Download - QDot Biolabels Talk From Invitrogen
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Qdot Conjugates:
Sensitive, Multicolor,Stable Fluorescence
Patricia Whaley, Ph.D.
Molecular Probes Labeling andDetection Technologies
Invitrogen Corporation
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Outline
Quantum Dot Basics Materials Spectral Properties
Microscopy Applications Immunofluorescent Cell Biology Molecular Pathology Cellular Assays
Flow Cytometry In-vivo imaging Western Blotting
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What Are Quantum Dots?
655 605 585 565 525 nm
25nm
Size of the nanocrystal determines the color
Size is tunable from ~2-10 nm (3%)
Size distribution determines the spectral width
Highly fluorescent, nanometer-size, single crystals of semiconductor materials
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Qdot Conjugates are Engineered
Core Nanocrystal (CdSe)- Determines color
Inorganic Shell (ZnS) Improves brightness and stability
Organic Coating
- Provides water solubility andfunctional groups forconjugation
Biomolecule
-Covalently attached to polymershell
- Immuoglobulins- Streptavidin, Protein A- Receptor ligands
- Oligonucleotides
15 - 18 nm
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Size of Qdot Nanocrystals
Images provided by Mark Ellisman, National Center for Microscopy and Imaging Research, UCSD, San Diego, CA
QD 525
QD 605
QD 585QD 565
QD 6555nm + 20 nm Gold
~4nm ~5nm~5nm
~5 x 12nm ~8 x 15nm
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Quality CountsZStem Shows It
Quantum Dot Corp Materials Literature Methods
Data courtesy Steve Penneycook (ORNL), James McBride, and Sandy Rosenthal (Vanderbilt)
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Qdot Conjugates vs. Competitors
Not Created Equal: Immunosorbent Results
0
500
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4500
-0.50 0.00 0.50 1.00 1.50 2.00 2.50
Log (Conj Conc nM)
RFU
onP
lateReader
Qdot 605 Anti-Rabbit Lot 1
Qdot 605 Anti-Rabbit Lot 2
Competitor 600 (Fort Orange) Anti-Rabbit
10x competitor data
NO measurable binding
Clear Dose-Response
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Excitation Spectra of Qdot Conjugates
High extinction >> High brightness
All colors can be excited at the same wavelength, 425DF45
0.0E+00
5.0E+05
1.0E+06
1.5E+06
2.0E+06
2.5E+06
3.0E+06
3.5E+06
4.0E+06
4.5E+06
5.0E+06
350 450 550 650 750 850
Wavelength (nm)
Extinctio
nCoefficient(M-1cm-1)
Qdot 525 Conjugate AbsorbanceQdot 565 Conjugate AbsorbanceQdot 585 Conjugate AbsorbanceQdot 605 Conjugate AbsorbanceQdot 655 Conjugate AbsorbanceQdot 705 Conjugate AbsorbanceQdot 800 Conjugate Absorbance
Organic Dyes {
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Emission Spectra of Qdot Conjugates
400 500 600 700 800 900
Wavelength (nm)
525525
605605
655655
705705
800800
565565
Minimal (> simplified multiplex labeling
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Exp. Time: 0.44 seconds 8.12 seconds
Exp. Time: 0.019 seconds 1.22 seconds
High level Her 2/neuexpression in SK-BR-3 Cells
Quantum dots up to 50x
brighter
Low level of Her 2/neu
expression in MDA-MB-
231 cells
Quantum dots easy todetect but dye
undetectable
Excellent Brightness Provides High Sensitivity
Quantum dot Organic dye
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525 (Mouse) Mitochondria 565 (Rat) Tubulin
605 (Rabbit) Ki-67 655 (Human) Nuclear antigen 705 (Streptavidin) Actin
Detection of More Parameters in a Single Experiment
More information from every sample using Qdot Conjugates
Partial informationfrom single color
experiments
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Five Color Multiplexed Cell Labeling
OverlayedPseudocolor
5-color labeling with dyes would be extremely difficult.Multiplexing gives more information from a single experiment.Multiplexing gives much more information than 5 single experiments.
