Defect height estimation via model-less TSOM under optical resolution.

We propose a new method of through-focus scanning optical microscopy (TSOM) without a reference database, i.e., a model-less TSOM method. Building a TSOM reference database is time-consuming or even impractical in some TSOM applications that involve complex structures, such as 3D NAND, or irregular shapes such as defects. The proposed model-less TSOM method was used to determine just the height of defect particles, for the first time as far as we are aware. Defect height is the only relevant dimension for the display panel application. Specifically, we analyzed 40 organic light-emitting diode (OLED) surface defects using a lab-developed motion-free TSOM tool consisting of a 50× objective lens (numerical aperture (NA) 0.55), a 532-nm light source, an imaging detector with a 7.5-µm pitch, and a deformable mirror. The tool is in-line and capable of achieving high throughput non-destructively, both relevant features for industrial applications. We investigated linear regression relations between newly defined TSOM parameters (TSOM height, TSOM area and TSOM volume) and the defect heights, which were first measured by atomic force microscopy (AFM). Following defect classification based on in-focus images, we successfully found that the AFM height has a linear correlation with 50% TSOM height (H50%) within ± 20.3 nm (1σ) error over the range of 140 to 950 nm. The one-sigma error, i.e., 20.3 nm, was approximately λ/26 or 1/43 of the depth of focus (DOF) of the applied microscope.

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.

Characterization of early-stage cutaneous radiation injury by using optical coherence tomography angiography.

Cutaneous radiation injury (CRI) is a skin injury caused by exposure to high dose ionizing radiation (IR). Diagnosis and treatment of CRI is difficult due to its initial clinically latent period and the following inflammatory bursts. Early detection of CRI before clinical symptoms will be helpful for effective treatment, and various optical methods have been applied with limitations. Here we show that optical coherence tomography angiography (OCTA) could detect changes in the skin during the latent period in CRI mouse models non-invasively. CRI was induced on the mouse hindlimb with exposure to various IR doses and the injured skin regions were imaged longitudinally by OCTA until the onset of clinical symptoms. OCTA detected several changes in the skin including the skin thickening, the dilation of large blood vessels, and the irregularity in vessel boundaries. Some of OCTA findings were confirmed by histology. The study results showed that OCTA could be used for early CRI detection.

Fast and sensitive delineation of brain tumor with clinically compatible moxifloxacin labeling and confocal microscopy.

Delineation of brain tumor margins during surgery is critical to maximize tumor removal while preserving normal brain tissue to obtain optimal clinical outcomes. Although various imaging methods have been developed, they have limitations to be used in clinical practice. We developed a high-speed cellular imaging method by using clinically compatible moxifloxacin and confocal microscopy for sensitive brain tumor detection and delineation. Moxifloxacin is a Food and Drug Administration (FDA) approved antibiotic and was used as a cell labeling agent through topical administration. Its strong fluorescence at short visible excitation wavelengths allowed video-rate cellular imaging. Moxifloxacin-based confocal microscopy (MBCM) was characterized in normal mouse brain specimens and visualized their cytoarchitecture clearly. Then, MBCM was applied to both brain tumor murine models and two malignant human brain tumors of glioblastoma and metastatic cancer. MBCM detected tumors in all the specimens by visualizing dense and irregular cell distributions, and tumor margins were easily delineated based on the cytoarchitecture. An image analysis method was developed for automated detection and delineation. MBCM demonstrated sensitive delineation of brain tumors through cytoarchitecture visualization and would have potentials for human applications, such as a surgery-guiding method for tumor removal.

Moxifloxacin Labeling-Based Multiphoton Microscopy of Skin Cancers in Asians.

