The core we envision will be centralized but otherwise perform as extensions of individual labs: places where scientists come to do their experiments with the only one major difference being that the equipment will be shared and technical expertise is immediately available. This model is standard in such fields as high energy physics, astronomy, oceanography where the accelerators, telescopes and ships are always shared. As biomedical neuroscience moves into similarly sophisticated and expensive technologies the same transition must take place. MRI and PET are two fields where from the beginning scientists shared equipment. It is now time for microscopy to make the same transition. Accordingly, the Advanced Imaging Core will provide users with access to state-of-the art tools, as well as the training needed to use them effectively. /'. Tools for optical imaging: Optical imaging devices will be housed in a multiuser optical imaging laboratory under the aegis of the CBS. The facility will be housed in over 1500 square feet in the new Northwest Building on the Cambridge campus of Harvard University, which will open in the spring of 2008. The optical imaging facility will be in vertically adjacent space on the two floors occupied by CBS faculty. A stairway connecting the two areas will assure good both flow between the two large rooms while the two floor facility assure researchers on both floors have easy access. Geographically the facility will be located at the nexus of the departments of life, physical, and engineering sciences on the Cambridge campus. The facility will have online sign-up for all major equipment. Neuroscientists throughout the university will be welcome to use the equipment. Equipment for this facility will be provided by Harvard University, through the Center for Brain Science. Equipment will include laser scanning confocals (Zeiss 510, Zeiss Pascal, Olympus FV1000), a Zeiss Axioplan for conventional epifluorescence microscopy, a total internal reflection fluorescence (TIRF) microscope with a sophisticated incubation housing for long lasting cell culture experiments, and a structured illumination scope (Apotome, Zeiss). For automated microscopy we will include motorized the stages on the three main laser scanning scopes and data acquisition software that allows automatic, large-scale, continuous laser scan PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page 60 Continuation Format Page Principal Investigator/Program Director (Last, first, middle): Sanes, Joshua R. montages. We expect to implement more sophisticated microscopes that surpass the diffraction limit of light in the near future and make them available. //. Tools for electron microscopy Through an initiative dubbed """"""""the Connectome Project,"""""""" which has been funded by a foundation gift and Harvard, we have been able to purchase both transmission and scanning electron microscopes, and, as described above, design and construct new ancillary devices. This equipment will be the foundation of the high-throughput microscopy core. It will be housed in more than 1,200 square feet of laboratory space in the lowest basement level of the Northwest Building in Cambridge, beginning in the spring of 2008. This basement level will be shared with the Neuro-engineering facility, the magnetic imaging facility, and possible expansion space for advanced optical imaging. ///'. Training: Hands on training in optical techniques will be available from the technicians we hire if this grant is funded. Another aspect of training is also critical: modern microscope technique depends heavily on an understanding of the concepts of optical microscopy, photochemistry, and digital image processing. Most neurobiologists have little formal education in any of these areas. We believe that these tools cannot be used to full advantage if the user is not familiar with the underlying principles. For example many users of laser scanning microscopes have no appreciation that fluorescence emission saturates due to the relatively long lifetime of the excited state. Thus they excite with far more laser intensity than the in-focus sample region can accommodate causing a disproportionate amount of out of focus light in the final image. To help users we have begun to offer a course in optical imaging that provides a clear (no calculus required) conceptual framework for understanding the behavior of light, microscope optics, fluorescence, confocal, multiphoton, image restoration, and nanoscopy. This one semester course (3 hrs per week) is open to all students, fellows, and faculty. Beginning last fall it has been presented by way of a live video feed to the medical campus to minimize the difficulty of commutes between the campuses. Training in conventional and high-throughput electron microscopy will also be provided. Our sense is that 3D electron microscopy in particular is such a powerful analytic tool that once a facility is up and running many scientists will consider using this approach for their studies. At present we know from discussions with neuroscientists here that there is widespread interest in obtaining ultrastructure within a three dimensional context but most neuroscientists imagine that this will be too burdensome to carry out. Thus, one of our goals will be to educate the community about the potential of using these approaches. As already set up for the optical imaging component, we think that presentations advertising the capabilities of this equipment will be useful. Lichtman?who already teaches a highly regarded graduate course on microscopy ?will use his syllabus as the basis of a """"""""mini-course"""""""" on the three main tools we are developing that we will present to the user community each fall.
| Sarin, Sumeet; Zuniga-Sanchez, Elizabeth; Kurmangaliyev, Yerbol Z et al. (2018) Role for Wnt Signaling in Retinal Neuropil Development: Analysis via RNA-Seq and In Vivo Somatic CRISPR Mutagenesis. Neuron 98:109-126.e8 |
| Hildebrand, David Grant Colburn; Cicconet, Marcelo; Torres, Russel Miguel et al. (2017) Whole-brain serial-section electron microscopy in larval zebrafish. Nature 545:345-349 |
| Duan, Xin; Qiao, Mu; Bei, Fengfeng et al. (2015) Subtype-specific regeneration of retinal ganglion cells following axotomy: effects of osteopontin and mTOR signaling. Neuron 85:1244-56 |
| Roberts, Mike; Jeong, Won-Ki; Vazquez-Reina, Amelio et al. (2011) Neural process reconstruction from sparse user scribbles. Med Image Comput Comput Assist Interv 14:621-8 |
| Jeong, Won-Ki; Schneider, Jens; Turney, Stephen G et al. (2010) Interactive histology of large-scale biomedical image stacks. IEEE Trans Vis Comput Graph 16:1386-95 |
