MOLECULAR IMAGING OF BIOMATERIALS - SINGLE CELLS

Project: Research project

Description

Our goal is to develop and apply mass spectrometric molecular imaging to examine phospholipid domain structure and spatial patterns of neurotransmitter release at single cells. This will be accomplished by use of mass spectrometry as a novel tool for acquiring molecule-specific images of single cells with subcellular spatial resolution. The specific focus of this proposal is to develop the necessary protocols to examine both neurotransmitters and membrane phospholipid chemistry at the subcellular level with a special emphasis on unraveling the mechanism of exocytosis. The instrumentation utilizes a liquid metal ion source which is focused to a size of 50 or 200 nanometers. Molecules are desorbed from this small area into a time-of-flight mas spectrometer. Images are recorded by scanning the beam across the target and acquiring mass spectra at each pixel. Our groups have recently demonstrated the capability of detecting several small molecules directly from single cells. The key to performing these measurements has been development of a special freeze-fracture protocol that allows a cell to be frozen in the laboratory and sectioned in the vacuum environment of the mass spectrometer. Three parallel strategies for implementation of our goal are proposed. First, we plan to continue efforts to enhance the information content associated with the mass spectra. These plans included expanded options for freeze fracture, laser positionization experiments to detect the desorbed neutral species, and the development of a unique cluster ion beam probe which would enhance molecular ion yields by a factor of 10 or more. Second, we plan to construct model systems consisting of Langmuir-Blodgett and liposome phospholipid layers that will define mass spectral parameters for characterization of the structure and dynamics of membrane chemistry. Preliminary data suggest that it is feasible to characterize domains of specific phospholipid molecules, to assess their molecular orientation and to monitor dynamic behavior all at the micrometer spatial dimension. Finally, we plan to develop aq series of experiments encompassing the above protocols and models to elucidate the molecular aspects that take place in and at the cellular membrane following exocytosis. Specifically, we propose to image phospholipids in subcellular domains of the cell and the spatial distribution of neurotransmitters. released during exocytosis at single cells. This work will provide the chemical basis for models of exocytosis (vesicle fusion with the cell membrane) and endocytosis (vesicle recycling).
StatusFinished
Effective start/end date9/30/922/28/14

Funding

  • National Institutes of Health: $341,705.00
  • National Institutes of Health: $413,589.00
  • National Institutes of Health: $411,826.00
  • National Institutes of Health: $502,630.00
  • National Institutes of Health: $330,900.00
  • National Institutes of Health: $411,856.00
  • National Institutes of Health: $563,804.00
  • National Institutes of Health: $344,205.00
  • National Institutes of Health: $480,110.00
  • National Institutes of Health: $341,750.00

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neurotransmitters
membranes
cells
histamines
molecules
mass spectrometers
mass spectra
lipids
mass spectroscopy
rafts
chemistry
dopamine
ion beams
cholesterol
Langmuir-Blodgett films
molecular ions
ice
brain
proposals
halos