hematoxylin and eosin histology. RESULTS: Combined RC and MB-TP microscopy visualized both cell and extracellular matrix structures of the skin specimens with multiple contrasts of reflection, moxifloxacin fluorescence, autofluorescence, and second harmonic generation. It distinguished normal cell structures in the skin dermis such as hair follicles, sebaceous and eccrine glands from BCC nests, and SCCs based on cell organization. Normal cell structures had organized cell arrangements for their functions, while cancer cell structures had dense and disorganized cell arrangements. Cellular features found by combined microscopy images were confirmed by both TP microscopy with nuclear labeling and histological examination. CONCLUSIONS: The imaging results showed the potential of combined microscopy for sensitive cancer detection and in vivo guiding of skin cancer surgery.

Deep learning enables reference-free isotropic super-resolution for volumetric fluorescence microscopy.

Volumetric imaging by fluorescence microscopy is often limited by anisotropic spatial resolution, in which the axial resolution is inferior to the lateral resolution. To address this problem, we present a deep-learning-enabled unsupervised super-resolution technique that enhances anisotropic images in volumetric fluorescence microscopy. In contrast to the existing deep learning approaches that require matched high-resolution target images, our method greatly reduces the effort to be put into practice as the training of a network requires only a single 3D image stack, without a priori knowledge of the image formation process, registration of training data, or separate acquisition of target data. This is achieved based on the optimal transport-driven cycle-consistent generative adversarial network that learns from an unpaired matching between high-resolution 2D images in the lateral image plane and low-resolution 2D images in other planes. Using fluorescence confocal microscopy and light-sheet microscopy, we demonstrate that the trained network not only enhances axial resolution but also restores suppressed visual details between the imaging planes and removes imaging artifacts.

Moxifloxacin-Based Extended Depth-of-Field Fluorescence Microscopy for Real-Time Conjunctival Goblet Cell Examination.

Conjunctival goblet cells (CGCs) are mucin-secreting cells in the eye and play essential roles for ocular surface homeostasis. Since various ocular surface pathologies are related to CGC dysfunction, CGC examination is important for the evaluation of ocular surface conditions. Recently we introduced moxifloxacin-based fluorescence microscopy (MBFM) for non-invasive CGC imaging. However, the imaging speed was up to 1 frame per second (fps) and needed to be improved for clinical applications. In this study, we developed a high-speed moxifloxacin-based, extended depth-of-field (EDOF) microscopy system that operates at a maximum imaging speed of 15 fps. The system used a deformable mirror for the high-speed axial sweeping of focal plane during single-frame acquisitions. The acquired images contained both in-focus and out-of-focus information, and deconvolution was used to filter the in-focus information. The system had a DOF of 800 [Formula: see text], field-of-view of 1.2 mm ×1.2 mm, and resolution of [Formula: see text]. Its performance was demonstrated by real-time, breathing-motion-insensitive CGC imaging of mouse and rabbit models, in vivo. High-speed EDOF microscopy has potentials for non-invasive, real-time CGC examinations of human subjects.

In vitro optical performance of multifocal and extended depth-of-focus intraocular lenses in spherical aberration conditions.

PURPOSE: To evaluate and compare the optical performances of 4 different types of intraocular lenses (IOLs) in various spherical aberration (SA) conditions. SETTING: POSTECH, Pohang, South Korea. DESIGN: In vitro laboratory study. METHODS: A custom optical bench system with adaptive optics was used. A monofocal IOL, a bifocal IOL, a trifocal IOL, and an extended depth-of-focus (EDoF) IOL from Zeiss were evaluated by measuring through-focus modulation transfer function (MTF) as a function of vergence. MTF changes with SA from -0.1 to +0.1 with 0.05 µm step size were analyzed and compared. RESULTS: In aberration-free conditions, the 4 IOLs showed different MTF curves consistent with their designs. In SA conditions, all the IOLs showed MTF value decreases and the decrease rates at the far focus varied from 28% to 38% per 0.1 µm SAs. The trifocal IOL had low MTF values at the intermediate focus in the noise level with ±0.1 µm SAs. CONCLUSIONS: All tested IOLs showed MTF decreases with SA in different levels. The trifocal and EDoF IOLs were the most and least sensitive to SA among the evaluated IOLs. The study results might be useful in the selection of IOLs for cataract patients with SA.

Multi-Focus Image Fusion Using Focal Area Extraction in a Large Quantity of Microscopic Images.

