Source: UNIV OF WISCONSIN submitted to
COLLOIDAL METAL PARTICLES FOR HIGH RESOLUTION BIOLOGICAL LABELING
 
PROJECT DIRECTOR: Albrecht, R. M.
 
PERFORMING ORGANIZATION
ANIMAL SCIENCES
UNIV OF WISCONSIN
MADISON,WI 53706
 
NON TECHNICAL SUMMARY: An understanding of cell function requires knowledge of the molecular organization within the cell. Cells are little machines and in order to understand how the machine works we need to know how the parts fit together. This project seeks to develop effective labeling strategies that will allow simultaneous visualization of multiple molecular species so the way in which they fit together in the cell can be determined.
 
OBJECTIVES: Objectives: The proposed studies are designed to develop novel methodologies for labeling of biological specimens at subcellular, molecular, and submolecular levels of resolution. The new technology will have the potential to substantially increase the number of labels that can be used simultaneously and will offer greater flexibility in cell labeling compared to existing techniques. The organization of molecules and macromolecular assemblies within the cell is a key factor in cell function. In order to help determine intracellular organization considerable effort has been directed toward the development of fluorescent probes having differing emission wavelengths. In this way specific cellular elements can be selectively stained with a dye of a unique color. Thus the relationships of different molecules and structures, stained by different colored fluorescent dyes, can be viewed simultaneously and spatial relationships determined. This approach is effective in fluorescent light or confocal fluorescent light (photon) based imaging systems and is limited to the resolution attainable with such systems. Determining relationships at molecular or submolecular levels of spatial resolution requires instrumentation capable of considerably higher levels of spatial resolution. Principal among these are electron based imaging systems. Until recently there has been no ability to easily recognize different wavelength (in essence different "color") electrons. Hence simultaneous labeling of multiple molecular species in electron microscopes has relied primarily on spherical colloidal gold nanoparticles of differing sizes. However the number of particles providing resolution in the molecular size range is generally limited to one or two; then particles become too large. The ability to use labels that are all of the same very small size but can be distinguished from on another based on properties other than size would be of considerable benefit to studies investigating molecular level organization within the cell. The specific aims of this study are as follows: Aim 1. To synthesize colloidal nanoparticles of uniform size from Au and other metals including Ag, Pd, Pt, Rh, Ru, Mo, Cd, Ni, Cu, and various of the lanthanides. Aim 2. To use electron energy loss spectroscopy to distinguish between particles of different metal composition. Aim 3. To synthesize colloidal metal particles of different, unique shapes which can be identified in conventional transmission electron microscopy, scanning electron microscopy, and scanned force microscopy. Aim 4. To conjugate antibodies, and other ligands or small active fragments of antibodies and ligands to colloidal particles in preparation for subsequent cell labeling.
 
APPROACH: Approach Aim 1. To synthesize colloidal nanoparticles of uniform size from Au and other metals. Colloidal phase preparations of Au, and other metals, will be synthesized via the reduction of a metal salt. The metal initially forms crystalline nuclei around which condenses additional reduced metal. Pursuant to this aim are the following goals: (1) we will attempt to form colloids of Ag, Cd, Cu, Mo, Ni, Pd, Pt, Rh, Ru and the lanthanides by using the citrate, tannic acid, white phosphorus, sodium borohydride, and thiocyanate methods used in the synthesis of cAu; (2) several other reduction methods that are specific to certain of the metals of interest will be used; and (3) we will attempt to control particle size and size distribution by altering the reducing agent and metal salt concentrations, along with reaction temperature, pH, and the presence of protecting polymers such as sodium polyacrylate and polyvinyl alcohol. Aim 2. To use electron energy loss spectroscopy (EELS) to distinguish between metal particles. EELS enables the detection of small numbers of atoms of a single element within a specimen. The metals under consideration as potential colloidal labels have been chosen based in part upon the unique energy loss spectrum of each. To date we have used EELS to detect cNi, cCu, cPt, cRh, cAu, cAg, and cPd particles. Current studies will be directed as follows: (1) techniques to identify particles synthesized from the various metals will be refined to permit particles in the 2nm to 30nm size range to be dentified; (2) studies will be pursued to enable the discrimination between individual metal particles in a mixture of two or more colloids; and (3) strategies will be devised to allow particle discrimination in cases where energy loss overlap occurs. Aim 3. To synthesize colloidal metal particles of different unique shapes. In this study we will pursue the synthesis of nanoparticles having unique shapes. The metallic particles will retain the properties which facilitate detection in the TEM, SEM, SFM and AFM but additionally the different shapes will permit differentiation of similarly sized particles attached to different identifier molecules. Studies will be directed toward the following: (1) production of nanoparticles of cAu in uniform small sizes but with different morphologies including spherical, polygonal, and irregular or lobate, and (2) production of nanoparticles of Pt, Pd, and Rh with unique polygonal and rough or lobate morphologies. Aim 4. To conjugate antibodies and other ligands to colloidal particles for cell labeling. The utility of any of the colloidal metal nanoparticles for labeling of biological systems requires that identifier molecules attach tightly to the surface and remain active. The binding of molecular species to metallic surfaces via hydrophobic interactions provides a virtually irreversible conjugation that, in theory, and generally in practice, retains the activity of the conjugated species. Our specific goals toward this aim are (1) to develop methodologies for conjugating proteins to the other colloidal metals, and (2) to use these conjugates to label model cellular systems.
 
