BACKGROUND: Development of a potent vaccine adjuvant without introduction of any side effects remains an unmet challenge in the field of the vaccine research.
METHODOLOGY/PRINCIPAL FINDINGS: We found that laser at a specific setting increased the motility of antigen presenting cells (APCs) and immune responses, with few local or systemic side effects. This laser vaccine adjuvant (LVA) effect was induced by brief illumination of a small area of the skin or muscle with a nondestructive, 532 nm green laser prior to intradermal (i.d.) or intramuscular (i.m.) administration of vaccines at the site of laser illumination. The pre-illumination accelerated the motility of APCs as shown by intravital confocal microscopy, leading to sufficient antigen (Ag)-uptake at the site of vaccine injection and transportation of the Ag-captured APCs to the draining lymph nodes. As a result, the number of Ag(+) dendritic cells (DCs) in draining lymph nodes was significantly higher in both the 1° and 2° draining lymph nodes in the presence than in the absence of LVA. Laser-mediated increases in the motility and lymphatic transportation of APCs augmented significantly humoral immune responses directed against a model vaccine ovalbumin (OVA) or influenza vaccine i.d. injected in both primary and booster vaccinations as compared to the vaccine itself. Strikingly, when the laser was delivered by a hair-like diffusing optical fiber into muscle, laser illumination greatly boosted not only humoral but also cell-mediated immune responses provoked by i.m. immunization with OVA relative to OVA alone.
CONCLUSION/SIGNIFICANCE: The results demonstrate the ability of this safe LVA to augment both humoral and cell-mediated immune responses. In comparison with all current vaccine adjuvants that are either chemical compounds or biological agents, LVA is novel in both its form and mechanism; it is risk-free and has distinct advantages over traditional vaccine adjuvants.
Figure 2. Laser increases the mobility of dermal APCs. Increased motility of individual cells after laser illumination. Representative time-lapse images demonstrated migratory behaviors of dermal APCs within a 20 minute period of recording: arrows indicate the original location of the cell.
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.
METHOD&RESULT: 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.
The early detection of prostate cancer is a life-saving event in patients harboring potentially aggressive disease. With the development of malignancy, there is a dramatic reduction in the zinc content of prostate tissue associated with the inability of cancer cells to accumulate the ion. In the current study, we used endogenous zinc as an imaging biomarker for prostate cancer detection and progression monitoring. We employed a novel fluorescent sensor for mobile zinc (ZPP1) to detect and monitor the development of prostate cancer in a transgenic mouse model of prostate adenocarcinoma, using in vivo optical imaging correlated with biological fluid-based methods. We showed that the progression of prostate cancer could be monitored in vivo judging by the decreasing zinc content in the prostates of tumor-bearing mice in an age-dependent manner. In a novel quantitative assay, we determined the concentration of mobile zinc in both prostate cell lysates and mouse prostate extracts through simple titration of the ZPP1 sensor. Our findings fulfill the promise of zinc-based prostate cancer diagnostics with the prospect for immediate clinical translation.
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 (Teff cells) and discrimination between natural and induced regulatory T cells (nTreg and iTreg 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 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.
Skeletal muscle is an interesting target for gene therapy. To achieve effective gene introduction in skeletal muscle, a hydrodynamic approach by intravenous injection of plasmid DNA (pDNA) with transient isolation of the limb has attracted attention. In this study, we demonstrated that polyplex nanomicelle, composed of poly(ethyleneglycol) (PEG)-block-polycation and pDNA, showed excellent capacity of gene introduction to skeletal muscle. The evaluation of luciferase expression in the muscle revealed that the nanomicelle provided higher and sustained profiles of transgene expression compared with naked pDNA. Real-time in vivo imaging using a video-rate confocal imaging system suggested that the nanomicelle showed tolerability in the intracellular environment, resulting in the slow but sustained transgene expression. The nanomicelle induced less TNFα induction in the muscle than naked pDNA, indicating the safety of nanomicelle-based gene delivery into the skeletal muscle. Moreover, the nanomicelle showed significant tumor growth suppression for almost a month by introducing a pDNA expressing a soluble form of vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1) to skeletal muscle to obtain anti-angiogenic effect on tumor growth. This feature of sustained effect gives an important advantage of gene therapy, especially on the points of cost effectiveness and high compliance. These results suggest that the hydrodynamic gene introduction to skeletal muscle using polyplex nanomicelle system possesses the potential for effective gene therapy.
