Previously, we used gold nanoparticles suspended in a buffer. However, metallic particles are not stable in suspension making homogeneous deposition challenging Jackson SN, Ugarov M, Post J, Egan T, Langlais D, Schultz JA and Woods AS. A study of Phospholipids by Ion Mobility TOFMS. JASMS 19, 16551662 (2008). An alternative deposition technique is the implantation of nanoparticles. Implantation is a dry and uniform deposition technique, which initially employed a massive cluster ion source. The gold cluster ion currents were insufficient for implanting large areas, and therefore not suited for full organ imaging Fernandez-Lima FA, Post J, DeBord JD, Eller MJ, Verkhoturov SV, Della-Negra S, Woods AS, and Schweikert EA. Analysis of native biological surfaces using a 100kV Massive Gold Cluster Source. Anal Chem. 83, 8448-8453 (2011). Tissue was implanted with a NPlanter (Ionwerks, Houston, TX). A silver target is inserted into a particle source NanoGen50. These nanoparticles (NP) are generated by expulsion from the metals surface using magnetron sputtering, and then allowed to grow by condensation in a refinement zone (particles are 0.5-15 nm in diameter). The size is selected with a quadrupole mass filter coupled to the magnetron, so that we can insure that the NP size is reproducible. Presently we are using AgNP 6-7 nm in diameter, so we always have the same size nanoparticles implanted in different tissue sections, making implantation reproducible. Finally, the particle beam can be deviated with electro-optics over a minimum of 400 square millimeters (20 x 20), allowing implantation of a whole tissue section at once. We use the equivalent of 2-4 monolayers. It takes 18 minutes to implant each tissue section. By using the same size NP particles and the same number of monolayers. We have standardized the implantation procedure and made it reproducible. Thus making comparison of tissue implanted at various time possible, as they are more likely to give reproducible data. In addition a tissue section can be reimplanted for in-depth profiling. So far we have been able to reimplant and reimage a tissue section nine times in a row. Each image is of an area 10-15 nanometer deeper. However we still are in the process of confirming the depth. We also were able to image the same tissue section five times in a row after a single implantation. The implanter reduces the inherent variability between operators and reduces the influence of humidity and temperature fluctuations, which makes it consistent across time, samples, and operators. Silver implantation is a dry method, and therefore does not cause any blurring or analyte migration which may occur with solvent dissolved matrix. We found that hand spraying with an artistic airbrush is a fairly reproducible method for depositing organic matrices with minimal lipid disruption. However it can be a work intensive and time consuming process that requires an experienced hand. While in implantation, the rapid automated rastering step achieves excellent uniformity in matrix implantation by precisely covering the whole implanted areas. Total control of the amount of particles implanted at each point also contributes to the improved uniformity and reproducibility of the implantation. The quantity of silver and quality of coverage can be validated through measurement.

Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2013
Total Cost
$181,357
Indirect Cost
Name
National Institute on Drug Abuse
Department
Type
DUNS #
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State
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Tovo-Rodrigues, L; Roux, A; Hutz, M H et al. (2014) Functional characterization of G-protein-coupled receptors: a bioinformatics approach. Neuroscience 277:764-79
Woods, Amina S; Jackson, Shelley N; Lewis, Ernest K et al. (2013) MALDI/Post Ionization-Ion Mobility Mass Spectrometry of Noncovalent Complexes of Dopamine Receptors' Epitopes. J Proteome Res :
Woods, A S; Jackson, S N (2013) How adenylate cyclase choreographs the pas de deux of the receptors heteromerization dance. Neuroscience 238:335-44