The non-invasive examination of conjunctival goblet cells using a microscope is a novel procedure for the diagnosis of ocular surface diseases. However, it is difficult to generate an all-in-focus image due to the curvature of the eyes and the limited focal depth of the microscope. The microscope acquires multiple images with the axial translation of focus, and the image stack must be processed. Thus, we propose a multi-focus image fusion method to generate an all-in-focus image from multiple microscopic images. First, a bandpass filter is applied to the source images and the focus areas are extracted using Laplacian transformation and thresholding with a morphological operation. Next, a self-adjusting guided filter is applied for the natural connections between local focus images. A window-size-updating method is adopted in the guided filter to reduce the number of parameters. This paper presents a novel algorithm that can operate for a large quantity of images (10 or more) and obtain an all-in-focus image. To quantitatively evaluate the proposed method, two different types of evaluation metrics are used: "full-reference" and "no-reference". The experimental results demonstrate that this algorithm is robust to noise and capable of preserving local focus information through focal area extraction. Additionally, the proposed method outperforms state-of-the-art approaches in terms of both visual effects and image quality assessments.

Longitudinal Label-Free Two-Photon Microscopy of Cellular Healing Processes in Non-Ablative Fractional Laser Wounds.

BACKGROUND AND OBJECTIVES: Wound healing is an important biomedical problem with various associated complications. Although cutaneous wound healing has been studied in vivo extensively using various optical imaging methods, early-stage cellular healing processes were difficult to study due to scab formation. The objective of this study is to demonstrate that minimal laser wounds and optical microscopy can access the detailed cellular healing processes of cutaneous wounds from the early stage. STUDY DESIGN/MATERIALS AND METHODS: A non-ablative fractional laser (NAFL) and label-free two-photon microscopy (TPM) were used to induce minimal cutaneous wounds and to image the wounds in three-dimension. Sixteen hairless mice and a single human volunteer were used. NAFL wounds were induced in the hindlimb skin of the mice and in the forearm skin of the human subject. The NAFL wounds were longitudinally imaged during the healing period, starting from an hour post wound induction in the earliest and until 21 days. Cells in the wound and surrounding normal skin were visualized based on two-photon excited auto-fluorescence (TPAF), and cellular changes were tracked by analyzing longitudinal TPM images both qualitatively and quantitatively. Damage and recovery in the skin dermis were tracked by using the second harmonic generation (SHG) signal of collagen. Immunofluorescence and hematoxylin and eosin histology analysis were conducted to validate the TPM results of the murine skin. RESULTS: Cellular healing processes in NAFL wounds and surroundings could be observed by longitudinal TPM. In the case of murine skin, various healing phases including inflammation, re-epithelization, granulation tissue formation, and late remodeling phase including collagen regeneration were observed in the same wounds owing to minimal or no scab formation. The re-epithelization process was analyzed quantitatively by measuring cell density and thickness of the epithelium in the wound surroundings. In the case of the human skin, the access inside the wound was blocked for a few days post wound induction due to scabs but the cellular changes in the wound surroundings were observed from the early stage. Cellular healing processes in the NAFL wound of the human skin were similar to those in murine skin. CONCLUSIONS: The minimal NAFL wound model and label-free TPM demonstrated the cell level assessment of wound healing processes with applicability to human subjects. © 2021 Wiley Periodicals LLC.

Open-top axially swept light-sheet microscopy.

Open-top light-sheet microscopy (OT-LSM) is a specialized microscopic technique for high throughput cellular imaging of large tissue specimens including optically cleared tissues by having the entire optical setup below the sample stage. Current OT-LSM systems had relatively low axial resolutions by using weakly focused light sheets to cover the imaging field of view (FOV). In this report, open-top axially swept LSM (OTAS-LSM) was developed for high-throughput cellular imaging with improved axial resolution. OTAS-LSM swept a tightly focused excitation light sheet across the imaging FOV using an electro tunable lens (ETL) and collected emission light at the focus of the light sheet with a camera in the rolling shutter mode. OTAS-LSM was developed by using air objective lenses and a liquid prism and it had on-axis optical aberration associated with the mismatch of refractive indices between air and immersion medium. The effects of optical aberration were analyzed by both simulation and experiment, and the image resolutions were under 1.6µm in all directions. The newly developed OTAS-LSM was applied to the imaging of optically cleared mouse brain and small intestine, and it demonstrated the single-cell resolution imaging of neuronal networks. OTAS-LSM might be useful for the high-throughput cellular examination of optically cleared large tissues.

Neural stem cells derived from human midbrain organoids as a stable source for treating Parkinson's disease: Midbrain organoid-NSCs (Og-NSC) as a stable source for PD treatment.