BACKGROUND AND OBJECTIVES: Although multiphoton microscopy (MPM) can visualize both cell and extracellular matrix (ECM) structures of the skin in high-contrast without exogenous labeling, label-free MPM is usually too slow to image clinically relevant large regions. A high-speed MPM method would be beneficial for evaluating clinical skin specimens by increasing the imaging area. In this study, moxifloxacin labeling-based MPM (moxifloxacin MPM) was characterized in various human skin cancer specimens. STUDY DESIGN/MATERIALS AND METHODS: Moxifloxacin ophthalmic solution was used for cell-labeling and MPM imaging was conducted afterwards. Moxifloxacin MPM was characterized in ex vivo normal human skin and skin cancer specimens in comparison with the label-free MPM and fluorescence confocal microscopy (FCM) using acridine orange as a labeling agent. Then, moxifloxacin MPM was applied to various ex vivo human skin cancer specimens including basal cell carcinoma (BCC), squamous cell carcinoma (SCC), dermatofibrosarcoma protuberans (DFSP). Results of moxifloxacin MPM were compared with bright-field clinical and histopathologic findings. RESULTS: Moxifloxacin MPM imaged both cells and collagen in the skin, similarly to label-free MPM, but with enhanced fluorescence intensities in cells and enhanced imaging speeds. Moxifloxacin MPM imaged cells in the skin similarly to acridine orange-based FCM. Moxifloxacin MPM of various human skin cancer specimens imaged their specific cellular features. The microscopic features detected in moxifloxacin MPM were confirmed with histological images. CONCLUSIONS: This observational pilot study demonstrated that moxifloxacin MPM could detect specific cellular features of various skin cancers in good correlation with histopathological images in Asian patients at the higher imaging speed than label-free MPM. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Two-photon microscopy of Paneth cells in the small intestine of live mice.

Paneth cells are one of the principal epithelial cell types in the small intestine, located at the base of intestinal crypts. Paneth cells play key roles in intestinal host-microbe homeostasis via granule secretion, and their dysfunction is implicated in pathogenesis of several diseases including Crohn's disease. Despite their physiological importance, study of Paneth cells has been hampered by the limited accessibility and lack of labeling methods. In this study, we developed a simple in vivo imaging method of Paneth cells in the intact mouse small intestine by using moxifloxacin and two-photon microscopy (TPM). Moxifloxacin, an FDA-approved antibiotic, was used for labeling cells and its fluorescence was strongly observed in Paneth cell granules by TPM. Moxifloxacin labeling of Paneth cell granules was confirmed by molecular counterstaining. Comparison of Paneth cells in wild type, genetically obese (ob/ob), and germ-free (GF) mice showed different granule distribution. Furthermore, Paneth cell degranulation was observed in vivo. Our study suggests that TPM with moxifloxacin labeling can serve as a useful tool for studying Paneth cell biology and related diseases.

Erratum: Visualization of laser tattoo removal treatment effects in a mouse model by two-photon microscopy: erratum.

[This corrects the article on p. 3735 in vol. 8, PMID: 28856046.].

Three-photon tissue imaging using moxifloxacin.

Moxifloxacin is an antibiotic used in clinics and has recently been used as a clinically compatible cell-labeling agent for two-photon (2P) imaging. Although 2P imaging with moxifloxacin labeling visualized cells inside tissues using enhanced fluorescence, the imaging depth was quite limited because of the relatively short excitation wavelength (<800 nm) used. In this study, the feasibility of three-photon (3P) excitation of moxifloxacin using a longer excitation wavelength and moxifloxacin-based 3P imaging were tested to increase the imaging depth. Moxifloxacin fluorescence via 3P excitation was detected at a >1000 nm excitation wavelength. After obtaining the excitation and emission spectra of moxifloxacin, moxifloxacin-based 3P imaging was applied to ex vivo mouse bladder and ex vivo mouse small intestine tissues and compared with moxifloxacin-based 2P imaging by switching the excitation wavelength of a Ti:sapphire oscillator between near 1030 and 780 nm. Both moxifloxacin-based 2P and 3P imaging visualized cellular structures in the tissues via moxifloxacin labeling, but the image contrast was better with 3P imaging than with 2P imaging at the same imaging depths. The imaging speed and imaging depth of moxifloxacin-based 3P imaging using a Ti:sapphire oscillator were limited by insufficient excitation power. Therefore, we constructed a new system for moxifloxacin-based 3P imaging using a high-energy Yb fiber laser at 1030 nm and used it for in vivo deep tissue imaging of a mouse small intestine. Moxifloxacin-based 3P imaging could be useful for clinical applications with enhanced imaging depth.

