Abstract-The field of molecular imaging has recently seen rapid advances in the development of novel contrast agents and the implementation of insightful approaches to monitor biological processes non-invasively. In particular, superparamagnetic iron oxide nanoparticles (SPIO) have demonstrated their utility as an important tool for enhancing magnetic resonance contrast, allowing researchers to monitor not only anatomical changes, but physiological and molecular changes as well. Applications have ranged from detecting inflammatory diseases via the accumulation of non-targeted SPIO in infiltrating macrophages to the specific identification of cell surface markers expressed on tumors. In this article, we attempt to illustrate the broad utility of SPIO in molecular imaging, including some of the recent developments, such as the transformation of SPIO into an activatable probe termed the magnetic relaxation switch.

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In vivo bioluminescence imaging has been used to monitor Staphylococcus aureus infections in preclinical models by employing bacterial reporter strains possessing a modified lux operon from Photorhabdus luminescens. However, the relatively short emission wavelength of lux (peak 490 nm) has limited tissue penetration. To overcome this limitation, the gene for the click beetle (Pyrophorus plagiophtalamus) red luciferase (luc) (with a longer >600 emission wavelength), was introduced singly and in combination with the lux operon into a methicillin-resistant S. aureus strain. After administration of the substrate D-luciferin, the luc bioluminescent signal was substantially greater than the lux signal in vitro. The luc signal had enhanced tissue penetration and improved anatomical co-registration with infected internal organs compared with the lux signal in a mouse model of S. aureus bacteremia with a sensitivity of approximately 3 × 104 CFU from the kidneys. Finally, in an in vivo mixed bacterial wound infection mouse model, S. aureus luc signals could be spectrally unmixed from Pseudomonas aeruginosa lux signals to noninvasively monitor the bacterial burden of both strains. Therefore, the S. aureus luc reporter may provide a technological advance for monitoring invasive organ dissemination during S. aureus bacteremia and for studying bacterial dynamics during mixed infections.

Over the past decade, firefly Luciferase (fLuc) has been used in a wide range of biological assays, providing insight into gene regulation, protein-protein interactions, cell proliferation, and cell migration. However, it has also been well established that fLuc activity can be highly sensitive to its surrounding environment. In this study, we found that when various cancer cell lines (HeLa, MCF-7, and 293T) stably expressing fLuc were treated with staurosporine (STS), there was a rapid loss in bioluminescence. In contrast, a stable variant of Renilla luciferase (RLuc), RLuc8, exhibited significantly prolonged functionality under the same conditions. To identify the specific underlying mechanism(s) responsible for the disparate sensitivity of RLuc8 and fLuc to cellular stress, we conducted a series of inhibition studies that targeted known intracellular protein degradation/modification pathways associated with cell death. Interestingly, these studies suggested that reactive oxygen species, particularly hydrogen peroxide (H2O2), was responsible for the diminution of fLuc activity. Consistent with these findings, the direct application of H2O2 to HeLa cells also led to a reduction in fLuc bioluminescence, while H2O2 scavengers stabilized fLuc activity. Comparatively, RLuc8 was far less sensitive to ROS. These observations suggest that fLuc activity can be substantially altered in studies where ROS levels become elevated and can potentially lead to ambiguous or misleading findings.