Full informationfrom multiplex
experiments
Wu, X., et al., Methods in Cell Biology, 752004.
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Direct Conjugates Provide Ultimate Flexibility
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HeLa cells fixed in paraformaldehyde and permeablized with Triton
No significant differences in cytoskeletal protein labeling
Qdot 655 Conjugate Alexa 594 conjugate
Qdot Conjugate Label Quality
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Qdot Conjugates and Pathology
Molecular Pathology Patient diagnosis and prognosis Patient stratification for best treatment regimen
Biomarkers for preclinical/clinical drug evaluation Traditional pathology loses all cellular/molecular correlations
Morphological correlation rather than molecular correlation Multiple markers are becoming the norm
Gene expression data Protein analysis Qdot conjugate stability, brightness and multiplex capability areideal.
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Obtain More Information From Each Specimen
Typical pathology: Single color, no relative measurements. Slices have no relative orientation.
Marker information available is based on morphological correlations.
Slice 1 Slice 2 Slice 3
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Pathology: See What Youve Been Missing
Courtesy ofJason AdamsUCSF
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Estrogen Receptor: monoclonal Rb clone SP1 RM-9101-SEstrogen Receptor: monoclonal Rb clone SP1 RM-9101-S
Peroxidase -DAB Qdot 655 Conjugate
RGB images acquired a single gray scale images at 655/20, 565/20, and 450/58 nm and mergedRGB images acquired a single gray scale images at 655/20, 565/20, and 450/58 nm and merged
Conventional IHC vs Fluorescence
Images courtesy of
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Her-2 in Breast CancerHer-2 in Breast Cancer
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Qdot Primary Conjugates
WGA Qdot 655 Conjugate
Phalloidin Qdot 525 Conjugate
Ki-67 Qdot 585 Conjugate
Vimentin Qdot 525 Conjugate (Clone V9)
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Correlative Light and Electron Microscopy
Glial fibrillar acidic protein - Qdot 655
Inositol triphosphate Receptor Qdot 525
Images provided by Mark Ellisman, National Center for Microscopy and Imaging Research, UCSD, San Diego, CA
Giepmans, et al.
Nature Methods
2(10), 2005.
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Correlative Light and Electron Microscopy
Better EM labels than colloidal gold because of superior penetration
Qdot 655
Qdot 525
Glial fibrillar acidic protein - Qdot 655Inositol triphosphate Receptor Qdot 525
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Qdot Conjugates and Spectral Imaging
Data Courtesy of CRI-Inc on a Nuance Camera System
Standard color image (500-720 nm)
Unmixed composite Nuance image
655 nm QD
565 nm QD
DAPI
Autofluorescence
You can see clearly now.
Ph t t bilit f Qd t C j t
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605
605
Top panel (a-e): Nucleus labeled with Qdotconjugates and microtubules labeled with AlexaFluor 488
Bottom panel (f-j): Nucleus labeled with AlexaFluor 488 and microtubules labeled with Qdot
conjugates
Left: quantitative data showing effect of anti-fade medium
Photostability of QdotConjugates
Wu, X., et al. Nature Biotechnology, 21(1)2003.
