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We present a versatile extended depth-of-field (EDOF) wide-field fluorescence microscopy using a new, to the best of our knowledge, active device, micro-mirror array lens system (MALS) for calibration-free and orientation-insensitive EDOF imaging. The MALS changed the focal plane during image acquisition, and the system could be operated in any orientation. Two EDOF imaging modes of high-speed accumulation and low-speed surface sectioning were implemented. The performance was demonstrated in non-contact imaging of conjunctival goblet cells in live mice and depth-resolved cellular examination of ex-vivo human cancer specimens. MALS-based EDOF microscopy has potential for versatile cellular examination.
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Lentes , Microscopía Fluorescente , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Animales , Ratones , Humanos , Células Caliciformes/citología , Conjuntiva/citología , Conjuntiva/diagnóstico por imagenRESUMEN
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.
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Encéfalo , Neuronas , Ratones , Animales , Microscopía Fluorescente/métodosRESUMEN
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.
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Adenoma , Proflavina , Humanos , Moxifloxacino , Estudios Prospectivos , Estudios de Factibilidad , Adenoma/patología , Imagen ÓpticaRESUMEN
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.
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Carcinoma Basocelular , Carcinoma de Células Escamosas , Neoplasias Cutáneas , Carcinoma Basocelular/patología , Carcinoma de Células Escamosas/diagnóstico por imagen , Eosina Amarillenta-(YS) , Hematoxilina , Humanos , Microscopía Confocal/métodos , Moxifloxacino , Soluciones Oftálmicas , Neoplasias Cutáneas/patologíaRESUMEN
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.
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Linfocitos B/citología , Membrana Celular/metabolismo , Colorantes Fluorescentes/química , Linfocitos T/citología , Animales , Linfocitos B/química , Linfocitos B/inmunología , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Membrana Celular/química , Citometría de Flujo , Lipidómica , Ratones , Linfocitos T/química , Linfocitos T/inmunologíaRESUMEN
The microbiota regulate hematopoiesis in the bone marrow (BM); however, the detailed mechanisms remain largely unknown. In this study, we explored how microbiota-derived molecules (MDMs) were transferred to the BM and sensed by the local immune cells to control hematopoiesis under steady-state conditions. We reveal that MDMs, including bacterial DNA (bDNA), reach the BM via systemic blood circulation and are captured by CX3CR1+ mononuclear cells (MNCs). CX3CR1+ MNCs sense MDMs via endolysosomal Toll-like receptors (TLRs) to produce inflammatory cytokines, which control the basal expansion of hematopoietic progenitors, but not hematopoietic stem cells, and their differentiation potential toward myeloid lineages. CX3CR1+ MNCs colocate with hematopoietic progenitors at the perivascular region, and the depletion of CX3CR1+ MNCs impedes bDNA influx into the BM. Moreover, the abrogation of TLR pathways in CX3CR1+ MNCs abolished the microbiota effect on hematopoiesis. These studies demonstrate that systemic MDMs control BM hematopoiesis by producing CX3CR1+ MNC-mediated cytokines in the steady-state.
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Células de la Médula Ósea/metabolismo , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/metabolismo , Leucocitos Mononucleares/metabolismo , Microbiota/fisiología , Animales , Receptor 1 de Quimiocinas CX3C/metabolismo , Citocinas/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
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.
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Microscopía , Cicatrización de Heridas , Animales , Colágeno , Rayos Láser , Ratones , PielRESUMEN
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.
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Antibacterianos , Quinolinas , Córnea , Fluoroquinolonas , Microscopía Fluorescente , MoxifloxacinoRESUMEN
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.
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Algoritmos , Procesamiento de Imagen Asistido por Computador , MicroscopíaRESUMEN
Intraocular islet transplantation was investigated as a new procedure to treat diabetes. The development of this procedure requires close monitoring of the function of both eye and islet graft. We developed a soft, smart contact lens to monitor the intraocular pressure and applied this for noninvasive monitoring in association with the intraocular islet transplantation in diabetes. A strain sensor inside the lens can detect detailed changes in intraocular pressure by focusing the strain only in the desired, selective area of the contact lens. In addition, this smart contact lens can transmit the real-time value of the intraocular pressure wirelessly using an antenna. The wireless measurement of intraocular pressure that was obtained using this contact lens had a high correlation with the intraocular pressure measured by a rebound tonometer, thereby proving the good accuracy of the contact lens sensor. In the initial period, a slight elevation of intraocular pressure was observed, but the pressure returned to normal in the initial period after the transplantation. This type of monitoring will provide important information on potential changes in the intraocular pressure associated with the transplantation procedure, and it enables appropriate clinical safety steps to be taken, if needed.
