High Resolution Microscopy and Imaging for Biological Materials
Thursday, January 29, 2009
Presented by the Soft Materials Discussion Group
The Soft Materials Discussion Group regularly convenes investigators in the New York region with an interest in soft materials research and development, and provides a forum for scientists, engineers, and other key stakeholders working in academia, industry, and not-for-profit entities to exchange ideas and discuss advances. To ensure impact globally, the meeting proceedings will be disseminated electronically through the Academy's eBriefings program. The interdisciplinary topics include a range of technologically important materials in colloids, polymers, emulsions, liquid and organic crystals, membranes, proteins, cells, and tissue.
Speakers: Jennifer Lippincott-Schwartz, National Institutes of Health; Maribel Vazquez, The City College of the City University of New York; Charles D. Humphrey, U.S. Centers for Disease Control and Prevention
Advances in Super-resolution Imaging Technologies
Jennifer Lippincott-Schwartz, National Institutes of Health
Superresolution techniques such as photoactivated localization microscopy (PALM) enable the imaging of fluorescent protein chimeras to reveal the organization of genetically-expressed proteins on the nanoscale with a density of molecules high enough to provide structural context. Various applications of this new technology are now possible. One application is for in cellula pulse-chase analysis to follow protein turnover and diffusion of photoactivated fluorescent proteins. Another approach combines the techniques of PALM and single particle tracking to resolve the dynamics of individual molecules by tracking them in live cells. Called single particle tracking PALM (sptPALM), the technique involves activating, localizing and bleaching many subsets of photoactivatated fluorescent protein chimeras in live cells. Spatially-resolved maps of single molecule motions can be obtained by imaging membrane proteins with this technique, providing several orders of magnitude more trajectories per cell than by traditional single particle tracking. By probing distinct subsets of molecules, including Gag and VSVG, sptPALM can provide a powerful means for exploring the origin of spatial and temporal heterogeneities in membranes. Examples such as these will be presented to illustrate the value of super-resolution imaging in providing quantitative insights into protein organization and dynamics at the nanoscale.
A Nano-microfluidic Approach to Understanding Medulloblastoma Dispersal
Maribel Vazquez, The City College of the City University of New York
Medulloblastoma is the most common malignant brain tumor affecting children worldwide. The ability of this disease to rapidly invade healthy brain tissue underscores the need to identify the mechanisms that regulate cell locomotion in order to develop potential new methods of therapeutic intervention. Our laboratory has taken an integrative approach to the study of tumor dispersal by developing an effective model system able to correlate observed extracellular motilities, with activation of the intracellular signal cascades that regulate tumor cell migration. The current study describes the use of nanoprobes to evaluate the translocation of extracellular and cytosolic proteins during medulloblastoma locomotion within controlled microfluidic environments. Results of confocal and electron microscopy illustrate highly specific intracellular binding within live cells, as well as measurements of cell velocity in real-time within microfluidic networks in response to extracellular signaling from known chemoattractant cytokines.
High Resolution Microscopy of Viruses and Virus-like Particles by Traditional Negative Stain Electron Microscopy
Charles D. Humphrey, U.S. Centers for Disease Control and Prevention
Virus cultures and recombinant virus-like particles (VLPs) are produced to make diagnostic immunoassay reagents, vaccines, pharmacological vectors, or biological control agents. These products also may be used for basic studies of virus structure and formation. Monitoring their structural character and purity is crucial to their successful use. The quality of viruses and VLPs cannot always be determined by visual interpretation of protein band locations after gradient centrifugation or gel electrophoresis. Electron microscopy (EM) including scanning EM, transmission EM (TEM), and scanning probe microscopy provide additional methods to evaluate the quality of virus/VLP production and purification. TEM may include vitreous ice embedding, chemical fixation-plastic embedding and thin-section, or negative stain processing prior to TEM. Evaluations are done for preparation purity, particle structural quality, and quantity. TEM of viruses and VLPs prepared by negative staining for quality assessment of viruses and VLPs is performed routinely within our laboratory. Examples, primarily focused on viral gastroenteritis and respiratory disease viruses, will be presented to demonstrate the value of negative stain TEM for these evaluations.