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Frontiers in Bioimaging Masaki Takeuchi and Takeaki Ozawa*
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nder the international collaboration between the National Institute for Basic Biology (NIBB) and the European Molecular Biology Laboratory (EMBL), the second Frontiers in Bioimaging symposium was held March 22–23, 2006, in Okazaki, Japan. This symposium highlighted emerging and innovative technologies for bioimaging and their practical applications in biology. Distinguished scientists offered 26 oral presentations and 20 poster sessions on the state of the art in various biological fields. More than 150 participants took the opportunity to exchange views and share experiences. The meeting covered cutting-edge topics such as the development of fluorescent and bioluminescent probes, analytical techniques with novel concepts, new microscopic systems for 3D imaging of living subjects, and biological progress with imaging techniques. Newly developed probes, analytical techniques, and microscopic systems were the main focus of the symposium. Existing fluorescent proteins are powerful tools for multicolor labeling of different proteins in a single living cell. However, the difficulty of aligning several lasers and the emission crossover between the fluorophores make multicolor imaging a challenge. Atsushi Miyawaki (RIKEN) introduced newly developed fluorescent proteins with various spectral properties (1). For example, the protein Keima experiences a large shift between excitation and emission spectra: the fluorescent protein is excited at 440 nm and emits light at 620 nm. Combining Keima with cyan fluorescent protein allows quantification and imaging of protein–pro-
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tein interactions by fluorescence crosscorrelation spectroscopy with a single 458-nm laser line. Labeling a target protein with synthetic chemical probes is also important for detection, purification, and functional studies. For practical applications, the labeling reaction must be highly specific. Kai Johnsson of the Swiss Federal Institute for Technology (Ecole Polytechnique Fédérale de Lausanne) talked about new methods for the covalent and specific labeling of fusion proteins with chemically diverse compounds. He demonstrated several applications, including labeling with spectroscopic and caged probes, selectively immobilizing protein microarrays, and manipulating membrane proteins. These new fluorescent and chemical probes will offer innovative answers to biological questions that traditional approaches have been unable to address. Another topic of interest included the latest developments of analytical techniques for examining protein–protein interactions, protein modification, and protein transport. In the past few decades, genetic and biochemical approaches have led to the identification of thousands of potential protein interactions. But the cell specificity and the subcellular localization of most of these interactions remain unknown. Tom Kerppola (University of Michigan) a talk
Image courtesy of Corbis
Department of Molecular Structure, Institute for Molecular Science, 38 Nishigonaka, Myodaiji, Okazaki, 444-8585 Japan
*Corresponding author,
[email protected].
Published online July 21, 2006 10.1021/cb600266h CCC: $33.50 © 2006 by American Chemical Society
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about protein-interaction analyses by complementation of split fragments of fluorescent proteins. Transcriptional protein complexes and protein modification by ubiquitinfamily peptides were visualized by the complementation technique. Tarik Issad (Centre National de la Recherche Scientifique) discussed protein-interaction analyses based on bioluminescence-resonance energy transfer. The technique makes it possible to visualize the interactions that occur in the insulin-signaling cascade of an insulin receptor and its intracellular adapters. Spatiotemporal visualization of RNA is also important for understanding the complex function of these molecules. Robert Singer (Albert Einstein College of Medicine) presented an analysis of intracellular messenger RNA trafficking by the fluorescence recovery after photobleaching technique. It was demonstrated that nuclear RNA mobility was not directed by other macromolecules but rather governed by rules of simple diffusion. These new analytical techniques will help us design new experimental systems for a variety of purposes and obtain novel biological data about living cells. The third topic of interest was discussed in presentations about microscopes based on new concepts. The development of microscopes with highly spatiotemporal resolution is now one of the most important and challenging areas of the bioimaging field. Ernst Stelzer (EMBL) reported on selective plane illumination microscopy (SPIM), a new technique that allows the observation of large (up to a few millimeters) and even living specimens. The illumination of a light sheet instead of a single laser line, along with the rotation of a sample embedded in agarose, has enabled the rapid capture of 3D images. Because excitation by the light sheet minimizes photobleaching, 4D imaging of moving specimens such as a beating heart of a fish was also made possible. John Sedat (California, San Francisco) talked about OMX, an advanced microscopic system. It is the first practical imple334
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mentation of structured illumination (SI), in which the grid is superimposed onto the sample (fringe projection) by the insertion of a grid structure into the plane diaphragm. After acquisition of several images with different grid positions and combination of those raw images, the grid lines and the image that are out of focus become invisible, and the contrast and image sharpness are much improved. The system was designed to record rapid 3D multiwavelength imaging on live samples. Stelzer also reported that this SI technology was incorporated into the SPIM to improve resolution at a wide range of magnifications. In addition to new chemical and proteinaceous probes, the development of novel hardware for microscopes appears to be a key challenge for the study of complex biological phenomena. Such technological advances in bioimaging contribute significantly to the growing body of biological information. Thus, the bioimaging field is in a position to compile and integrate the data to elucidate new biological information. Jan Ellenberg (EMBL) discussed the MitoCheck project (www. mitocheck.org), which is a genome-wide screening analysis of mitotic genes and is run by various European research groups. Transfected-cell arrays, automated timelapse fluorescence imaging, and computational phenotype analysis of a chromosomesegregation assay were used to develop a fully automated method for microscopybased small interfering RNA screening. The automated imaging system is powerful enough to analyze complex spatiotemporal processes of target proteins in living cells. Scientists can obtain new quantitative and qualitative insights into the dynamic intracellular events of eukaryotic cells. In the near future, many researchers will apply such systematic analyses to the study of the biologically complex eukaryotic cells. In addition to the above-mentioned presentations, other speakers reported on impresTAKEUCHI AND OZAWA
sive recent results and perspectives from various fields in which bioimaging is used. We agree with many participants that this symposium was a fruitful exchange of cutting-edge information about the rapid developments in imaging science, particularly new probes, analytical techniques, and microscopic systems. We would like to express our gratitude to the organizing committee for this successful meeting. In 3 years, NIBB plans to hold the next symposium, which promises even more remarkable advances. REFERENCE 1. Kogure, T., Karasawa, S., Araki, T., Saito, K., Kinjo, M., and Miyawaki, A. (2006) Nat. Biotechnol. 24, 577–581.
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