Comparison between reflectance confocal microscopy and 2-photon microscopy in early detection of cutaneous radiation injury in a mouse model in vivo.

Cutaneous radiation injury (CRI) is a skin injury caused by high-dose exposure of ionizing radiation (IR). For proper treatment, early detection of CRI before clinical symptoms is important. Optical microscopic techniques such as reflectance confocal microscopy (RCM) and 2-photon microscopy (TPM) have been tested as the early diagnosis method by detecting cellular changes. In this study, RCM and TPM were compared in the detection of cellular changes caused by CRI in an in vivo mouse model. CRI was induced on the mouse hindlimb skin with various IR doses and the injured skin regions were imaged longitudinally by both modalities until the onset of clinical symptoms. Both RCM and TPM detected the changes of epidermal cells and sebaceous glands before clinical symptoms in different optical contrasts. RCM detected changes of cell morphology and scattering property based on light reflection. TPM detected detail changes of cellular structures based on autofluorescence of cells. Since both RCM and TPM were sensitive to the early stage CRI by using different contrasts, the optimal method for clinical CRI diagnosis could be either individual methods or their combination.

Mono-allyloxylated Cucurbit[7]uril Acts as an Unconventional Amphiphile To Form Light-Responsive Vesicles.

Serendipitously, mono-allyloxylated cucurbit[7]uril (AO1 CB[7]) was discovered to act as an unconventional amphiphile which self-assembles into light-responsive vesicles (AO1 CB[7]VC) in water. Although the mono-allyloxy group, directly tethered on the periphery of CB[7], is much shorter (C4) than the hydrophobic tails of conventional amphiphiles, it played an important role in vesicle formation. Light-activated transformation of the allyloxy group by conjugation with glutathione was exploited as a remote tool to disrupt the vesicle. The vesicle showed on-demand release of cargo upon irradiation by a laser, after they were internalized into cancer cells. This result demonstrated the potential of AO1 CB[7]VC as a new type of light-responsive intracellular delivery vehicle for the release of therapeutic cargo, within cells, on demand.

Erratum: Visualization of laser tattoo removal treatment effects in a mouse model by two-photon microscopy: publisher's note.

[This corrects the article on p. 3735 in vol. 8, PMID: 28856046.].

Visualization of laser tattoo removal treatment effects in a mouse model by two-photon microscopy.

Laser tattoo removal is an effective method of eliminating tattoo particles in the skin. However, laser treatment cannot always remove the unwanted tattoo completely, and there are risks of either temporary or permanent side effects. Studies using preclinical animal models could provide detailed information on the effects of laser treatment in the skin, and might help to minimize side effects in clinical practices. In this study, two-photon microscopy (TPM) was used to visualize the laser treatment effects on tattoo particles in both phantom specimens and in vivo mouse models. Fluorescent tattoo ink was used for particle visualization by TPM, and nanosecond (ns) and picosecond (ps) lasers at 532 nm were used for treatment. In phantom specimens, TPM characterized the fragmentation of individual tattoo particles by tracking them before and after the laser treatment. These changes were confirmed by field emission scanning electron microscopy (FE-SEM). TPM was used to measure the treatment efficiency of the two lasers at different laser fluences. In the mouse model, TPM visualized clusters of tattoo particles in the skin and detected their fragmentation after the laser treatment. Longitudinal TPM imaging observed the migration of cells containing tattoo particles after the laser treatment. These results show that TPM may be useful for the assessment of laser tattoo removal treatment in preclinical studies.

Dermoscopy guided dark-field multi-functional optical coherence tomography.

