Technical Reports in 2011

Ali Ertürk and colleagues present a novel tetrahydrofuran-based histological tissue clearing procedure that renders fixed and unsectioned adult CNS tissue (spinal cord and brainstem) transparent and, as such, fully amenable to a range of different optical imaging techniques. This three-dimensional imaging method can be used for studying axon regeneration and glial reactions.

New methods for analyzing ligand-receptor binding under physiologic conditions in cell-based assays and living animals are much needed for the testing and validation of candidate therapeutic agents targeting ligand-receptor binding. Here, Kathryn Luker and her colleagues developed a molecular imaging assay for ligand-receptor binding based on Gaussia luciferase complementation, focusing on chemokine CXCL12 and its chemokine receptors CXCR4 and CXCR7, although the assay could be applied more broadly to any receptor with a protein ligand.

The future of imaging is the integration of function and anatomy. Hongki Yoo et al. have successfully done just that by combining two existing intravascular imaging techniques into a single catheter-based system. Their dual-modality intra-arterial catheter uses a combination of optical frequency domain imaging and near-infrared fluorescence imaging to simultaneously provide molecular information in the context of the surrounding three-dimensional microanatomy of the artery wall.

Makoto Mitsunaga et al. have developed a new form of molecular-targeted cancer therapy that provides an alternative to current photodynamic approaches where damage to surrounding healthy cells and tissues can be a problem. They use a target-specific photosensitizer based on a near-infrared phthalocyanine dye, which is conjugated to monoclonal antibodies targeting human epidermal growth factor receptors (HER1 and HER2). Selective treatment using this approach was shown in vivo in subcutaneous cancer xenografts in mice.

The prognosis for patients with advanced stage ovarian cancer is poor. Here, Gooitzen van Dam and colleagues demonstrate the first human application of a tumor-specific intraoperative fluorescence imaging methodology using a folate receptor-α (FR-α)-targeted fluorescent agent that exploits the overexpression of FR-α in the majority of epithelial ovarian cancers. It is hoped this approach may lead to improved intraoperative staging and more radical cytoreductive surgery.

By exploiting the thermoreversible properties of the US Food and Drug Administration–approved poloxamer 407 (triblock polymer) and 2-octylcyanoacrylate bioadhesive, Edward Chang et al. have developed a new method of sutureless vascular anastomosis, even in vessels with a diameter of less than 1.0 mm. This nonmechanical, sutureless approach compared favorably to the standard hand-sewn approach in long-term (two-year) rat studies.

Infection by coagulase-positive Staphylococcus aureus (S. aureus) is the most common cause of acute endocarditis, a destructive and progressive condition of heart valves. Here, Peter Panizzi and his colleagues have developed a targeted, noninvasive fluorescence or positron emission technology imaging strategy that uses an engineered analog of prothrombin that can detect S. aureus in vivo in the endocarditic vegetations that form as a result of bacterial colonization.

Ready access to diagnostic tests that work well under remote field conditions is a major barrier to improving the health of people in the developing world. Here, Curtis Chin and his colleagues have developed a chip-based, microfluidic device and handheld reader for the simultaneous and rapid diagnosis of HIV and syphilis that uses only 1 μl of unprocessed whole blood and that was successfully field tested in Rwanda.

Progress in understanding coronary artery disease has been hampered by the inability of current approaches to interrogate the human coronary wall at cellular-level resolution. Here, Liu and colleagues introduce a second-generation form of OCT, called ↘OCT, that provides three-dimensional images of human coronary atherosclerosis at an axial resolution of only 1 ↘m—an order of magnitude greater than that provided by standard OCT systems.

Using the recently developed phosphorescent probe PtP-C343, in combination with two-photon phosphorescence lifetime microscopy, Lecoq and his colleagues offer a method for mapping oxygen levels in both microvascular and extravascular compartments with high spatial and temporal resolution. They used this set-up to make micron-scale simultaneous measurements of partial pressure of oxygen (PO₂) and blood flow in the rat olfactory bulb vasculature and neuropil.

Current approaches for analyzing the functional heterogeneity of immune cells include ELISPOT and flow cytometry. Chao Ma et al. have introduced an antibody-barcode microchip platform that enables the measurement of a large panel of secreted proteins from several hundred single cells or small cell colonies in parallel. The platform offers advantages over existing technologies in terms of cost, multiplexing capacity, types of proteins that can be measured and experiments performed.

Whether graft rejection occurs by direct or indirect presentation of antigen is still controversial. However, using a mouse ear skin allograft model and a two-photon intravital imaging approach, Susanna Celli and her colleagues have been able to dissect some of the dynamic processes involved in graft rejection, including early- and late-stage events at the transplant site, as well as intermediate events in the draining lymph node.

Studies of the recently discovered human rhinovirus species C (HRV-C) virus have been hampered by an inability to propagate the virus in standard cell culture. Yury Bochkov et al. have now developed a tissue culture system for HRV-C using human organ culture of sinus mucosa. This, together with development of a reverse genetics system for HRV-C, should provide a better understanding of HRV-C biology and facilitate discovery of an HRV-C receptor.

Kristoffer Weber et al. describe a new lentiviral vector-mediated RGB (red, green and blue) multicolor cell marking technique for analyzing clonal cell fates in vitro and in vivo. Here they use RGB marking to assess clonality after regeneration of injured livers by transplanted primary hepatocytes, to mark hematopoietic stem/progenitor cells and to assess the clonality of tumor cells. The approach can potentially be adapted to various cell types and other vector systems.

Noninvasive testing for Down's syndrome (trisomy 21) would greatly reduce the risks associated with the more invasive techniques used currently. Earlier identification of differentially methylated regions between fetal DNA and maternal peripheral blood has now enabled Elisavet Papageorgiou and her colleagues to develop a strategy involving methylated DNA immunoprecipitation in combination with real-time quantitative PCR that discriminates normal from trisomy 21 cases in maternal peripheral blood with high sensitivity.

The invasive nature of intravascular ultrasound and other approaches used to screen for atherosclerosis has prompted Marcus Makowski and his colleagues to look for a more noninvasive means of assessing plaque burden. Their new elastin–specific, gadolinium-based MR contrast takes advantage of changes in elastin content that occur during plaque development. Use of the new contrast agent is shown in an Apoe transgenic mouse model of accelerated atherosclerosis.

The current diagnosis of acute kidney injury involves the measurement of renal biomarkers, such as serum creatinine, which provide a crude means of detecting cellular stress and injury. To determine whether Ngal expression provides an alternate renal biomarker capable of detecting the initial phases of renal injury, Paragas et al. have developed an Ngal reporter mouse that offers a noninvasive and real-time method for the continuous and quantitative reporting of cell stress and injury at the injury site.

Tony Ko and his colleagues introduce a fluorescence microendoscopy imaging approach for time-lapse studies of deep brain tissue previously inaccessible to conventional optical imaging techniques. It can be used to study the cellular effects of brain disease over weeks to months with comparable resolution to light microscopy. They use the approach to monitor individual hippocampal neurons, neuronal dendrites and blood vessels and to follow the process of glioma angiogenesis.