Live Images of Donor Dendritic Cells Trafficking via CX3CR1 Pathway.

BACKGROUND: A number of studies have demonstrated the role of CX3CR1 in regulating the migration of monocytes into peripheral tissue and their transformation into dendritic cell (DC). No data are yet available on the importance of chemokine pathways in regulating homeostasis of DC in heart transplants. Recently, we showed that recipients of heart allografts from CX3CR1-/- donors show longer survival. To assess the trafficking of dDC, we have developed and tested a novel in vivo imaging tool in CX3CR1GFP/+ DC (B6 background) heart graft into BALB/c recipient model. RESULTS: Majority of GFP+ cells were noted in the middle of cardiac myocyte. However few hours post transplant, they experienced morphological changes including stretching their extensions (3 and 24 h). However, images from 72 h at cardiac graft showed many of GFP+ cells moved to vessel areas. GFP+ cells were detected in near vessel wall. Only one GFP+ cell was observed in three lymph nodes (two mesenteric and one inguinal) (72 h). CONCLUSION: Our data indicate that immediately post transplant dDC undergo morphological changes and traffic out of the organs via systemic circulation. While, we still noted presence of dDC in the transplanted organs, their trafficking to lymphoid tissue remains to be fully explored.

High-finesse sub-GHz-resolution spectrometer employing VIPA etalons of different dispersion.

We report a novel configuration of a two-stage virtually imaged phased array spectrometer that enables high-throughput sub-GHz spectroscopy at a high finesse (>750). Two etalons with different free spectral range and different dispersion are arranged in an orthogonal direction and spread the spectrum across two dimensions, with a greatly improved rejection ratio of white-light background noise. A proof-of-concept application for Brillouin spectroscopy is demonstrated.

Observation of sound-induced corneal vibrational modes by optical coherence tomography.

The mechanical stability of the cornea is critical for maintaining its normal shape and refractive function. Here, we report an observation of the mechanical resonance modes of the cornea excited by sound waves and detected by using phase-sensitive optical coherence tomography. The cornea in bovine eye globes exhibited three resonance modes in a frequency range of 50-400 Hz. The vibration amplitude of the fundamental mode at 80-120 Hz was ~8 µm at a sound pressure level of 100 dB (2 Pa). Vibrography allows the visualization of the radially symmetric profiles of the resonance modes. A dynamic finite-element analysis supports our observation.

High Speed Sub-GHz Spectrometer for Brillouin Scattering Analysis.

The goal of this protocol is to build a parallel high-extinction and high-resolution optical Brillouin spectrometer. Brillouin spectroscopy is a non-contact measurement method that can be used to obtain direct readouts of viscoelastic material properties. It has been a useful tool in material characterization, structural monitoring and environmental sensing. In the past, Brillouin spectroscopy has usually employed scanning Fabry-Perot etalons to perform spectral analysis. This process requires high illumination power and long acquisition times, making the technique unsuitable for biomedical applications. A recently introduced novel spectrometer overcomes this challenge by employing two VIPAs in a cross-axis configuration. This innovation enables sub-Gigahertz (GHz) resolution spectral analysis with sub-second acquisition time and illumination power within the safety limits of biological tissue. The multiple new applications facilitated by this improvement are currently being explored in biological research and clinical application.

Translating ocular biomechanics into clinical practice: current state and future prospects.

Biomechanics is the study of the relationship between forces and function in living organisms and is thought to play a critical role in a significant number of ophthalmic disorders. This is not surprising, as the eye is a pressure vessel that requires a delicate balance of forces to maintain its homeostasis. Over the past few decades, basic science research in ophthalmology mostly confirmed that ocular biomechanics could explain in part the mechanisms involved in almost all major ophthalmic disorders such as optic nerve head neuropathies, angle closure, ametropia, presbyopia, cataract, corneal pathologies, retinal detachment and macular degeneration. Translational biomechanics in ophthalmology, however, is still in its infancy. It is believed that its use could make significant advances in diagnosis and treatment. Several translational biomechanics strategies are already emerging, such as corneal stiffening for the treatment of keratoconus, and more are likely to follow. This review aims to cultivate the idea that biomechanics plays a major role in ophthalmology and that the clinical translation, lead by collaborative teams of clinicians and biomedical engineers, will benefit our patients. Specifically, recent advances and future prospects in corneal, iris, trabecular meshwork, crystalline lens, scleral and lamina cribrosa biomechanics are discussed.

