MRI: Development of FPALM-STORM for Live Cell Single Molecule Microscopy

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This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Proteins are nanometer-scale (one billionth of a meter) machines found inside of cells. In order to see such tiny objects we need microscopy. An electron microscope has good resolution to allow visualization of such small objects, but the samples (usually cells or other biological elements) must be fixed or frozen, so visualizing live cells is not possible. On the other hand, light microscopy, such as fluorescence microscopy, allows for proteins to be localized in living tissue. But, the gains in motion dynamics are lost in resolution, since light makes nanometer objects appear as fuzzy dots that are 100s of nanometers across. In addition, there are thousands of proteins of the same type inside cells, so fuzzy dots give way to fuzzy blobs that are even more difficult to resolve. This proposal aims to greatly enhance the optical resolution using Fluorescence Photoactivation Localization Microscopy (PaLM) and Stochastic Optical Reconstruction Microscopy (StORM). In these techniques, we use special fluorescent molecules that can be switched on to visualize a very few proteins at a time. Each individual fluorescently labeled protein can be localized with high accuracy using mathematical fitting routines. The motions can also be tracked inside cells. These molecules are switched off, and a new set of molecules are switched on, localized, and tracked. The analysis routines will build a map of the locations of all the molecules to reveal detailed structural data. This new instrument will unlock unprecedented new views of cells and scientific revelations of cellular workings. The research scientists and students at the University of Massachusetts Amherst will use this instrument to delve deeper into some basic biological questions. For instance, several groups are interested in the cell division process, which is the underlying process for organ development, tissue and organ regeneration, and cancer cell growth. A few groups focus on plant cell growth, which may have future implications for bio-energy. Another group studies neuron-receptor dynamics to understand memory formation. These new glimpses of cells will result in new information for the study of basic science and reveal amazing images of life inside a cell. Researchers will display these images obtained on a gallery webpage hosted by the University of Massachusetts Department of Physics (www.umass.edu/physics). In addition the University will share these images with the Amherst community in a display of large-format prints of the cells. The prints will be displayed in and around the Amherst area, including the Department of Motor Vehicles and Libraries through the VISUAL program. The prints will include descriptions of the proteins being imaged and the overall science being presented by the image.