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Translational Medicine Molecular Imaging Dr. Braeken also presented a lens-free imaging method that is cheaper than conventional microscopy, while providing detailed information and a large feld of view. "Lens-free imaging makes it possible to perform bright-feld microscopy of cells with high resolution—to 1.4 µm—and a large feld of view—up to 20 mm," he elaborates. With this approach, the sample under investigation is illuminated by a coherent light source, and diffraction patterns are captured on a CMOS imager chip positioned underneath the sample. Images from cell cultures are obtained using dedicated image reconstruction algorithms. "The lens-free imaging system is an optical component-free, compact, and cheap imaging system with high resolution and a large feld of view," Dr. Braeken claimed. The resulting images are comparable to those taken with a conventional phase contrast microscope. The technique also enables reconstruction of a holographic image of cells. It is portable inside a cell incubator for direct time-lapse imaging. Integrating the lens-free imaging system into cell bioreactors helps to monitor the ki- A C B D Continued from page 34 netics of cell growth and differentiation, for example, in human stem cell cultures. Determining PK/PD Different imaging modalities are being used to monitor disease progression and to analyze therapeutic effcacy in preclinical stages of drug development. According to Werner Scheuer, Ph.D., group leader, preclinical optical imaging, pharmacology TR-PD, and pharmaceutical research at Roche Diagnostics, fuorescence and bioluminescence technologies are best because of their sheer simplicity, fast scanning times, nonhazardous radiation, and nonradioactive isotopes. In pharmaceutical drug development, long acquisition times can hinder efforts to determine pharmacodynamics and pharmacokinetics in preclinical models. Speaking with GEN, Dr. Scheuer explained that optical imaging has very short acquisition times, ranging from one second to two minutes. Using fuorescence-labeled antibodies targeting a tumor-associated surface antigen, accumulation in tumor tissue can be accomplished within six to 24 hours post-injection. A detailed evaluation of treatment response shows an axial FATSAT T1W MRI image (A) of a mouse tumor (red outline) and corresponding speckled transformation of the pixels within the region of interest (B). For a human tumor treated with experimental therapy (C & D), the shape and amplitude of the histogram curve has little difference on precontrast CT scans but a different shape and amplitude with contrast following therapy suggesting a favorable treatment response and change in tumor morphology. Imaging Endpoints The World's Brightest Luminescent Protein An Osaka University professor has taken up a common challenge with optogenetic imaging: How do you use light to both control and analyze the activity of individual neurons while avoiding interference between the two? Fluorescent indicators ofer a means to analyze what happens inside the cell after optogenetic manipulation. But because of their common dependence on light, problems can arise when optogenetics and fuorescent indicators are used together in the same cell at the same time. The excitation light that's used to "see" the signal from the indicator might misactivate what's under optogenetic control. For instance, the blue light that excites a fuorescent-based calcium indicator may also activate an optogenetically controlled photosensitive receptor. To address this problem, Osaka University's Takeharu Nagai set out to develop an optoge- 36 | netically compatible indicator that does not require light illumination. His strategy was to reengineer a chemiluminescent probe—which produces its own light through a chemical reaction but is too weak for use in optogenetic studies—to make it as bright as a fuorescent probe. As reported in a recent issue of Nature Communications, Dr. Nagai's lab fused a luminescent protein from a sea pansy with another fuorescent protein. The result is the "Nanolantern," the world's brightest luminescent protein, with a brightness and spatial resolution on par with fuorescence. To test the protein in an optogenetics scheme, Dr. Nagai's group modifed it into a calcium sensor and co-expressed it with a light-sensitive photoreceptor in rat neurons. To visualize Nanolantern signals, they turned to Photometrics' Evolve 512 EMCCD camera. August 2013 | GENengnews.com | Genetic Engineering & Biotechnology News As such, it is possible to monitor the biodistribution of the therapeutic antibodies in