Ira Mellman - US grants

A field emission scanning electron microscope will be acquired to allow direct imaging of individual macromolecules. The molecular imaging technology will be applied to lung and lens tissues as well as to individual cells, subcellular components, and the extracellular matrix. Receptor and channel protein molecules and polymerizable lipid films will be directly imaged and their structures compared with images obtained by scanning tunnelling microscopy and x-ray diffraction.

ABSTRACT 9730059 Michael Nathanson Yale University A Two-Photon Imaging System for Biological Research and Education This proposal will fund the purchase of a two-photon excitation imaging system for the Center for Cell Imaging at Yale University School of Medicine. Specifically, the Center's confocal microscope will be upgraded to have two- photon capability. Two photon laser excitation imaging has several properties that make it unique, even relative to point scanning confocal imaging. Two photon imaging permits more highly localized excitation of fluorophores, excitation of dyes that excite at lower wavelengths, much deeper sample penetration, less photobleaching, and less cytotoxicity than conventional confocal microscopy. These features improve spatial resolution, especially in thicker tissue preparations such as intact organs, and make it possible for the first time to photolyse caged compounds or photobleach dyes in highly localized, submicron-sized intracellular regions. No two-photon imaging system is currently available at the School of Medicine, although a number of investigators require this technology to advance their research efforts. The range of projects that will make immediate use of this technology include flash photolysis of caged second messengers to examine regulation of Cai2+ signals at the subcellular level, photolysis of caged oligonucleotides to perform in situ PCR at the single cell level, immunochemistry of intact embryos and larvae, examination of acid production in intact gastric glands, examination of intercellular Cai2+ waves in intact, perfused livers, and distribution and trafficking of GFP-labeled proteins in tissues (including brain slices) and in whole brains of transgenic animals. In addition, the Center for Cell Imaging is the only such imaging resource available to all investigators at the School of Medicine. Therefore, the acquisition of the two-photon imaging system by the Center will insure that any investigator at the school will have access to this technology now and in the future.

A grant has been awarded to Yale University under the direction of Drs. Ira Mellman and Derek Toomre for the acquisition of a state-of-the-art multicolor spinning disk confocal microscope for the imaging and analysis of living cells. A central problem facing modern biology is to understand how cellular activities are integrated in space and time; this challenge applies to every branch of cell biology ranging from fundamental aspects of cellular organization and biogenesis to immunology, neurobiology, developmental biology, and microbiology. Increasingly, high resolution live cell imaging is emerging as a key tool in helping one understand how molecules, vesicles, organelles and whole cells are (re)organized in response to internal and external cues. New technologies that enable the rapid analysis of intact cells is needed to give a complete picture of these processes, and to study objects that occupy or dynamically traverse several optical planes as a function of time. Such imaging is therefore referred to as being "four-dimensional", capturing the behavior of a single cell in three physical dimensions plus time. The instrument will be the first of its kind at Yale University enabling experiments that were impossible using slower conventional confocal microscopes. In summary, this unique microscope will enable new applications in cellular imaging and analysis, permitting quantitative analysis of biochemical reactions in living cells and tissues.

This instrumentation will enable researchers from diverse fields to address an array of basic cell biological questions using model systems including yeast, worms, flies, and mammalian cells, all united by a need to investigate the complex spatial-temporal dynamics of complex cellular processes. Not only will data be visualized but a key element of the project, is the involvement of expert biomedical imaging engineers. Under the leadership of Dr. James Duncan, new software for the analysis of cell imaging datasets will be implemented and will foster new exchanges between life scientists and those in the engineering and computational fields. Private corporate partnerships will further enhance the capabilities of the instrument.

In addition to enabling research for a diverse group of students, post-docs and visiting scientists, the instrumentation and supporting infrastructure will provide relevant training and education through seminars and courses. Underrepresented minorities will participate under the auspices of Yale's well established "STARS" summer internship program, and a new biology educational mentorship program for underprivileged New Haven students.

Proposal Title: NIRT: Molecular Nanodevices for Creation of Smart, Adaptable Vaccine Delivery Vehicles
CTS-0609326
Principal Investigator: Tarek Fahmy
Institution: Yale University

This proposal was received in response to Nanoscale Science and Engineering Initiative,
NSF 05-610, category NIRT.

The proposed work, which will be conducted by an interdisciplinary team of established investigators (engineers, cell biologists, and immunologists), will lead to improvements in the state of the art in the preparation of a new generation of vaccine systems, and will provide the first systematic study of the interactions of polymer nanoparticles with both immune system cells such as dendritic cells and tissues in live animals. Our team has considerable expertise with each individual element of the proposed smart nanoparticle system; a dedicated effort in combining these elements will be the major goal of the proposal. The proposed research program will have broad impact because it will explore new methods for synthesis of modular nanoparticles, which may be useful in a wide variety of settings. By focusing on use of these new materials as vaccine vectors, the work will provide an improved understanding of the mechanisms of nanoparticle interaction with dendritic cells, and will therefore contribute understanding that can be applied to significant problems in world health. In addition, this work will lead to a better understanding and better design of materials needed to address important challenges in biology. Unlike approaches relying on the use of engineered recombinant antibodies, our strategy should produce a stable, easy to fabricate vaccine product that might be useful as an agent useful for the challenges posed by global health problems. This research team will approach an important technological problem in nanoscale science within the context of an educational and outreach program that seeks to train young scientists and engineers in a new area of interdisciplinary science that is of immediate societal relevance and public interest: vaccination against viral and biothreat agents. In addition, this novel nanoscale science project, which addresses a problem of biomedical significance, provides an opportunity to attract the attention of young people; the investigators will work with high school students and educators in the great New Haven area through well-established and successful outreach and education programs. This proposal addresses the following research and education theme listed in the program announcement: Biosystems at the Nanoscale.