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Cámara Anterior , Lentes de Contacto Hidrofílicos , Presión Intraocular , Trasplante de Islotes Pancreáticos , Animales , Cámara Anterior/fisiopatología , Cámara Anterior/cirugía , Monitoreo Fisiológico , Ratas , Ratas Endogámicas LewRESUMEN
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.
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Pueblo Asiatico , Carcinoma/diagnóstico por imagen , Microscopía de Fluorescencia por Excitación Multifotónica , Moxifloxacino/uso terapéutico , Neoplasias Cutáneas/diagnóstico por imagen , Inhibidores de Topoisomerasa II/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma/etnología , Carcinoma/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Cutáneas/etnología , Neoplasias Cutáneas/patología , Técnicas de Cultivo de TejidosRESUMEN
Two-photon microscopy (TPM) is a three dimensional (3D) microscopic technique based on nonlinear two-photon fluorescence, which has been tested as an alternative to reflectance confocal microscopy (RCM) for detecting fungal keratitis via optical imaging. Although TPM provided images with better contrast than RCM for fungal keratitis, its imaging speed was relatively low because of weak intrinsic signal. Moxifloxacin, a Food and Drug Administration (FDA)-approved antibiotic, was recently used as a cell-labeling agent for TPM. In this study, moxifloxacin was used to label fungal cells for TPM imaging of fungal keratitis models. Fungal cell suspensions and ex vivo fungal keratitis-affected rabbit corneas were prepared using two types of fungal pathogens, Aspergillus fumigatus and Candida albicans, and TPM imaging was performed both with and without moxifloxacin treatment. Fungal cells with enhanced fluorescence were clearly visible by TPM of moxifloxacin-treated fungal cell suspensions. TPM of moxifloxacin-treated fungal keratitis rabbit corneas revealed both the infecting fungal cells and corneal cells similar to those observed in TPM without moxifloxacin treatment, albeit with approximately 10-times enhanced fluorescence. Fungal cells were distinguished from corneal cells on the basis of their distinct morphologies. Thus, TPM with moxifloxacin labeling might be useful for the detection of fungal keratitis at the improved imaging speed.
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Aspergilosis/diagnóstico por imagen , Candidiasis/diagnóstico por imagen , Técnicas de Diagnóstico Oftalmológico , Infecciones Fúngicas del Ojo/diagnóstico por imagen , Queratitis/diagnóstico por imagen , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Moxifloxacino/administración & dosificación , Coloración y Etiquetado/métodos , Animales , Modelos Animales de Enfermedad , Infecciones Fúngicas del Ojo/microbiología , Imagenología Tridimensional/métodos , Queratitis/microbiología , ConejosRESUMEN
Vesicles exchange their contents through membrane fusion processes, kiss-and-run and full-collapse fusion. Indirect observation of these fusion processes using artificial vesicles enhanced our understanding on the molecular mechanisms involved. Direct observation of the fusion processes in a real biological system, however, remains a challenge owing to many technical obstacles. We report a ratiometric two-photon probe offering real-time tracking of lysosomal ATP with quantitative information for the first time. By applying the probe to two-photon live-cell imaging, the lysosomal membrane fusion process in cells has been directly observed and the concentration of its content, lysosomal ATP, has been measured. Results show that the kiss-and-run process between lysosomes proceeds through repeated transient interactions with gradual content mixing, whereas the full-fusion process occurs at once. Furthermore, it is confirmed that both the fusion processes proceed with conservation of the content. Such a small-molecule probe exerts minimal disturbance and hence has potential for studying various biological processes associated with lysosomal ATP.
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Adenosina Trifosfato/análisis , Colorantes Fluorescentes/química , Membranas Intracelulares/química , Lisosomas/química , Fotones , Adenosina Trifosfato/metabolismo , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Lisosomas/metabolismo , Estructura Molecular , Imagen ÓpticaRESUMEN
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.
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Fluorescence imaging of tissues offer an essential means for studying biological systems. Autofluorescence becomes a serious issue in tissue imaging under excitation at UV-vis wavelengths where biological molecules compete with the fluorophore. To address this critical issue, a novel class of fluorophores that can be excited at â¼900 nm under two-photon excitation conditions and emits in the red wavelength region (≥600 nm) has been disclosed. The new π-extended dipolar dye system shows several advantageous features including minimal autofluorescence in tissue imaging and pronounced solvent-sensitive emission behavior, compared with a widely used two-photon absorbing dye, acedan. As an important application of the new dye system, one of the dyes was developed into a fluorescent probe for amyloid-ß plaques, a key biomarker of Alzheimer's disease. The probe enabled in vivo imaging of amyloid-ß plaques in a disease-model mouse, with negligible background signal. The new dye system has great potential for the development of other types of two-photon fluorescent probes and tags for imaging of tissues with minimal autofluorescence.