Dermoscopy is a skin surface microscopic technique allowing specular reflection free observation of the skin, and has been used to examine pigmented skin lesions. However, dermoscopy has limitations in providing depth information due to lack of 3D resolution. In order to overcome the limitations, we developed dermoscopy guided multi-functional optical coherence tomography (MF-OCT) providing both high-contrast superficial information and depth-resolved structural, birefringent, and vascular information of the skin simultaneously. Dermoscopy and MF-OCT were combined by using a dichroic mirror, and dark-field configuration was adapted for MF-OCT to reduce specular reflection. After characterization, dermoscopy guided MF-OCT was applied to several human skin lesions such as the scar, port-wine stain (PWS) as well as the normal skin for demonstration. Various features of the scar and PWS were elucidated by both dermoscopy and MF-OCT. Dermoscopy guided MF-OCT may be useful for evaluation and treatment monitoring of skin lesions in clinical applications.

Visualization of prostatic nerves by polarization-sensitive optical coherence tomography.

Preservation of prostatic nerves is critical to recovery of a man's sexual potency after radical prostatectomy. A real-time imaging method of prostatic nerves will be helpful for nerve-sparing radical prostatectomy (NSRP). Polarization-sensitive optical coherence tomography (PS-OCT), which provides both structural and birefringent information of tissue, was applied for detection of prostatic nerves in both rat and human prostate specimens, ex vivo. PS-OCT imaging of rat prostate specimens visualized highly scattering and birefringent fibrous structures superficially, and these birefringent structures were confirmed to be nerves by histology or multiphoton microscopy (MPM). PS-OCT could easily distinguish these birefringent structures from surrounding other tissue compartments such as prostatic glands and fats. PS-OCT imaging of human prostatectomy specimens visualized two different birefringent structures, appearing fibrous and sheet-like. The fibrous ones were confirmed to be nerves by histology, and the sheet-like ones were considered to be fascias surrounding the human prostate. PS-OCT imaging of human prostatectomy specimens along the perimeter showed spatial variation in the amount of birefringent fibrous structures which was consistent with anatomy. These results demonstrate the feasibility of PS-OCT for detection of prostatic nerves, and this study will provide a basis for intraoperative use of PS-OCT.

Moxifloxacin: Clinically compatible contrast agent for multiphoton imaging.

Multiphoton microscopy (MPM) is a nonlinear fluorescence microscopic technique widely used for cellular imaging of thick tissues and live animals in biological studies. However, MPM application to human tissues is limited by weak endogenous fluorescence in tissue and cytotoxicity of exogenous probes. Herein, we describe the applications of moxifloxacin, an FDA-approved antibiotic, as a cell-labeling agent for MPM. Moxifloxacin has bright intrinsic multiphoton fluorescence, good tissue penetration and high intracellular concentration. MPM with moxifloxacin was demonstrated in various cell lines, and animal tissues of cornea, skin, small intestine and bladder. Clinical application is promising since imaging based on moxifloxacin labeling could be 10 times faster than imaging based on endogenous fluorescence.

In vivo characterization of early-stage radiation skin injury in a mouse model by two-photon microscopy.

Ionizing radiation (IR) injury is tissue damage caused by high energy electromagnetic waves such as X-ray and gamma ray. Diagnosis and treatment of IR injury are difficult due to its characteristics of clinically latent post-irradiation periods and the following successive and unpredictable inflammatory bursts. Skin is one of the many sensitive organs to IR and bears local injury upon exposure. Early-stage diagnosis of IR skin injury is essential in order to maximize treatment efficiency and to prevent the aggravation of IR injury. In this study, early-stage changes of the IR injured skin at the cellular level were characterized in an in vivo mouse model by two-photon microscopy (TPM). Various IR doses were applied to the mouse hind limbs and the injured skin regions were imaged daily for 6 days after IR irradiation. Changes in the morphology and distribution of the epidermal cells and damage of the sebaceous glands were observed before clinical symptoms. These results showed that TPM is sensitive to early-stage changes of IR skin injury and may be useful for its diagnosis.