Perspectives of mid-infrared optical coherence tomography for inspection and micrometrology of industrial ceramics.

Optical coherence tomography (OCT) is a promising tool for detecting micro channels, metal prints, defects and delaminations embedded in alumina and zirconia ceramic layers at hundreds of micrometers beneath surfaces. The effect of surface roughness and scattering of probing radiation within sample on OCT inspection is analyzed from the experimental and simulated OCT images of the ceramic samples with varying surface roughnesses and operating wavelengths. By Monte Carlo simulations of the OCT images in the mid-IR the optimal operating wavelength is found to be 4 µm for the alumina samples and 2 µm for the zirconia samples for achieving sufficient probing depth of about 1 mm. The effects of rough surfaces and dispersion on the detection of the embedded boundaries are discussed. Two types of image artefacts are found in OCT images due to multiple reflections between neighboring boundaries and inhomogeneity of refractive index.

Optical fine-needle imaging biopsy of the brain.

We demonstrate optical fine-needle imaging biopsy (FNIB), combining a fine needle (22 gauge) and a high-resolution side-view probe (350-µm diameter) for minimally invasive interrogation of brain tissue in situ. We apply this technique to examine pathogenesis in murine models of neurodegeneration, brain metastasis of melanoma, and arterial occlusion, respectively. The demonstrated ability to obtain cellular images in the deep brain without craniotomy may be useful in the longitudinal studies of brain diseases.

Subnanometer optical coherence tomographic vibrography.

The ability to quantify and visualize submicrometer-scale oscillatory motions of objects in three dimensions has a wide range of application in acoustics, materials sciences, and medical imaging. Here we demonstrate that volumetric snapshots of rapid periodic motion can be captured using optical coherence tomography (OCT) with subnanometer-scale motion sensitivity and microsecond-scale temporal resolution. This technique, termed OCT vibrography, was applied to generate time-resolved volumetric vibrographs of a miniature drum driven acoustically at several kilohertz.

Harmonic mode locking in a sliding-frequency fiber laser.

We demonstrate a sliding-frequency mode-locked (SFM) erbium fiber laser generating 20 ps pulses with center wavelengths rapidly sweeping across a spectral range of 50 nm. Excess optical nonlinearity in the laser cavity leads to multipulsing, with a tendency to tight pulse bunching (<3 ns) at the fundamental cavity frequency of 25 MHz. The addition of a parallel optical delay line, with a path difference equal to a rational fraction of the cavity length, distributes the pulses uniformly across the entire cavity and achieves a harmonic SFM up to 1 GHz. The result establishes cavity nonlinearity as a critical design parameter for picosecond wavelength-swept lasers.

Triggered optical coherence tomography for capturing rapid periodic motion.

Quantitative cross-sectional imaging of vocal folds during phonation is potentially useful for diagnosis and treatments of laryngeal disorders. Optical coherence tomography (OCT) is a powerful technique, but its relatively low frame rates makes it challenging to visualize rapidly vibrating tissues. Here, we demonstrate a novel method based on triggered laser scanning to capture 4-dimensional (4D) images of samples in motu at audio frequencies over 100 Hz. As proof-of-concept experiments, we applied this technique to imaging the oscillations of biopolymer gels on acoustic vibrators and aerodynamically driven vibrations of the vocal fold in an ex vivo calf larynx model. Our results suggest that triggered 4D OCT may be useful in understanding and assessing the function of vocal folds and developing novel treatments in research and clinical settings.

Podoplanin-expressing cells derived from bone marrow play a crucial role in postnatal lymphatic neovascularization.

