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8/3/2019 Bio Medical Tracers Particles
1/19
Particle Tracers
Use of natural or synthetic particles astracers includes at least 4 primaryapproaches:
Use of particles to visually demonstrate
molecular aggregates Attachment of particles to molecules or
cells to mark their locations
Attachment of particles to molecules orcells to allow them to be counted
Attachment of particles to molecules or
cells to allow them to be isolated
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Molecular Aggregation
Many early immunologic assays werebased on molecular agglutination,
flocculation, or precipitation of molecular
aggregates to produce macroscopicallyvisible changes in previously uniform,
clear solutions. The aggregates were
formed by antigens interacting with
polyclonal antisera, e.g., the preciptinreaction, or spot - plate testing for blood
group substances or syphilis.
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Molecular/Cellular Aggregation
Extension of the idea of aggregation to amore 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 carbonparticles, bacterial cells, white blood
cells, or chemically conjugated latex
(polystyrene) spheres.
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Localization with Particles
Microscopic visualization also allowedlocalization 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 wasoften marked as a color change (reddish with
gold colloids) rather than direct visualization of
individual particles.
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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=rk3USdGGBYbglQf4y
sC-DA&sa=X&oi=book_result&ct=result&resnum=3
http://www.ias.ac.in/matersci/bmsjun2000/165.pdf
Characterization & modifications,
noble metal colloids:
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Localization with Particles (cont.)
Localization & counting of individual small
particles, including small viruses like lamdaphage, 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 beintroduced 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.
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HCGBinding on MA-10 Cells by SEM
Problem: villi
& particles of
similar size.
MA-10cells in
culture
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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
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Automated Particle Counting
Counting of particle - tagged moleculesor cells may also be done automatically
so long as the particles can be detected
by size, color, fluorescence, electricalconductivity, or magnetic properties. The
Coulter counter evaluates size & number.
The Fluorescence Activated Cell Sorter
(FACS) detects laser - activatedfluorescence (often in attached latex
beads). Imaging software tracks
contrast, color, or dimensional profiles.
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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 ofBiomedical
Engineering, Schools ofVeterinary Medicine & Engineering, Purdue University
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Automated Particle Counting
http://www.spectrex.
com/html_files2/pc2
200.php
All the FACS & nothingbut the FACS:
http://www.cyto.purdue.edu/fl
owcyt/educate/pptslide.htm
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www.bio.davidson.edu/COURSES/
Bio111/FACS1.html
http://www.bio.davidson.edu/cour
ses/genomics/method/FACS.gif
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Some modern FACS systems such as theBD Aria can examine as many as 9 or
even 13 different signals simultaneously.
Dye Sensed Laser - Ex/Em Laser Detector
FSC FSC Blue FSC
SSC SSC Blue F
FITC B-530/30 Blue E
PE B-576/26 Blue DPE-Texas Red B-610/20 Blue C
PE-Cy5.5 B-695/40 Blue B
PE-Cy7 B-780/60 Blue A
APC R-660/20 Red C
Alexa 680 R-710/20 Red B
APC-Cy7 R-780/60 Red A
Alexa 405 V-450/40 Violet BAlexa 430 V-530/30 Violet A
www.microbiology.emory.edu/.../p_pfc.shtml
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Magnetic & latex particles from BangsLaboratories:
http://www.bangslabs.com/learning
Formation of mini-emulsionscontaining 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
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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 ofQdot
output by
adjustment of
particle sizewww.immunology.utoronto.ca/..
./FlowIntro.htm
www.aist.go.jp/.../hot_line/
hot_line_22.html
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www.ceac.aston.ac.uk/research/staff/as/research/
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a,b, Quantumdotshavingdifferentmoleculesfortarget-specificinteraction, and, attachedtothesurface,
paramagneticlipids (a, basedondescriptionsinref.15; themoleculesareshownattachedtopartofthedot
forclarity, butextendoverthewholesurface)andchelators (b, basedondescriptionsin Refs5,18,19)for
nuclear-spinlabelling.c, Basedonthedescriptioninref.3.thesilicaspherehasQDsandparamagnetic
nanoparticlesinsideandtarget-specificgroupsattachedtotheoutside.d, ThestructureofamultimodalQDprobe, basedonsilica-shelledsingle-QDmicelles.Adaptedfromref.20, copyright (2006)ACS.
Figure 1 - Various designs of multimodal QD probes.
From the article
Designing quantum-dot
probes
Rumiana Bakalova, Zhivko
Zhelev, IchioAoki & Iwao
Kanno
Nature Photonics 1, 487 -
489 (2007)
doi:10.1038/nphoton.2007.
150
www.nature.com
/.../nphoton.2007
.150_F1.html
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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
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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.