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RESEARCH FUNDING

Centers for the Development of High Resolution Probes for Cellular Imaging

OVERVIEW

The Centers for the Development of High Resolution Probes for Cellular Imaging support multi-investigator teams to develop new technologies to enable higher sensitivity biological imaging in living cells. Each of the nine centers will focus on different strategies for probe development, cellular delivery, probe targeting, and signal detection to improve detection schemes by a factor of 10 to 100. A major emphasis of this initiative is to apply novel, high-risk approaches to create fundamentally new probes with enhanced spectral characteristics. The ultimate goal is to develop probes and imaging systems that can be used to routinely achieve single molecule sensitivity for imaging dynamic processes in living cells.

The centers are funded in conjunction with the NIH Roadmap for Medical Research as part of the “New Pathways to Discovery,” an effort to advance our knowledge of biological systems by building a better toolbox for medical research. This initiative originated in NIGMS and was later adopted by the Roadmap.  NIGMS currently supports seven of the centers as Roadmap-affiliated grants.  Funding for all nine centers is expected to total approximately $25 million over four years ($6.8 million the first year).

Copyright 2002 Wiley-Liss, a subsidiary of John Wiley & Sons, Inc. Image by permission of John Wiley & Sons, Inc. and Edward Salmon, Ph.D., University of North Carolina.


Mammalian cell in the last stage of mitosis showing microtubules (green) pulling the chromosomes (red) apart to form two daughter cells.

Image used by permission of John Wiley & Sons, Inc. and Edward Salmon, Ph.D., University of North Carolina.
                               

 

 

 

 

For more information on the Centers for the Development of High Resolution Probes for Cellular Imaging, contact Catherine Lewis Ph.D., Division of Cell Biology and Biophysics, National Institute of General Medical Sciences, 45 Center Drive, Room 2AS.13C, Bethesda MD 20892-6200; tel. (301) 594-0828 or e-mail lewisc@nigms.nih.gov.

SUMMARIES OF THE NINE FUNDED GRANTS

1. Fluorescent Probes for Multiplexed Intracellular Imaging
 
Researchers from Texas A&M University and the University of Pennsylvania plan to create novel probe sets composed of  multiplexed “through-bond energy transfer cassettes,” using multiple, linked donor-acceptor dye pairs that are optimized for cellular imaging. These probes, which efficiently absorb light at one wavelength, emit amplified fluorescent signals at different, resolvable wavelengths close to the red-infrared region, far removed from celullar autofluorescence. The dye cassettes will be specifically adapted for tracking interactions of proteins in cells, ultimately with single molecule detection.
 
Kevin Burgess, Ph.D., Principal Investigator, Texas A&M University
 
 
2. Sub-nm Dendrimer-Metal Nanoclusters as Ultrabright, Modular Targeted in vivo Single Molecule Raman and Fluorescence Labels
 
Metal nanoclusters, composed of silver and gold atoms stabilized on organic dendrimers, exhibit strong, size-dependent emission throughout the visible and near infrared spectrum. The spectral characteristics of these clusters--their small size (< 1 nm), short and highly radiative lifetimes–create signals that have the potential to be several orders of magnitude higher than conventional labels. Grantees from the Georgia Institute of Technology and Emory University plan to functionalize the nanoclusters for attachment to different biological targets and to develop single molecule imaging methods to facilitate detection of the signal inside cells.
 
Robert M. Dickson, Ph.D., Principal Investigator, Georgia Institute of Technology
 
3. Single-Molecule Fluorophores for Cellular Imaging

A group from Stanford and Kent State University plans to synthesize and characterize a new class of highly emissive (dicyanodihydrofuran) fluorophores that exhibit large increases in signal when bound to rigid surfaces. The strategy for incorporating the probes into cells will be based upon the genetically encoded tetracysteine-biarsenical targeting system and then tested for single molecule specificity and detection in bacteria.