BACKGROUND: Emerging evidence has suggested a contribution of bone marrow (BM) cells to lymphatic vessel formation; however, the exact phenotype of the cells with lymphatic endothelial progenitor cell function has yet to be identified. Here, we investigate the identity of BM-derived lymphatic endothelial progenitor cells and their role in lymphatic neovascularization. METHODS AND RESULTS: Culture of BM-mononuclear cells in the presence of vascular endothelial growth factors A and C and endothelial growth factor resulted in expression of lymphatic endothelial cell markers. Among these cells, podoplanin(+) cells were isolated by magnetic-activated cell sorting and characterized by fluorescence-activated cell sorter analysis and immunocytochemistry. These podoplanin(+) cells highly express markers for lymphatic endothelial cells, hematopoietic lineages, and stem/progenitor cells; on further cultivation, they generate lymphatic endothelial cells. We further confirmed that podoplanin(+) cells exist in small numbers in BM and peripheral blood of normal mice but are significantly (15-fold) augmented on lymphangiogenic stimuli such as tumor implantation. Next, to evaluate the potential of podoplanin(+) cells for the formation of new lymphatic vessels in vivo, we injected culture-isolated or freshly isolated BM-derived podoplanin(+) cells into wound and tumor models. Immunohistochemistry demonstrated that the injected cells were incorporated into the lymphatic vasculature, displayed lymphatic endothelial cell phenotypes, and increased lymphatic vascular density in tissues, suggesting lymphvasculogenesis. Podoplanin(+) cells also expressed high levels of lymphangiogenic cytokines and increased proliferation of lymphatic endothelial cells during coculture, suggesting a lymphangiogenic or paracrine role. CONCLUSIONS: Our results provide compelling evidence that BM-derived podoplanin(+) cells, a previously unrecognized cell type, function as lymphatic endothelial progenitor cells and participate in postnatal lymphatic neovascularization through both lymphvasculogenesis and lymphangiogenesis.

In vivo tracking of 'color-coded' effector, natural and induced regulatory T cells in the allograft response.

Here we present methods to longitudinally track islet allograft-infiltrating T cells in live mice by endoscopic confocal microscopy and to analyze circulating T cells by in vivo flow cytometry. We developed a new reporter mouse whose T cell subsets express distinct, 'color-coded' proteins enabling in vivo detection and identification of effector T cells (T(eff) cells) and discrimination between natural and induced regulatory T cells (nT(reg) and iT(reg) cells). Using these tools, we observed marked differences in the T cell response in recipients receiving tolerance-inducing therapy (CD154-specific monoclonal antibody plus rapamycin) compared to untreated controls. These results establish real-time cell tracking as a powerful means to probe the dynamic cellular interplay mediating immunologic rejection or transplant tolerance.

In vivo wide-area cellular imaging by side-view endomicroscopy.

In vivo imaging of small animals offers several possibilities for studying normal and disease biology, but visualizing organs with single-cell resolution is challenging. We describe rotational side-view confocal endomicroscopy, which enables cellular imaging of gastrointestinal and respiratory tracts in mice and may be extensible to imaging organ parenchyma such as cerebral cortex. We monitored cell infiltration, vascular changes and tumor progression during inflammation and tumorigenesis in colon over several months.

Two-photon microscopy by wavelength-swept pulses delivered through single-mode fiber.

Nonlinear microscopy through flexible fiber-optic catheters has potential in clinical diagnostic applications. Here, we demonstrate a new approach based on wavelength-swept narrowband pulses that permits simple fiber-optic delivery without need of the dispersion management and allows nonmechanical beam scanning. Using 0.86 ps pulses rapidly tuned from 789 nm to 822 nm at a sweep rate of 200 Hz, we demonstrate two-photon fluorescence and second-harmonic generation imaging through a 5-m-long standard single-mode fiber.

Long-wavelength optical coherence tomography at 1.7 microm for enhanced imaging depth.

