Superfund Research Program
Multiscale Imaging and Proteomics Core
Final Progress Reports
Year: 2016 2009
For 2009, the Imaging Core added two new capabilities under the thrust of fluorescent tags and reporters, directed by Roger Tsien. One new capability overcomes the limitation of fluorescent proteins for deep tissue imaging in animals because of absorbance by hemoglobin, resulting in the opacity of tissues to excitation light below 600 nm. Tsien’s group created far-red fluorescent proteins with peak excitation at 600 nm or above (Lin et al., 2009). The brightest one of these, Neptune, performs well in imaging deep tissues in living mice. The other new capability is along the same lines and involves the engineering of a bacteriophytochrome from Deinococcus radiodurans, incorporating biliverdin as the chromophore, into monomeric, infrared-fluorescent proteins, producing excitation and emission maxima of 684 and 708 nm (Shu et al., 2009). These proteins are thus suitable for whole-body imaging.
Under the thrust of advanced instrumentation, directed by Mark Ellisman, the Imaging Core added two confocal microscopes, three scanning electron microscopes (SEM), a transmission electron microscope (TEM) and a scanning transmission electron microscope (S/TEM). The Olympus FluoView1000 is a confocal microscope with automated computer control for producing very large field 3-D mosaic images. It is also equipped with an environment chamber for long-time, live-cell imaging. The Olympus spinning disk confocal microscope is equipped with a high-resolution, highly sensitive CCD camera for high-speed mosaic acquisition using commercial mosaic acquisition software from MicroBrightField. The JEOL JSM SEM is a low vacuum SEM for high-performance, surface-scanning of fine structures and has a resolution of 3.0 nm at 30 kV. The Hitachi variable pressure SU6600 SEM is used for the high-resolution analysis of challenging samples without the need for metal coating. The Technai Spirit TEM offers the machine intelligence needed for optimal and automated 2D and 3D imaging of cells and organelles or even macromolecules. The FEI Titan is a S/TEM providing energy filtering, electron energy loss spectroscopy, elemental mapping, and high angle annular dark field (HAADF) capabilities. It is an ideal tool for obtaining high content three-dimensional information about biological tissues, cells and organelles down to the scale resolving individual macromolecules. The Gatan 3view is a new type of SEM that automatically operates a high-precision ultramicrotome that removes thin slices after the block face has been imaged. In this way, ultrastructural features can be followed through thousands of image slices.
Within the mitochondrion microanatomy thrust, Guy Perkins reported on new insights of mitochondrial structural remodeling during cell death (Ju et al., 2009; Perkins et al., 2009a; Yamaguchi and Perkins, 2009). Recent work demonstrates that crista junctions play a key role in the sequestration and subsequent release of pro-apoptotic effectors during apoptosis. These events are regulated in part by OPA1 and the time course of mitochondrial remodeling and pro-apoptotic protein release was characterized by correlated confocal light microscopy and electron microscopy, including a 3D analysis using electron tomography (Perkins et al., 2009b). Separate work showed that cerium oxide nanoparticles are neuroprotective through the scavenging of nitric oxide and peroxynitrite (Dowding et al., 2009). The Imaging Core aided Karin’s group in a study of sestrins. Sestrins are conserved proteins that accumulate in cells exposed to stress. They activate adenosine monophosphate-activated protein kinase (AMPK) and inhibit target of rapamycin (TOR). It was found that sestrin-dependent control of AMPK-TOR signaling is essential for prevention of mitochondrial dysfunction and for maintaining muscle homeostasis during aging. This year, the Imaging Core also aided the Tukey group through increased sampling of retinas to confirm that there is indeed a large increase in the abundance of centrioles in the kernicterus retina between the outer and inner segments. As part of the microtubule organizing center, centrioles play a role in transport, including organellar transport along microtubules. Further work is needed to sort out what role centrioles actually play in this disease.