 
William E. Moerner, Ph.D., Principal Investigator, Stanford University
 
4. Bioaffinity Nanoparticle Probes for Molecular/Cellular Imaging
A collaborative group will develop a new class of polymer encapsulated bioconjugated luminescent nanoparticles with enhanced optical properties, cellular delivery, and targeting/binding functions for real-time and multicolor imaging in living cells. The focus will be on core-shell semiconductor quantum dots because of their improved brightness, resistance against photobleaching, and simultaneous multicolor excitation.  The researchers will test the probes and their ability to detect them in studies aimed at finding the subcellular locations of p53, nuclear factor B, and androgen receptor in living cells.
 
Shuming Nie, Ph.D., Principal Investigator, Emory University and Georgia Tech
 
5. Probes for Quantitative Optical and Electron Microscopy
A group from Vanderbilt will develop new fluorescent probes in the visible and infrared spectral regions based on three approaches: genetically-encoded proteins, lanthanide chelates, and nanocrystals (quantum dots). Each approach will be tested for imaging of a protein in the plasma membrane as well as an intracellular target. Subcellular resolution fluorescence imaging by widefield, deconvolution, confocal, and multi-photon excitation microscopy will be used to implement and test the new detection schemes based on spectral and time-gated resolution. To reach the highest resolution, the researchers will determine the utility and limitations of using the new probes for direct detection by electron microscopy for correlative imaging.
 
David W. Piston, Ph.D., Principal Investigator, Vanderbilt University Medical Center
 
6. Imaging Single Proteins in vivo with Quantum Dots
Researchers from the Rockefeller University plan to extend and optimize an in vivo trans-splicing and expressed protein ligation approach to ligate quantum dot derivatives to cytosolic or integral membrane proteins.  Their strategy includes development of a  conditional protein trans-splicing approach that will allow probes to be ligated to the target following a designated functional interaction.  The cellular fate of “activated” proteins will thus be monitored by a change in the signal emitted by the probe. The team intends to use these tools to study exocytosis and transport through nuclear pores.
 
Sanford Simon, Ph.D., Principal Investigator, Rockefeller University
 
 
7. Light-Activated Gene Expression in Single Cells
 
Investigators from the Albert Einstein College of Medicine will develop a photoactivatable gene that, upon exposure to light, begins transcription of visible nascent chains of RNA. The ecdysone response element and a caged, photoactivatable ecdysone gene into which an RNA reporter has been inserted, will be used.  Gene expression will be initiated by uncaging the ecdysone in vivo by conventional and two-photon microscopy. The system will be engineered into cancer cells and then imaged intravitally in tumors.  The dynamics of single RNA molecule movements and distribution will be monitored.
 
Robert H. Singer, Ph.D., Principal Investigator, Albert Einstein College of Medicine
 
8. Library-Based Development of New Optical Imaging Probes
The investigators plan three parallel approaches to generate small-molecule and genetically encoded probes that can be targeted to specific RNA or protein sequences inside living cells. In the first, libraries of fluorophores will by synthesized in a combinatorial fashion and then screened for their ability to label small peptide motifs or RNA aptamers with high specificity. In the second approach, the natural bacterial enzyme biotin transferase will be re-engineered to catalyze covalent labeling of fluorescent probes to peptides inside cells. Third, a systematic approach using a combination of rational design and screening of mutant libraries will be used to create green fluorescent proteins with improved photophysical properties.
 
Alice Ting, Ph.D., Principal Investigator, Massachusetts Institute of Technology
 
 
9. Genetically Targetable Labels for Light and EM
 
A team from the University of California and the University of Illinois plan a series of approaches to generate fluorescent proteins with increased photostability and higher quantum yield, to explore quantum dot construction and targeting, and to further develop tetracysteine labeling techniques for light and electron microscopy. Their plans also include exploring genetically targetable labels with long excited state lifetimes based on lanthanide and transition metal luminescence as well as directed evolution of fluorescent proteins to improve their photophysical properties. A major goal of this team is to enable direct visualization in the electron microscope of the same molecules that have been tagged, observed, and dynamically tracked in the light microscope.
 
Roger Y. Tsien, Ph.D., Principal Investigator, University of California, San Diego
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Last reviewed: October 13, 2004

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