d j f l b li
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QdotConjugates for Fluorescent LabelingQdotConjugates for Fluorescent Labeling
Extremely bright for sensitive detection
High photostability provides:
Ability to monitor signal over long periods of time
Ease of use (time for focusing and image collection) Enables pathology and live cell imaging applications
Narrow emission peaks for simple multiplexing
Easily illuminated by many excitation sources
Easily conjugated to a variety of biomolecules
Key issues: Fixation Protocol, Filter Selection, PAP PenQuenching, Photobrightening
Extremely bright for sensitive detection
High photostability provides:
Ability to monitor signal over long periods of time
Ease of use (time for focusing and image collection) Enables pathology and live cell imaging applications
Narrow emission peaks for simple multiplexing
Easily illuminated by many excitation sources
Easily conjugated to a variety of biomolecules
Key issues: Fixation Protocol, Filter Selection, PAP PenQuenching, Photobrightening
More data faster
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Cellular Assays
N l T l ti A
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Nuclear Translocation Assay
Unstimulated
EGF (10 min)
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200400
600
800
1000
1200
1400
1600
1800
Unstimulated EGF
(100ng/ml)
RFU
(Meannuclearintensity)
Nuclear expression of phosphorylated
Erk in starved HeLa cells following EGF
stimulation
Pl t b d C ll l I B fit
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High information content in high throughput Lower cost and simpler instrumentation requirements
Plate readers are readily available in all target labs
More valid biology More flexibility in assay configuration More controls or reference markers may be included
Lower level of expertise required
Instant data reduction Image analysis and storage not required Label reagents can be transferred to tissue characterization in
preclinical models
High information content in high throughput Lower cost and simpler instrumentation requirements
Plate readers are readily available in all target labs
More valid biology More flexibility in assay configuration More controls or reference markers may be included
Lower level of expertise required Instant data reduction
Image analysis and storage not required Label reagents can be transferred to tissue characterization in
preclinical models
Plate-based Cellular Immunoassays Benefits
d j b li f i h i llQd t C j t L b li f MAP Ki P th i 3T3 ll
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Qdot Conjugate Labeling of MAP Kinase Pathway in 3T3 cellsQdot Conjugate Labeling of MAP Kinase Pathway in 3T3 cells
pErk
pAkt
pMEK
NSB
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150
200
250
0 10 25 40 70 115
Time in PDGF (50ng/ml)
RFU
MEK 1
pAkt
pErk
Time 0 0 0 10 10 25 25 40 40 70 70 115
pErk
pAkt
pMEK
Serum starved cells were stimulated with 50 ng/ml PDGF for the
times indicated. Cell (left) were stimulated for 25 min for fixation.
Phospho Erk Assay Results
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At left: Images of the same set of wells acquired at two colors assaying pan Erk and phosphoErk. The red circle represents the area used for measuring mean intensity in each well.
At right: Results plotted showing time course of PDGF induced phosphorylation of Erk
Z values at 20 through 80 minutes are 0.63-0.77
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Minute
MeanRFU,
(n=3)
pERK_PDGF-
pERK_PDGF+
panERK_PDGF-
panERK_PDGF+
Induction of phosphorylated Erk
by PDGF in NIH3T3 cellspan Erk
605 QdotpErk
565 QdotMin
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Phospho-Erk Assay Results
M C t l L d t M R b t D t
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0
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0.1 1 10 100
Log PDGF (ng/mL)
Norma
lized
RFUR
atio
o
pErk
to
Tubulin
3T3
Kodak MM2000 gel imager
0
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3
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89
10
0.1 1 10 100
Log PDGF/EGF (ng/mL)
Normalize
dRFU
RatioofpE
rkto
Tubulin
3T3
BMG PolarStar Plate Reader
p42/44 MAPK phosphorylation was indexed to tubulin but can be
normalized to pan protein expression or wheat germ agglutinin (Qdot WGA
Conjugate) expression.
Multiplexing with Qdot Conjugates allows extensive information andreferencing in a single well readout.
More Controls Leads to More Robust Data
Respiratory Syncitial Virus Progression
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1 day after infection
3 days after infection
1 hour after infection
2 days after infection
E. Bentzen,Nano Letters, 52005.
Respiratory Syncitial Virus Progression
Syncitia
formation
Cellular
infection
Dose Response for Viral Infection in Culture
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Dose-Response for Viral Infection in Culture
Data collected on a Bio-Tek Synergy HT
Ex:250 nm
Em:598+/-18 nm
Qdot 605 Streptavidin Conjugate
Biotinylated anti-RSV F-Protein
Detection limit of 35-50 PFU in 24 hours
Whole well F-Protein cell intensity vs initial infection load
Progression can be monitored by image analysis or
by quantitative analysis of the cell population.