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.
We demonstrate a method of cascading multiple diffractive elements for improving the purity of spectral dispersion. The cross-axis cascade was implemented in a two-stage grating spectrograph, resulting in a hundredfold reduction of stray light and a high dynamic range up to −75 dB. The technique can be used for parallel spectral measurements and processing.
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.
Peer-Reviewed Journal - Photonic Device and Technology for Communication
Figure 2. Lymphvasculogenesis from pod+ cells in animal model. In vivo live confocal microscopic image from an ear wound model showed that multiple pod+ cells (DiI) were clearly incorporated into lymphatic vessels and colocalized with LYVE-1. Arrows indicate cells positive for Dil and LYVE-1. Scale bar 20 μm
Figure 3. In vivo detection of zinc in the mouse prostate. Imaging at 30 min after i.v. injection.
Figure 3.Serial endomicroscopy of infiltrating T cells within islet allografts. Representative endomicroscopy images within islet allografts on days 7, 10, 12 and 14 after transplantation in untreated hosts and hosts treated with CD154-specific mAb plus rapamycin
Figure 3.Longitudinal imaging of colorectal tumorigenesis. (a–c) Fluorescence image of colorectal vasculature in a floxed Apc mouse at 11 weeks (a) and 13 weeks (b) after adeno-Cre administration, and of a large lesion at week 17 in another adeno-Cre–treated mouse (c). (d) Fluorescence images of Apc-knockout GFP+ cells (green) and blood vessels (red) at the same site in the descending colon, observed at days 10, 12, 14 and 28. Each image is a projection view of 50-μm z-dimension stack. The images show a GFP+ lesion that appeared to grow (*). Other GFP+ nodules shrunk (arrowhead) or vanished at day 28 (dashed circle). Blood vessels were visualized by intravenously injected FITC-dextran in a–c and by tetramethylrhodamine (TAMRA)-dextran in d. Scale bars, 200 μm.
Figure 5. Distribution of Cy5-labeled pDNA in the muscle fibers after hydrodynamic injection of naked pDNA or polyplex nanomicelle. In vivo confocal images of Cy5-labeled pDNA in histone-GFP mice, in which every cell stably expresses GFP signal in the nuclei. Scale bars 100 μm.
Figure 4. Two-photon imaging by wavelengthswept picosecond pulses. a, Fluorescence image of lens tissue stained with rhodamine B. The horizontal line scan rate is 200 Hz, determined by the wavelength sweep rate. b, Mosaic image of the lens tissue. c, Second-harmonicgeneration mosaic image of murine tail tendon. Scale bar 30 μm.
Figure 4. Images of the intact ear skin in anesthetized mouse. (a) Confocal image showing Langerhans cells expressing GFP+ major histocmpatibility complex (MHC) class II molecules in a genetically engineered mouse. Excitation: 491 nm; emission: 520/35 nm. (b) Two-photon fluorescence image of blood vessels with free-flowing rhodamine-B-dextran conjugates after tail-vein injection [2,000,000 MW, 200 μg/200 μl). Excitation: 800 nm, 30 mW; emission: 590/80 nm. (c) Collagen fibrillar structure visualized by SHG. Excitation: 800 nm, 30 mW; emission: 417/60 nm. The images in (a) to (c) were averaged over 30 consecutive frames acquired in 1 s. (d) to (f) A sequence of frames showing ovarian cancer cells (OVCAR-1) in blood circulation, superimposed on a green fluorescence image of blood vessels in a GFP+ Tie2 mouse. The OVCAR-1 cells were labeled in vitro using DiD (Vybrant@ Invitrogen) and injected at the tail vein [2 million cells in phosphate-buffered saline (PBS) 200 μl]. Scale bar is 50 μm.