Single-channel stereoscopic video imaging modality based on transparent rotating deflector.

In this study, we developed a single-channel stereoscopic video imaging modality based on a transparent rotating deflector (TRD). Sequential two-dimensional (2D) left and right images were obtained through the TRD synchronized with a camera, and the components of the imaging modality were controlled by a microcontroller unit. The imaging modality was characterized by evaluating the stereoscopic video image generation, rotation of the TRD, heat generation by the stepping motor, and image quality and its stability in terms of the structural similarity index. The degree of depth perception was estimated and subjective analysis was performed to evaluate the depth perception improvement. The results show that the single-channel stereoscopic video imaging modality may: 1) overcome some limitations of conventional stereoscopic video imaging modalities; 2) be a potential economical compact stereoscopic imaging modality if the system components can be miniaturized; 3) be easily integrated into current 2D optical imaging modalities to produce a stereoscopic image; and 4) be applied to various medical and industrial fields.

Dark-field polarization-sensitive optical coherence tomography.

Polarization-sensitive optical coherence tomography (PS-OCT) is a functional OCT providing both structural and birefringent information of the sample, and it has been applied to the studies of various organs having polarization properties. Fiber-based PS-OCT is sensitive to specular reflection from the sample surface, because signal saturation due to the strong specular reflection can make the polarization measurement difficult. We developed a dark-field PS-OCT which can avoid the specular reflection problem. Dark-field PS-OCT was implemented by adapting a hybrid method of Bessel-beam illumination and Gaussian-beam detection, and a PS-OCT method based on passive delay unit (PDU). The new system was characterized in comparison with the conventional Gaussian-beam based method in both polarization components and various samples including the human skin. Dark-field PS-OCT performed as good as the conventional PS-OCT without the specular reflection artifact. Dark-field PS-OCT may be useful in practical situations where the specular reflection is unavoidable.

In vivo wide-field reflectance/fluorescence imaging and polarization-sensitive optical coherence tomography of human oral cavity with a forward-viewing probe.

We report multimodal imaging of human oral cavity in vivo based on simultaneous wide-field reflectance/fluorescence imaging and polarization-sensitive optical coherence tomography (PS-OCT) with a forward-viewing imaging probe. Wide-field reflectance/fluorescence imaging and PS-OCT were to provide both morphological and fluorescence information on the surface, and structural and birefringent information below the surface respectively. The forward-viewing probe was designed to access the oral cavity through the mouth with dimensions of approximately 10 mm in diameter and 180 mm in length. The probe had field of view (FOV) of approximately 5.5 mm in diameter, and adjustable depth of field (DOF) from 2 mm to 10 mm by controlling numerical aperture (NA) in the detection path. This adjustable DOF was to accommodate both requirements for image-based guiding with high DOF and high-resolution, high-sensitivity imaging with low DOF. This multimodal imaging system was characterized by using a tissue phantom and a mouse model in vivo, and was applied to human oral cavity. Information of surface morphology and vasculature, and under-surface layered structure and birefringence of the oral cavity tissues was obtained. These results showed feasibility of this multimodal imaging system as a tool for studying oral cavity lesions in clinical applications.

Three-dimensional stereoscopic imaging modality based on a single optical channel and detector.

A three-dimensional stereoscopic imaging modality (3D-SIM) based on a single optical channel and detector was developed to overcome some of the limitations of conventional 3D-SIM. It produces 3-D stereoscopic images by adjusting the angle of a transparent rotating deflector (TRD) to generate disparity between left and right images. The angular effect of the TRD was demonstrated to investigate the feasibility of the proposed method in 3-D stereoscopic image generation. Results indicate that image disparity increased as a function of the rotation angles of the TRD, while maintaining adequate 3-D perception. These results are expected to facilitate the practical use of a 3D-SIM in medicine.