Faster and easier to look at the whole population.
Qtracker Cell Labeling Kits
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Qtracker Cell Labeling Kits
Lagerholm, B.C., et al., Nano Letters, 4(10) 2004.
Three Color Qtracker Cell Labeling
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Three Color Qtracker Cell Labeling
10x
3T3(green), HeLa(red), and U188(white) cells labeled with Qtracker565, 655, and 705 respectively.
Co-cultured in 8-well chambers for 24 hrs. Images captured with aLeica Confocal microscope (ex. = 488nm).
Multiplexed Cellular Assays
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Multiplexed Cellular Assays
TTP Acumen Explorer for single-excitation, multiplexed cellular analysis.
Real-time proliferation readouts from multiple cell-lines within a single well.
Other multiplexed cellular readouts possible (Internalization, Calcium, etc.)
CHO-655 SH-SY5Y-705 Mixed
0 4
0
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1000
1500
2000
2500
CHO
SH-SY5Y
Day
NumberofC
ells
0 4
0
500
1000
1500
2000
2500CHO
SH-SY5Y
Day
NumberofC
ells
Single cell proliferation over 4 days Mixed cell proliferation over 4 days
Cell Fusion Assays and Stem Cells
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Cell Fusion Assays and Stem Cells
Murasawa, et al. Arterioscler Thromb Vasc Bio, 25(7) 2005, 1388-1394
Endothelial Progenitor CellsQtracker 565
H9C2 Cells (Cardiac Lineage)
Qtracker 655
Cell Fusion Rate:Both colors, one cell0.50 +/- 0.23%
EPC committed to Cardiac Lineage30-50% Transdifferentiation
Conclusion: Cell Fusion cannotaccount for differentiation behavior
Multiplexed Cellular Assays
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Multiplexed Cellular Assays
Qtracker Cell Labeling Kits are non-toxic
Analysis of phenotype, metabolism, proliferation, differentiation
Qtracker Reagents do not transfer between cells
Qtracker Reagents are passed to daughter cells for 6-8 generationstypically
Ideal tools for studying cell-cell interactions
Ideal tools for tracking cell fate in living systems
Qdot Conjugates in Cytometry
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Qdot Conjugates in Cytometry
Flexible excitation Usable with all common platforms
Perfect for single laser, multicolor systems
High brightness
Comparable to or better than best dye molecules Very low cross-talk
No or minimal compensation required from single laser.
Improving platform
QDC R&D producing ongoing improvements to brightness, width,and NSB.
Unlimited colors available
6 colors from 525-800 nm with < 5% cross-talk
Photostability allows for imaging and resorting
Flow Cytometry: Qdot Streptavidin Sampler Kit
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Qdot 525 streptavidin525/20
Qdot 565 streptavidin565/20
Qdot 585 streptavidin585/20
Qdot 605 streptavidin605/20
Qdot 655 streptavidin655/20
Qdot 705 streptavidin695/40
CD4-biotin + Streptavidin Sampler Kit on human PBMCs, 405 nm excitation
Flow Cytometry: Qdot Streptavidin Sampler Kit
Single Laser Multicolor Flow Without Compensation
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Qdot 585 CD8
Qdot
655CD4
Qdot 525 CD3
Qdot
585CD8
Single Laser, Multicolor Flow Without Compensation
Reagent Intensity off 488 nm Spectral Overlap
Qdot 525 crystal ~ Fluorescein 2% into FL2
Qdot 585 crystal ~ 50% RPE 11% into FL3 (>620 nm)
Qdot 655 ~ RPE/Cy5 0.2% into FL2
PBMCs + direct
conjugate cocktail
Modified BD FACScan:
FL1: 530/30
FL2: 585/42
FL3: 620 LP
Run without
compensation
Clean Spectral Signals from Qdot Conjugates
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Clean Spectral Signals from Qdot Conjugates
Other dyes dont cross-overinto Qdot Conjugate channels
Qdot Conjugates dontcross-over into other dyechannels substantially(in spite of broad excitation)
17 colors withmanageable compensation
Perfetto, S.P., Chattopadhyay, P. K., Roederer,M.; Nature Reviews Immunology; V.4 (2004);
648-655.
