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Jun 1, 2013 (Vol. 33, No. 11)

Lights, Camera, and Lots of Cellular Action

  • More Quantitative Information

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    Researchers at Imperial College London are working to provide more quantitative information with noval approaches in fluorescence lifetime imaging on an automated multiwell plate reader. [J. Biophotonics 6(5), 2013.]

    Fluorescence lifetime-based readouts of protein interactions can provide more quantitative information for fluorescence assays compared to intensity-based readouts. Lifetime-based readouts are independent of probe concentration and of the spectral transmission properties of the instrument, or the sample, and thus can be readily translated between different instruments, or from cells in culture to live disease models.

    The Photonics Group at Imperial College London has developed its third prototype of an automated fluorescence lifetime imaging microscopy-Förster resonance energy transfer (FLIM-FRET) multiwell plate reader based on time-gated imaging. They are applying this prototype to probe protein interactions in cell-signaling networks.

    “We are also investigating the application of FLIM and FRET to read out biomolecular interactions in live zebrafish, which can serve as disease models, such as for cancer and inflammation. Since they are relatively transparent, they are optically accessible for longitudinal studies. This means that fewer animals are needed to study disease progression,” commented Paul French, Ph.D., professor of physics at Imperial College London.

    “Of course, murine studies are still necessary at some stage because their physiology is closer to humans but here the strong scattering and attenuation of optical radiation present challenges for fluorescence assays.”

    “Because fluorescence lifetime-based readouts are more robust in the presence of scattering and absorption than intensity-based readouts, they could provide more quantitative in vivo functional information, for example of protein interactions, and longitudinal studies could again reduce the number of animals required for testing. However, it will still be difficult to obtain high-spatial resolution in vivo.”

    In the future, Dr. French believes that high-content analysis of 3D cell and tissue cultures could become an important approach, including for FLIM and FRET multiwell plate assays, and could replace live disease models for some studies.

  • Tracking DNA Repair Kinetics

    Sylvain Costes, Ph.D., principal scientist in the biocomputational modeling and imaging group at Lawrence Berkeley National Laboratory, spoke about his interest in the dose dependence of the response to ionizing radiation, and the repair kinetics of human cells. “We believe it holds the key to a better understanding of the risk from ionizing radiation,” he said.

    “A DNA damage-sensing protein, p53 binding protein (p53BP1) labeled with GFP was monitored as an indirect measure of the incidence of breaks over time. With such an assay, one can monitor the induction and movement of radiation-induced foci (RIF) in live cells, and calculate repair kinetics.

    “At first, we would locate and image a few fluorescent cells, take the specimen from the fluorescence microscope to an x-ray machine, expose the cell, and then run back to the microscope, try to find the initial location, and start imaging again.

    “Even with the specimens on a warm pack, temperature fluctuations occurred and the first time point achieved was often 10 minutes past exposure. Temperature re-equilibration was required when the specimen was returned to the microscope to limit focal changes due to the warming effect of the media. In addition, large cellular movement made acquisition and foci quantification difficult,” Dr. Costes said.

    His team has developed novel RIF counting and cell-tracking algorithms along with custom instrumentation. The researchers also mounted a very small x-ray machine, typically used for material composition detection, directly on the microscope. Aligned with the microscope’s objective, the x-ray device allows irradiation of the specimen on the stage. Microfluidics approached help to keep human cells in eight independent chambers under perfect physiological conditions.

    Stage controls are used to look at wells, expose different wells to different doses, and to control the time points.

    Software allows time-lapse experiments. The software is paused to run an external routine, in this case, irradiation of the cells and allows pauses for different wells, and different dosage levels, providing a precise kinetic experiment.

    The patented technology, including the DNA repair kinetic assay, is in the process of commercialization by Exogen Biotechnology, a company that intends to provide services to track genetic repair capacity.

    One of the causes of an acute reaction to radiotherapy is slower than normal DNA repair. Using DNA repair kinetic assays could help identify patients with different radiation sensitivities. The company is also investigating the correlation of breast cancer risk and DNA repair kinetics to determine if DNA repair kinetics can be used as an initial risk indicator to warrant additional genetic testing.

    “In live-cell imaging, the biggest problem is that we grow cells in a dish, which is far from the real environment. We are really only looking at one aspect of a much bigger problem,” Dr. Costes explained.

    “Microfluidics, combined with tissue engineering to create the complexity of a tissue, will play an important role in the future. Microfluidics is becoming very sophisticated and could provide the means to work with cells while mimicking the micro-environment, the scaffold, that cells live in,” he said. “This is an integral part of the puzzle. With microfluidics we have the tools to design these scaffolds, artificially. They could be used in live-cell imaging in a very elegant way.”

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