Multiple scattering in a sample presents a significant limitation to achieve meaningful structural information at deeper penetration depths in optical coherence tomography (OCT). Previous studies suggest that the spectral region around 1.7 microm may exhibit reduced scattering coefficients in biological tissues compared to the widely used wavelengths around 1.3 mum. To investigate this long-wavelength region, we developed a wavelength-swept laser at 1.7 microm wavelength and conducted OCT or optical frequency domain imaging (OFDI) for the first time in this spectral range. The constructed laser is capable of providing a wide tuning range from 1.59 to 1.75 microm over 160 nm. When the laser was operated with a reduced tuning range over 95 nm at a repetition rate of 10.9 kHz and an average output power of 12.3 mW, the OFDI imaging system exhibited a sensitivity of about 100 dB and axial and lateral resolution of 24 mum and 14 mum, respectively. We imaged several phantom and biological samples using 1.3 mum and 1.7 microm OFDI systems and found that the depth-dependent signal decay rate is substantially lower at 1.7 microm wavelength in most, if not all samples. Our results suggest that this imaging window may offer an advantage over shorter wavelengths by increasing the penetration depths as well as enhancing image contrast at deeper penetration depths where otherwise multiple scattered photons dominate over ballistic photons.

Entangled-photon coincidence fluorescence imaging.

We describe fluorescence imaging using the second-order correlation of entangled photon pairs. The proposed method is based on the principle that one photon of the pair carries information on where the other photon has been absorbed and has produced fluorescence in a sample. Because fluorescent molecules serve as "detectors" breaking the entanglement, multiply-scattered fluorescence photons within the sample do not cause image blur. We discuss experimental implementations.

In vivo confocal and multiphoton microendoscopy.

The ability to conduct high-resolution fluorescence imaging in internal organs of small animal models in situ and over time can make a significant impact in biomedical research. Toward this goal, we developed a real-time confocal and multiphoton endoscopic imaging system. Using 1-mm-diameter endoscopes based on gradient index lenses, we demonstrate video-rate multicolor multimodal imaging with cellular resolution in live mice.

Comprehensive esophageal microscopy by using optical frequency-domain imaging (with video).

BACKGROUND: Optical coherence tomography (OCT) has been used for high-resolution endoscopic imaging and diagnosis of specialized intestinal metaplasia, dysplasia, and intramucosal carcinoma of the esophagus. However, the relatively slow image-acquisition rate of the present OCT systems inhibits wide-field imaging and limits the clinical utility of OCT for diagnostic imaging in patients with Barrett's esophagus. OBJECTIVE: This study describes a new optical imaging technology, optical frequency-domain imaging (OFDI), derived from OCT, that enables comprehensive imaging of large esophageal segments with microscopic resolution. DESIGN: A prototype OFDI system was developed for endoscopic imaging. The system was used in combination with a balloon-centering catheter to comprehensively image the distal esophagus in swine. RESULTS: Volumetric images of the mucosa and portions of the muscularis propria were obtained for 4.5-cm-long segments. Image resolution was 7 microm in depth and 30 microm parallel to the lumen, and provided clear delineation of each mucosal layer. The 3-dimensional data sets were used to create cross-sectional microscopic images, as well as vascular maps of the esophagus. Submucosal vessels and capillaries were visualized by using Doppler-flow processing. CONCLUSIONS: Comprehensive microscopic imaging of the distal esophagus in vivo by using OFDI is feasible. The unique capabilities of this technology for obtaining detailed information of tissue microstructure over large mucosal areas may open up new possibilities for improving the management of patients with Barrett's esophagus.

Multimodality optical imaging of embryonic heart microstructure.

Study of developmental heart defects requires the visualization of the microstructure and function of the embryonic myocardium, ideally with minimal alterations to the specimen. We demonstrate multiple endogenous contrast optical techniques for imaging the Xenopus laevis tadpole heart. Each technique provides distinct and complementary imaging capabilities, including: 1. 3-D coherence microscopy with subcellular (1 to 2 microm) resolution in fixed embryos, 2. real-time reflectance confocal microscopy with large penetration depth in vivo, and 3. ultra-high speed (up to 900 frames per second) that enables real-time 4-D high resolution imaging in vivo. These imaging modalities can provide a comprehensive picture of the morphologic and dynamic phenotype of the embryonic heart. The potential of endogenous-contrast optical microscopy is demonstrated for investigation of the teratogenic effects of ethanol. Microstructural abnormalities associated with high levels of ethanol exposure are observed, including compromised heart looping and loss of ventricular trabecular mass.