We report the design, testing and in vivo application of pH sensitive contrast agents designed specifically for Cerenkov imaging. Radioisotopes used for positron emission tomography (PET) emit photons via Cerenkov radiation. The multispectral emission of Cerenkov radiation allows for selective bandwidth quenching, where a band of photons are quenched by absorption by a functional dye. Under acidic conditions, 18F-labeled derivatives emit the full spectrum of Cerenkov light. Under basic conditions, the dyes change color and a wavelength-dependent quenching of Cerenkov emission is observed. METHODS Mono and di-18F-labeled derivatives of phenolsulfonphthalein (phenol red) and meta-cresolsulfonphthalein (cresol purple) were synthesized by electrophilic fluorination. Cerenkov emission was measured at different wavelengths as a function of pH in vitro. Intramolecular response was measured in fluorinated probes; intermolecular quenching by mixing phenolphthalein with 18F FDG. Monofluorocresol purple (MFCP) was tested in mice treated with acetazolamide to cause urinary alkalinization and Cerenkov images compared to PET images. RESULTS Fluorinated pH indicators were produced with radiochemical yields of 4-11% at >90% purity. Selective Cerenkov quenching was observed intramolecularly with difluorophenol red or MFCP, and intermolecularly in phenolphthalein 18F-FDG mixtures. The probes were selectively quenched in the bandwidth closest to the indicator’s absorption maximum (λmax) at pHs above the indicator pKa. Addition of acid or base to the probes resulted in reversible switching from unquenched to quenched emission. In vivo, the bladders of acetazolamide-treated mice exhibited a wavelength-dependent quenching in Cerenkov emission, with the greatest reduction occurring near the λmax. Ratiometric imaging at two wavelengths showed significant decreases in Cerenkov emission at basic pH and allowed the estimation of absolute pH in vivo. CONCLUSIONS We have created contrast agents that selectively quench photons emitted during Cerenkov radiation within a given bandwidth. In the presence of a functional dye, such as a pH indicator, this selective quenching allows for a functional determination of pH in vitro and in vivo. This method can be used to obtain functional information from radiolabeled proves using multimodal imaging. It allows for the imaging of non-fluorescent chromophores and is generalizable to any functional dye that absorbs at suitable wavelengths.

In many clinical cases of radicular pain, no noticeable neuropathology is detected by conventional medical imaging strategies. Superparamagnetic iron oxide (SPIO) nanoparticles were evaluated as magnetic resonance contrast agents to specifically detect neuroinflammation at sites of painful injury in a rat model of cervical nerve root compression. Two separate groups of rats were used: an injury group that underwent controlled transient compression of the dorsal root and a sham group that received the same surgical procedures but no injury. Precontrast magnetic resonance imaging (MRI) was performed 6 days after surgery, followed by administration of SPIO via tail vein injection. After 24 hours, T2*-weighted imaging at the site of root injury revealed a postcontrast enhancement of 72.9 ± 31%. This was significantly greater than that of injured animals prior to SPIO administration (5.3 ± 12.9%). SPIO did not generate any significant postcontrast enhancement in the nerve roots of the sham group. Histology confirmed colocalization of SPIO with macrophage at the injury site. These findings suggest that SPIO-enhanced MRI may be a valuable tool to identify otherwise undetectable nerve root compression and enable improved patient management.

KPF report that they are employees of PerkinElmer, a manufacturer of optical and PET imaging equipment. LSM reports that he has received grant support from MedImmune, Pfizer, Moderna Therapeutics, Regeneron Pharmaceuticals, and Boehringer Ingelheim and consulting fees from Integrated BioTherapeutics, related to Staphylococcus aureus vaccines and therapeutics. JMVD reports that he has filed a patent application (WO2015/088346) on the use of 1D9, which is owned by his employer University Medical Center Groningen. NMB reports that he has received consulting fees from Zimmer Biomet, Bonesupport, Daiichi Sankyo, and Onkos and that he is a board or committee member of the Musculoskeletal Tumor Society and Orthopaedic Research and Education Foundation.

The fission yeast Schizosaccharomyces pombe serves as a good genetic model organism for the molecular dissection of the microtubule (MT) cytoskeleton. However, analysis of the number and distribution of individual MTs throughout the cell cycle, particularly during mitosis, in living cells is still lacking, making quantitative modelling imprecise. We use quantitative fluorescent imaging and analysis to measure the changes in tubulin concentration and MT number and distribution throughout the cell cycle at a single MT resolution in living cells. In the wild-type cell, both mother and daughter spindle pole body (SPB) nucleate a maximum of 23 ± 6 MTs at the onset of mitosis, which decreases to a minimum of 4 ± 1 MTs at spindle break down. Interphase MT bundles, astral MT bundles, and the post anaphase array (PAA) microtubules are composed primarily of 1 ± 1 individual MT along their lengths. We measure the cellular concentration of αβ-tubulin subunits to be ~5 µM throughout the cell cycle, of which one-third is in polymer form during interphase and one-quarter is in polymer form during mitosis. This analysis provides a definitive characterization of αβ-tubulin concentration and MT number and distribution in fission yeast and establishes a foundation for future quantitative comparison of mutants defective in MTs.