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Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/análisis , Colorantes Fluorescentes/química , Imagen Óptica , Fotones , Animales , Línea Celular Tumoral , Supervivencia Celular , Modelos Animales de Enfermedad , Colorantes Fluorescentes/síntesis química , Humanos , Ratones , Ratones Transgénicos , Estructura MolecularRESUMEN
Hydrogen sulfide has emerged as an exciting endogenous gasotransmitter in addition to nitric oxide and carbon dioxide. Noninvasive detection methods for hydrogen sulfide thus become indispensable tools for studying its diverse roles in biological systems. Accordingly, fluorescent probes for hydrogen sulfide have received great attention in recent years. A practically useful fluorescent probe for bioimaging of hydrogen sulfide should be selective, sensitive, fast-responsive, biocompatible, observable in the biological optical window, and capable of deep-tissue imaging. These sensing properties, however, are extremely difficult to achieve at the same time. Disclosed here is the two-photon fluorescent probe that meets all of these criteria. The probe belongs to a Michael acceptor system, which raised a serious selectivity issue over the competing biothiols such as cysteine and glutathione. We have addressed the selectivity issue by optimizing the electronic and steric interactions between biothiols and the probe, in addition to achieving very high sensitivity, fast-response, and biocompatibility. Also, the sensing mechanism suggested in the literature was revised. The probe thus enables us to image the endogenously produced hydrogen sulfide with negligible interference from other biothiols in live cells. The excellent sensing properties of the probe combined with its capability of bioimaging thus make it a practically useful tool for further studying biological roles of hydrogen sulfide.
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Benzaldehídos/química , Cisteína/análisis , Colorantes Fluorescentes/química , Glutatión/análisis , Sulfuro de Hidrógeno/análisis , Técnicas Biosensibles , Células HeLa , Humanos , Imagen Óptica , Compuestos de Sulfhidrilo/análisisRESUMEN
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.
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High-resolution imaging of the cornea is important for studying corneal diseases at cellular levels. Confocal microscopy (CM) has been widely used in the clinic, and two-photon microscopy (TPM) has recently been introduced in various pre-clinical studies. We compared the performance of CM and TPM in normal mouse corneas and neovascularized mouse corneas induced by suturing. Balb/C mice and C57BL/6 mice expressing green fluorescent protein (GFP) were used to compare modalities based on intrinsic contrast and extrinsic fluorescence contrast. CM based on reflection (CMR), CM based on fluorescence (CMF), and TPM based on intrinsic/extrinsic fluorescence and second harmonic generation (SHG) were compared by imaging the same sections of mouse corneas sequentially in vivo. In normal mouse corneas, CMR visualized corneal cell morphologies with some background noise, and CMF visualized GFP expressing corneal cells clearly. TPM visualized corneal cells and collagen in the stroma based on fluorescence and SHG, respectively. However, in neovascularized mouse corneas, CMR could not resolve cells deep inside the cornea due to high background noise from the effects of increased structural irregularity induced by suturing. CMF and TPM visualized cells and induced vasculature better than CMR because both collect signals from fluorescent cells only. Both CMF and TPM had signal decays with depth due to the structural irregularity, with CMF having faster signal decay than TPM. CMR, CMF, and TPM showed different degrees of image degradation in neovascularized mouse corneas.
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Córnea/anatomía & histología , Neovascularización de la Córnea/patología , Microscopía Confocal/métodos , Animales , Colágeno/análisis , Sustancia Propia/citología , Modelos Animales de Enfermedad , Epitelio Corneal/citología , Proteínas Fluorescentes Verdes/análisis , Imagenología Tridimensional/métodos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , MicroscopíaRESUMEN
We report a miniaturized probe-based combined two-photon microscopy (TPM) and optical coherence tomography (OCT) system. This system is to study the colorectal cancer in mouse models by visualizing both cellular and structural information of the colon in 3D with TPM and OCT respectively. The probe consisted of gradient index (GRIN) lenses and a 90° reflecting prism at its distal end for side-viewing, and it was added onto an objective lens-based TPM and OCT system. The probe was 2.2 mm in diameter and 60 mm in length. TPM imaging was performed by raster scanning of the excitation focus at the imaging speed of 15.4 frames/s. OCT imaging was performed by combining the linear sample translation and probe rotation along its axis. This miniaturized probe based dual-modal system was characterized with tissue phantoms containing fluorescent microspheres, and applied to image mouse colonic tissues ex vivo as a demonstration. As OCT and TPM provided structural and cellular information of the tissues respectively, this probe based multi-modal imaging system can be helpful for in vivo studies of preclinical animal models such as mouse colonic tumorigenesis.