3D media stabilizes membrane and prolongs lifespan of defolliculated Xenopus laevis oocytes

Xenopus laevis oocytes are commonly used in many fundamental biological studies. One of the major limitations of X. laevis oocytes is their short storage lifespan with most defolliculated oocytes physically deteriorating in 10 days or less. Herein, we identified a 3D Cultrex-based storage media that incorporates extracellular membrane-based hydrogels to maintain oocyte integrity. Under these treatments, the lifespan of the oocytes increased to more than 20 days compared to standard conditions. The treatment preserved the oocytes membrane integrity and did not interfere with mRNA- or cDNA-derived protein expression

Cell-free measurements of brightness of fluorescently labeled antibodies

Validation of imaging contrast agents, such as fluorescently labeled imaging antibodies, has been recognized as a critical challenge in clinical and preclinical studies. As the number of applications for imaging antibodies grows, these materials are increasingly being subjected to careful scrutiny. Antibody fluorescent brightness is one of the key parameters that is of critical importance. Direct measurements of the brightness with common spectroscopy methods are challenging, because the fluorescent properties of the imaging antibodies are highly sensitive to the methods of conjugation, degree of labeling, and contamination with free dyes. Traditional methods rely on cell-based assays that lack reproducibility and accuracy. In this manuscript, we present a novel and general approach for measuring the brightness using antibody-avid polystyrene beads and flow cytometry. As compared to a cell-based method, the described technique is rapid, quantitative, and highly reproducible. The proposed method requires less than ten microgram of sample and is applicable for optimizing synthetic conjugation procedures, testing commercial imaging antibodies, and performing high-throughput validation of conjugation procedures

Fluorescence lifetime imaging ophthalmoscopy.

Imaging techniques based on retinal autofluorescence have found broad applications in ophthalmology because they are extremely sensitive and noninvasive. Conventional fundus autofluorescence imaging measures fluorescence intensity of endogenous retinal fluorophores. It mainly derives its signal from lipofuscin at the level of the retinal pigment epithelium. Fundus autofluorescence, however, can not only be characterized by the spatial distribution of the fluorescence intensity or emission spectrum, but also by a characteristic fluorescence lifetime function. The fluorescence lifetime is the average amount of time a fluorophore remains in the excited state following excitation. Fluorescence lifetime imaging ophthalmoscopy (FLIO) is an emerging imaging modality for in vivo measurement of lifetimes of endogenous retinal fluorophores. Recent reports in this field have contributed to our understanding of the pathophysiology of various macular and retinal diseases. Within this review, the basic concept of fluorescence lifetime imaging is provided. It includes technical background information and correlation with in vitro measurements of individual retinal metabolites. In a second part, clinical applications of fluorescence lifetime imaging and fluorescence lifetime features of selected retinal diseases such as Stargardt disease, age-related macular degeneration, choroideremia, central serous chorioretinopathy, macular holes, diabetic retinopathy, and retinal artery occlusion are discussed. Potential areas of use for fluorescence lifetime imaging ophthalmoscopy will be outlined at the end of this review

Highly sensitive image-derived indices of water-stressed plants using hyperspectral imaging in SWIR and histogram analysis

The optical signature of leaves is an important monitoring and predictive parameter for a variety of biotic and abiotic stresses, including drought. Such signatures derived from spectroscopic measurements provide vegetation indices – a quantitative method for assessing plant health. However, the commonly used metrics suffer from low sensitivity. Relatively small changes in water content in moderately stressed plants demand high-contrast imaging to distinguish affected plants. We present a new approach in deriving sensitive indices using hyperspectral imaging in a short-wave infrared range from 800 nm to 1600 nm. Our method, based on high spectral resolution (1.56 nm) instrumentation and image processing algorithms (quantitative histogram analysis), enables us to distinguish a moderate water stress equivalent of 20% relative water content (RWC). The identified image-derived indices 15XX nm/14XX nm (i.e. 1529 nm/1416 nm) were superior to common vegetation indices, such as WBI, MSI, and NDWI, with significantly better sensitivity, enabling early diagnostics of plant health

Central memory CD8+ T lymphocytes mediate lung allograft acceptance

Memory T lymphocytes are commonly viewed as a major barrier for long-term survival of organ allografts and are thought to accelerate rejection responses due to their rapid infiltration into allografts, low threshold for activation, and ability to produce inflammatory mediators. Because memory T cells are usually associated with rejection, preclinical protocols have been developed to target this population in transplant recipients. Here, using a murine model, we found that costimulatory blockade–mediated lung allograft acceptance depended on the rapid infiltration of the graft by central memory CD8(+) T cells (CD44(hi)CD62L(hi)CCR7(+)). Chemokine receptor signaling and alloantigen recognition were required for trafficking of these memory T cells to lung allografts. Intravital 2-photon imaging revealed that CCR7 expression on CD8(+) T cells was critical for formation of stable synapses with antigen-presenting cells, resulting in IFN-γ production, which induced NO and downregulated alloimmune responses. Thus, we describe a critical role for CD8(+) central memory T cells in lung allograft acceptance and highlight the need for tailored approaches for tolerance induction in the lung

Shortwave-infrared (SWIR) emitters for biological imaging: a review of challenges and opportunities

Shortwave infrared radiation (SWIR) is the portion of the electromagnetic spectrum from approximately 900 nm to 2500 nm. Recent advances in imaging systems have expanded the application of SWIR emitters from traditional fields in materials science to biomedical imaging, and the new detectors in SWIR opened an opportunity of deep tissue imaging. Achieving deep photon penetration while maintaining high resolution is one of the main objectives and challenges in bioimaging used for the investigation of diverse processes in living organisms. The application of SWIR emitters in biological settings is, however, hampered by low quantum efficiency. So far, photoluminescent properties in the SWIR region have not been improved by extending concepts that have been developed for the visible (400–650 nm) and near-infrared (NIR, 700–900 nm) wavelengths, which indicates that the governing behavior is fundamentally different in the SWIR. The focus of this minireview is to examine the mechanisms behind the low efficiency of SWIR emitters as well as to highlight the progress in their design for biological applications. Several common mechanisms will be considered in this review: (a) the effect of the energy gap between the excited and ground state on the quantum efficiency, (b) the coupling of the excited electronic states in SWIR emitters to vibrational states in the surrounding matrix, and (c) the role of environment in quenching the excited states. General strategies to improve the quantum yields for a diverse type of SWIR emitters will be also presented

Multiphoton microscopy with near infrared contrast agents

While multiphoton microscopy (MPM) has been performed with a wide range of excitation wavelengths, fluorescence emission has been limited to the visible spectrum. We introduce a paradigm for MPM of near-infrared (NIR) fluorescent molecular probes via nonlinear excitation at 1550 nm. This all-NIR system expands the range of available MPM fluorophores, virtually eliminates background autofluorescence, and allows for use of fiber-based, turnkey ultrafast lasers developed for telecommunications

3D Media Stabilizes Membrane and Prolongs Lifespan of Defolliculated Xenopus laevis Oocytes

Xenopus laevis oocytes are commonly used in many fundamental biological studies. One of the major limitations of X. laevis oocytes is their short storage lifespan with most defolliculated oocytes physically deteriorating in 10 days or less. Herein, we identified a 3D Cultrex-based storage media that incorporates extracellular membrane-based hydrogels to maintain oocyte integrity. Under these treatments, the lifespan of the oocytes increased to more than 20 days compared to standard conditions. The treatment preserved the oocytes membrane integrity and did not interfere with mRNA- or cDNA-derived protein expression