preclinical cancer xenografts. By using fuorophores that differ in their emission spectra, it is possible to examine a combination of antibodies. Such multiplexing studies cannot be performed using radioactive isotopes. Further, in combination with luciferasetransfected tumor cells, it is possible to monitor binding kinetics and antitumoral effcacy noninvasively and simultaneously. Fluorescence-labeled antibodies are stable ranging from six to 12 months at -20°C, making them superior to radioactive isotopes. Furthermore, noninvasive fuorescence imaging in mice allows monitoring of blood peak levels, half-life, organ distribution and saturation kinetics. It improves the quality of data for pharmacokinetic and pharmacodynamic simulation. It also reduces the number of animals needed, reducing time and costs. Combination of fuorescence with bioluminescence and subsequent examination of explanted organs by 3D multispectral fuorescence histology enables the monitoring of primary tumor growth, metastasis, and angiogenesis. Dr. Scheuer and his colleagues have demonstrated the advantages of optical imaging in the combined measurement of pharmacodynamics and pharmacokinetics in cancer xenografts. based imaging group. Patients who are identifed using etarfolatide as overexpressing the folate receptor are then treated with vintafolide. The companion imaging agent is used to identify patients that overexpress the specifc receptor, so that only patients who are likely to respond to treatment will actually be given the drug. Vintafolide (MK-8109/EC145) and etarfolatide (EC20) are currently being studied in a Phase III trial involving patients with platinum-resistant ovarian cancer, and a Phase IIb study on patients with non-smallcell lung cancer (NSCLC). Dr. Leamon reported that analysis of Phase II data shows that etarfolatide can identify ovarian cancer and NSCLC patients who could beneft from vintafolide. Patients identifed with 100% FR+ target tumor lesions showed substantial progression-free survival compared to patients with 10% to 90% FR+ target lesions, when treated with single-agent vintafolide. "Due to the numerous benefts of companion imaging agents, including the ability to conduct a noninvasive, whole-body scan of a patient and the ability to decrease clinical risk through use early on in drug development, interest in developing companion imaging technologies will only continue to grow," he said. Predicting Therapeutic Response Translational R&D;, Clinical Trials Christopher P. Leamon, Ph.D., vp, research at Endocyte, presented a novel and personalized approach to identify patients who were most likely to beneft from folate receptor (FR)-targeted therapy. Dr. Leamon has invented small molecule drug conjugates to target receptors that are overexpressed in cancer or arthritis. Vintafolide is a small molecule targeting the folate receptor that is linked to a potent chemotherapy drug. Dr. Leamon also developed the companion imaging agent, etarfolatide, which consists of the same small molecule that targets the folate receptor, but is instead conjugated to a 99mTc- The increasing use of PET imaging has led to a dramatic increase in the number of novel F-18 tracers in preclinical studies, advancing into clinical development. The utility of PET imaging can be enhanced by creating new F-18 PET tracers that can pair with the ever expanding armamentarium of target drugs, said Scott Edwards, Ph.D., vp and GM, R&D;, SciFluor Life Sciences. Researchers at SciFluor are developing new methodologies for synthesizing F-18 tracers including innovative chemistry to incorporate F-18 into a wider array of small molecule drugs. "SciFluor has developed SF0034 as an im- First, though, they had to solve an imaging problem: The light used to stimulate optogenetic processes is so strong, it can "contaminate" the camera and lead to unacceptable background noise. To reduce the noise, they conducted light stimulation and erased the charges during the camera's "dead-time," a feature that is easily accessible in the Evolve. Using these techniques, they were able to track photoreceptor excitation by imaging the Ca2+ increase as reported by the Nano-lantern Ca2+ indicator. With the arrival of the Nano-lantern, imaging can now be performed in the absence of external light, enabling analysis of events that cascade from optogenetically controlled proteins. n Luminescence (left) and fluorescence (right) imaging of HeLa cells expressing Nano-lantern targeted to cytoplasm, mitochondria, and histone H2B. The reference fluorescence signal was captured by exciting Venus with light at 490 nm. Scale bars, 50 mm. Photometrics