Successful clinical translation of stem cell-based therapy largely relies on the scalable and reproducible preparation of donor cells with potent therapeutic capacities. In this study, midbrain organoids were yielded from human pluripotent stem cells (hPSCs) to prepare cells for Parkinson's disease (PD) therapy. Neural stem/precursor cells (NSCs) isolated from midbrain organoids (Og-NSCs) expanded stably and differentiated into midbrain-type dopamine(mDA) neurons, and an unprecedentedly high proportion expressed midbrain-specific factors, with relatively low cell line and batch-to-batch variations. Single cell transcriptome analysis followed by in vitro assays indicated that the majority of cells in the Og-NSC cultures are ventral midbrain (VM)-patterned with low levels of cellular senescence/aging and mitochondrial stress, compared to those derived from 2D-culture environments. Notably, in contrast to current methods yielding mDA neurons without astrocyte differentiation, mDA neurons that differentiated from Og-NSCs were interspersed with astrocytes as in the physiologic brain environment. Thus, the Og-NSC-derived mDA neurons exhibited improved synaptic maturity, functionality, resistance to toxic insults, and faithful expressions of the midbrain-specific factors, in vitro and in vivo long after transplantation. Consequently, Og-NSC transplantation yielded potent therapeutic outcomes that are reproducible in PD model animals. Collectively, our observations demonstrate that the organoid-based method may satisfy the demands needed in the clinical setting of PD cell therapy.

Clinically Compatible Fluorescence Microscopy Based on Moxifloxacin Antibiotic.

High-resolution fluorescence tissue imaging with the application of moxifloxacin as a cell-labelling agent is described. Moxifloxacin is an antibiotic used in the clinic to both treat and prevent bacterial infections, and it has both good pharmacokinetic properties for tissue penetration and intrinsic fluorescence under ultraviolet (UV) excitation. Alternative usage of moxifloxacin as the cell-labelling agent was discovered and its imaging applications have been explored. With moxifloxacin administration, fluorescence microscopy could visualize cells within tissues either in enhanced contrasts or at high imaging speeds. Both linear and nonlinear fluorescence microscopies could be used for moxifloxacin-based tissue imaging. High-contrast cellular imaging was demonstrated in various tissues including the cornea, skin, small and large intestines, and brain. Moxifloxacin-based fluorescence microscopy can be clinically compatible by using the FDA-approved moxifloxacin and it could be used for both diagnosis and surgery guidance. Moxifloxacin-based fluorescence microscopy has been tested in several preclinical studies, including the detection of infecting pathogens in fungal keratitis, and the delineation of tumor margin in brain tumor and skin cancer.

Lipid-Oriented Live-Cell Distinction of B and T Lymphocytes.

The identification of each cell type is essential for understanding multicellular communities. Antibodies set as biomarkers have been the main toolbox for cell-type recognition, and chemical probes are emerging surrogates. Herein we report the first small-molecule probe, CDgB, to discriminate B lymphocytes from T lymphocytes, which was previously impossible without the help of antibodies. Through the study of the origin of cell specificity, we discovered an unexpected novel mechanism of membrane-oriented live-cell distinction. B cells maintain higher flexibility in their cell membrane than T cells and accumulate the lipid-like probe CDgB more preferably. Because B and T cells share common ancestors, we tracked the cell membrane changes of the progenitor cells and disclosed the dynamic reorganization of the membrane properties over the lymphocyte differentiation progress. This study casts an orthogonal strategy for the small-molecule cell identifier and enriches the toolbox for live-cell distinction from complex cell communities.

Moxifloxacin based fluorescence imaging of intestinal goblet cells.

Goblet cells (GCs) in the intestine are specialized epithelial cells that secrete mucins to form the protective mucous layer. GCs are important in maintaining intestinal homeostasis, and the alteration of GCs is observed in inflammatory bowel diseases (IBDs) and neoplastic lesions. In the Barrett's esophagus, the presence of GCs is used as a marker of specialized intestinal metaplasia. Various endomicroscopic imaging methods have been used for imaging intestinal GCs, but high-speed and high-contrast GC imaging has been still difficult. In this study, we developed a high-contrast endoscopic GC imaging method: fluorescence endomicroscopy using moxifloxacin as a GC labeling agent. Moxifloxacin based fluorescence imaging of GCs was verified by using two-photon microscopy (TPM) in the normal mouse colon. Label-free TPM, which could visualize GCs in a negative contrast, was used as the reference. High-speed GC imaging was demonstrated by using confocal microscopy and endomicroscopy in the normal mouse colon. Confocal microscopy was applied to dextran sulfate sodium (DSS) induced colitis mouse models for the detection of GC depletion. Moxifloxacin based GC imaging was demonstrated not only by 3D microscopies but also by wide-field fluorescence microscopy, and intestinal GCs in the superficial region were imaged. Moxifloxacin based endomicroscopy has a potential for the application to human subjects by using FDA approved moxifloxacin.