The biophysical and biomechanical properties of the crystalline lens (e.g., viscoelasticity) have long been implicated in accommodation and vision problems, such as presbyopia and cataracts. However, it has been difficult to measure such parameters noninvasively. Here, we used in vivo Brillouin optical microscopy to characterize material acoustic properties at GHz frequency and measure the longitudinal elastic moduli of lenses. We obtained three-dimensional elasticity maps of the lenses in live mice, which showed biomechanical heterogeneity in the cortex and nucleus of the lens with high spatial resolution. An in vivo longitudinal study of mice over a period of 2 months revealed a marked age-related stiffening of the lens nucleus. We found remarkably good correlation (log-log linear) between the Brillouin elastic modulus and the Young's modulus measured by conventional mechanical techniques at low frequencies (∼1 Hz). Our results suggest that Brillouin microscopy is potentially useful for basic and animal research and clinical ophthalmology.
Figure 2. In vivo Brillouin imaging of the mouse eye. (c) Brillouin elasticity map of a murine eye in vivo.These cross-sectional images span areas of 1.7 × 2 mm2 (XY), 1.8 × 3.1 mm2 (YZ), and 2 × 3.5 mm2 (XZ). With a sampling interval of 100 μm, it took 20 min over an entire 3D volume. These images taken in vivo visualize the gradient of modulus increasing from the outer cortex to inner nucleus, consistent with previous mechanical and ultrasound measurements of excised lens tissues. Scale bar: 1 mm.
Nanoparticles for cancer therapy and imaging are designed to accumulate in the diseased tissue by exploiting the Enhanced Permeability and Retention (EPR) effect. This limits their size to about 100 nm. Here, using intravital microscopy and elemental analysis, we compare the in vivo localization of particles with different geometries and demonstrate that plateloid particles preferentially accumulate within the tumor vasculature at unprecedented levels, independent of the EPR effect. In melanoma-bearing mice, 1000 × 400 nm plateloid particles adhered to the tumor vasculature at about 5% and 10% of the injected dose per gram organ (ID/g) for untargeted and RGD-targeted particles respectively, and exhibited the highest tumor-to-liver accumulation ratios (0.22 and 0.35). Smaller and larger plateloid particles, as well as cylindroid particles, were more extensively sequestered by the liver, spleen, and lungs. Plateloid particles appeared well-suited for taking advantage of hydrodynamic forces and interfacial interactions required for efficient tumoritropic accumulation, even without using specific targeting ligands.
Figure 1. Intravital microscopy images of individual silicon particles. Time-dependent trajectories of individual 600 × 200 nm (red) and 1000 × 400 nm (blue) plateloid silicon particles in the ear venule of a Tie-2 GFP mouse. The triangles and the circle indicate relatively slow and fast moving particles, respectively. The square indicates particles adhered to the vessel wall. Endothelial cells are colored green, vessel walls are demarcated in yellow (scale bar, 50 μm).
Intravital fluorescence microscopy has emerged as a powerful technique to visualize cellular processes in vivo. However, owing to their size, the objective lenses required have limited physical accessibility to various tissue sites in the internal organs of small animals. The use of small-diameter probes using graded-index (GRIN) lenses expands the capabilities of conventional intravital microscopes to minimally invasive imaging of internal organs. In this protocol, we describe the detailed steps for the fabrication of front- and side-view GRIN probes and the integration and operation of the probes in a confocal microscope to enable visualization of fluorescent cells and microvasculature in various mouse organs. Some experience in building an optical setup is required to complete the protocol. We also present longitudinal imaging of immune cells in renal allografts and tumor development in the colon. Fabrication and integration can be completed in 5–7 h, and a typical in vivo imaging session takes 1–2 h.
Figure 1. GRIN optical probes. (a) A front-view optical probe. A 0.25-pitch (P) CL collects light at the input (from the left) and collimates it onto an RL (1.0P). The collimated output is directed onto an IL (0.25P), which serves as the imaging lens. (b) A side-view optical probe. Red lines illustrate ray tracing. A prism is attached onto the polished facet of the imaging lens to direct light onto the side. (c) Photograph of individual GRIN lenses and prism.
In Vivo Micro-Visualization Laboratory
Graduate School of Nanoscience and Technology (GSNT)
Korea Advanced Institute of Science and Technology (KAIST)