Particle Tracers

Use of natural or synthetic particles as tracers includes at least 4 primary approaches:

• Use of particles to visually demonstrate molecular aggregates

• Attachment of particles to molecules or cells to mark their locations

• Attachment of particles to molecules or cells to allow them to be counted

• Attachment of particles to molecules or cells to allow them to be isolated

Molecular Aggregation

Many early immunologic assays were based on molecular agglutination, flocculation, or precipitation of molecular aggregates to produce macroscopically visible changes in previously uniform, clear solutions. The aggregates were formed by antigens interacting with polyclonal antisera, e.g., the preciptin reaction, or spot - plate testing for blood group substances or syphilis.

Molecular/Cellular Aggregation

Extension of the idea of aggregation to a more quantitative format was provided by microscopic examination of cellular aggregates (rosettes) formed by washed, tannic acid treated red blood cells, coated with antibodies interacting with antigen molecules or antigen - containing cells. Other versions of this used carbon particles, bacterial cells, white blood cells, or chemically conjugated latex (polystyrene) spheres.

Localization with Particles

Microscopic visualization also allowed localization of particle tagged cells or molecules. As diffusion is inversely related to molecular/particle size, however, smaller particles were used in these contexts, e.g., noble metal colloids (5 - 150 nm), large viruses (TMV), bacterial cells (coccus sp.), small latex spheres, & even small atomic chelate collections such as decagold. Locale was often marked as a color change (reddish with gold colloids) rather than direct visualization of individual particles.

Adsorption of macromolecules to colloids

Colloid definition:

Conjugation of molecules to latex or bioparticles involves the same kinds of chemistries already discussed for linking biomolecules to antibodies, lectins, or similar markers.

http://en.wikipedia.org/wiki/Colloid

http://books.google.com/books?id=rlcLQmcTADEC&pg=PA1136&lpg=PA1136&dq=noble+metal+colloids&source=bl&ots=yEk7E3Lj_j&sig=Og5QFJqeoOt8o5K3LGeo9QM0tT8&hl=en&ei=rk3USdGGBYbglQf4ysC-DA&sa=X&oi=book_result&ct=result&resnum=3

http://www.ias.ac.in/matersci/bmsjun2000/165.pdf

Characterization & modifications, noble metal colloids:

Localization with Particles (cont.)

Localization & counting of individual small particles, including small viruses like lamda phage, can be done using transmission (TEM; resolution to 1 - 10 nm) or scanning electron microscopy (SEM; resolution to 10 - 50 nm). Particles or particle conjugates may be introduced into the live organism prior to fixation & embedding for TEM or fixation & carbon/metal coating for SEM. They may also be used after fixation & washing for surface structures in SEM. Or after fixation, embedding, & sectioning for TEM following etching with alkalis or treatment with microwave heat to unmask antigens.

HCG Binding on MA-10 Cells by SEM

Problem: villi & particles of similar size.

MA-10 cells in culture

TEM of various particles:

www.tanaka.co.jp/english/products/html/f_6.html

www.devicelink.com/ivdt/archive/00/03/004.html

www.ansci.wisc.edu/.../microscopy/colloid.html

www.liv.ac.uk/Chemistry/Staff/brust.html

Automated Particle Counting

Counting of particle - tagged molecules or cells may also be done automatically so long as the particles can be detected by size, color, fluorescence, electrical conductivity, or magnetic properties. The Coulter counter evaluates size & number. The Fluorescence Activated Cell Sorter (FACS) detects laser - activated fluorescence (often in attached latex beads). Imaging software tracks contrast, color, or dimensional profiles.

The first Coulter Counter

High Speed Automatic Blood Cell Counter and Cell Size Analyzer

Wallace H. Coulter

Coulter Electronics, Chicago, Illinois

:1034-1042, 1956

High Speed Automatic Blood Cell Counter and Cell Size Analyzer

Wallace H. Coulter

Coulter Electronics, Chicago, Illinois

:1034-1042, 1956

J.Paul Robinson, Professor of Immunopharmacology, Professor of Biomedical Engineering, Schools of Veterinary Medicine & Engineering, Purdue University

Automated Particle Counting

http://www.spectrex.com/html_files2/pc2200.php

All the FACS & nothing but the FACS:

http://www.cyto.purdue.edu/flowcyt/educate/pptslide.htm

www.bio.davidson.edu/COURSES/Bio111/FACS1.html

http://www.bio.davidson.edu/courses/genomics/method/FACS.gif

Some modern FACS systems such as the BD Aria can examine as many as 9 or even 13 different signals simultaneously.

Dye Sensed Laser - Ex/Em Laser DetectorFSC FSC Blue FSCSSC SSC Blue FFITC B-530/30 Blue EPE B-576/26 Blue DPE-Texas Red B-610/20 Blue CPE-Cy5.5 B-695/40 Blue BPE-Cy7 B-780/60 Blue AAPC R-660/20 Red CAlexa 680 R-710/20 Red BAPC-Cy7 R-780/60 Red AAlexa 405 V-450/40 Violet BAlexa 430 V-530/30 Violet A

www.microbiology.emory.edu/.../p_pfc.shtml

Magnetic & latex particles from Bangs Laboratories:

http://www.bangslabs.com/learning

Formation of mini-emulsions containing carbon or magnetic cores:

http://www.mpikg-golm.mpg.de/kc/landfester/

Microscopy of latex & latex films:

http://www.lehigh.edu/~ols0/intro.html

Magnetic particles from Seradyn:

http://www.thermo.com/com/cda/landingpage/0,,2348,00.html

Quantum Dots (Last but not least!)

QdotProteinAconjugatewithUV

http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/Qdot/Technology-Overview.html

Tunability of Qdot output by adjustment of particle size

www.immunology.utoronto.ca/.../FlowIntro.htm

www.aist.go.jp/.../hot_line/hot_line_22.html

www.ceac.aston.ac.uk/research/staff/as/research/

a,b, Quantum dots having different molecules for target-specific interaction, and, attached to the surface, paramagnetic lipids (a, based on descriptions in ref. 15; the molecules are shown attached to part of the dot for clarity, but extend over the whole surface) and chelators (b, based on descriptions in Refs 5,18,19) for nuclear-spin labelling. c, Based on the description in ref. 3. the silica sphere has QDs and paramagnetic nanoparticles inside and target-specific groups attached to the outside. d, The structure of a multimodal QD probe, based on silica-shelled single-QD micelles. Adapted from ref. 20, copyright (2006) ACS.

Figure 1 - Various designs of multimodal QD probes.

From the articleDesigning quantum-dot probesRumiana Bakalova, Zhivko Zhelev, Ichio Aoki & Iwao KannoNature Photonics 1, 487 - 489 (2007)doi:10.1038/nphoton.2007.150

www.nature.com/.../

nphoton.2007.150_F1.html

Quantum dot conjugates can simultaneously reveal the fine details of many cell structures. Here, the nucleus is blue, a specific protein within the nucleus is pink, mitochondria look yellow, microtubules are green, and actin filaments are red. Soon the technique may be used for speedy disease diagnosis, DNA testing, or analysis of biological samples.QUANTUM DOT CORP., HAYWARD, CA publications.nigms.nih.gov/.../chapter1.html

Other New Uses:

New uses for particles are modifications of those already mentioned.

• Uniform latex or gold particles can be used to calibrate electron or visual microscopes.

• Labeling of blots using latex or colloid tagged antibodies or nucleic acids generate colors without added enzyme reactions; gold or silver colloid stains can also be intensified using silver salts.

• Quantum dots are semiconductor aggregates with high optical activity & electron density.