CRIS NUMBER: 0187000 SUBFILE: CRIS
PROJECT NUMBER: WIS04452 SPONSOR AGENCY: CSREES
PROJECT TYPE: HATCH PROJECT STATUS: TERMINATED MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Oct 1, 2000 TERMINATION DATE: Sep 30, 2004

GRANT PROGRAM: (N/A)
GRANT PROGRAM AREA: (N/A)

CLASSIFICATION
Knowledge Area (KA)Subject (S)Science (F)Objective (G)Percent
206701010302.2100%

CLASSIFICATION HEADINGS
KA206 - Basic Plant Biology
S7010 - Biological Cell Systems
F1030 - Cellular biology
G2.2 - Increase Efficiency of Production and Marketing Systems


RESEARCH EFFORT CATEGORIES
BASIC 85%
APPLIED 15%
DEVELOPMENTAL (N/A)%

KEYWORDS: cell structure; colloidal gold; electron microscopy; scanning electron microscopy; light microscopy; heavy metals; particle size; labeling; cell biology; plant biology; nanotechnology; new technology; process development; fluorescent dyes; spatial analysis; wavelength; antibodies; ligands; synthesis; colloids; temperature; ph; reduction (chemistry); hydrophobic interactions

PROGRESS: Oct 1, 2000 TO Sep 30, 2004
The work here dealt specifically with the development of synthetic pathways and detection systems for a variety of nanoparticles principally for cell labeling purposes. The project was successful in meeting the goals originally put forward. A wide range of reducing agents and conditions were investigated. Nano-particles were generated in the molecular and sub-molecular size range (3nm to 30nm) having specific, narrowly defined, sizes, unique shapes, and different elemental compostions. For the first time this allows for simultaneous, in situ, labeling and co-localization of multiple (currently in the range of 5 to 7)different molecular species with both the scanning electron and transmission electron microscope. Particle shapes (spheres, popcorn, pyramid, square/trapezoid) can be clearly recognized in both high resolution field emission SEM and in TEM. Particles can be differentitated on the basis of composition( Au, Ag, Pd, Pt, Rh, Fe) via high resolution EDX-STEM and by energy filtering (EELS)TEM. Successful methodology for conjugation of identifier species such as the Fab fragment of antibody molecules or the active fragment of ligands was also developed. Most recently core-shell particles have been synthesized. Cores of spectrally distinct elements are surrounded by a thin but continuous layer of gold. Cores, of different elements, provide unique labels while the gold shell provides a stable, non-toxic surface which can be readily conjugated to identifier species such as antibody or ligand. The combined use of the different, unique particle labels, permits high resolution qualitative and quantitative analysis of molecule numbers and organization at the level of cell ultrastructure. The individual particles in the 10nm and greater range are detectable and trackable using interference base light microscopy so that labeling and tracking of specific molecular species is possible in living cells which can then be fixed and prepared for ultrastructural level studies. Tracking in whole animals or plants is also possible. Neutron activation can be used to measure bulk concentration of the particles at parts per trillion levels. This facilitates studies on distribution of particle-conjugated or labeled species in whole animals and in environmental studies. Samples with particles identified by neutron activation can subsequently be examined by electron microscopy to identify the exact tissue, cellular, or subcellular location of molecular species of interest.

IMPACT: 2000-10-01 TO 2004-09-30 The principal impact of the nanoparticles discovered in this study will be the ability of investigators to specifically and simultanously label different individual molecules and even specific parts of molecules for high resolution visualization in scanning and transmisson electron microscopes. The particles also have a variety of other potential uses from more effective drug delivery agents in animals and humans as anti-cancer agents and as tracking markers for molecules and organisms in living animals and in environmental studies.

PUBLICATION INFORMATION: 2000-10-01 TO 2004-09-30
Albrecht, R.M. and D. A. Meyer. The Potential for Correlative and Multiple Labeling With Colloidal Particles of differing Shapes. Microscopy and Microanalysis 6, Supplement 2: 318-319, 2000

PROJECT CONTACT INFORMATION
NAME: Albrecht, R. M.
PHONE: 608-263-3952
FAX: 608-262-7420