4 Qdot Tetramer Reagents
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4 Qdot Tetramer Reagents
CMV(Qdot 705)
EBV(Qdot 545)
Gag(Qdot 655)
Nef(Qdot 605)
Data courtesy Stephen DeRosa (FHCRC)and Mario Roederer (NIH-VRC)
More information per
sample possible
Phenotyping & Activation ina single sample context
Streptavidin Conjugatesused with Biotin-MHC-Peptide complexes toelucidate T-cell specificity
Alexa
Fluor680
CD8
(Qdot 705 streptavidin) (Qdot 655 streptavidin)
(Qdot 545 streptavidin) (Qdot 605 streptavidin)
LSR II: Trigon / Octagon Configurations
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690LP570LP
720/20
635LP555LP
505LP
655/20
585/4
2
525/50
440/50
605/
20
405 nmLaser
LSR II: Trigon / Octagon Configurations
525/5
0440/40 5
05LP
780/6
0
585/4
2
695/60
730LP5
50LP
787/4
2660/20 7
60LP
633 nm
Laser
532 nm
Laser
UV nm
Laser
735LP635LP
787/42
685LP545LP
5
05LP
xxxLP
720/20
585/4
2
5
15/10
xxx/x
x
488/10
670/
14
488 nm
Laser
5-Color Stain: Violet Excitation
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Pacific BlueTM CD4
Pac
ificOrangeTM
CD8
Qdot705CD56
Pacific Orange CD8Pacific Blue CD4
Qdot705CD56
Qdot 655 CD3
Qdot605CD19
Qdot705CD56
Qdot 655 CD3
5-Color Stain: Violet Excitation
CD3-b + Qdot 655 SA
CD56-b + Qdot 705 SA
CD19-b + Qdot 605 SA
Pacific Orange CD8 /Pacific Blue CD4
Summary
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Summary
Current Qdot nanocrystals are comparable to best organic dyesin intensity.
Narrow emission spectra of the Qdot nanocrystals result in verylow cross-talk.
Selected Qdot reagents exhibit minimal spectral overlap
Qdot reagents can be multiplexed for multicolor analysis insingle- and multi-laser systems
Current solutions available: Streptavidin and anti-immunoglobulin conjugates
Conjugation kits
Reactive Qdot nanocrystals Custom conjugation services
Direct Conjugates Give Excellent Results
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Direct Conjugates Give Excellent Results
At the right titration level
Chattopadyhay, et al. Methods in Molecular Biology (in press)
Importance of Optimal Titration and Clone
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p p
PBMC stained with Qdot 655 Leu3a/RPA-T4 Conjugate
Washed 2x with HBSS. 100 ul staining with 106 cells.
High affinity clones give high quality products.
Signal of Positives and Negatives (Qdot 655 Leu3a/RPA-T4)
1
10
100
1000
10000
0.1 1 10 100
Concentration (nM)
FL3
-H
PopMedia
n
Leu3a Positives
Leu3a Negatives
RPA-T4 Positives
RPA-T4 Negatives
Direct Antibody Conjugate ComparisonQdot 655 vs PE Cy5 Antibody Conjugates
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Qdot 655 Antibody Conjugate PE-Cy5 Antibody Conjugate
CD3
CD4
CD8
Qdot 655 vs PE-Cy5 Antibody Conjugates
Current (488 based) and Future Performance
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( )
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g , p
Buffy coat stained
Direct Qdot Conjugatereagent cocktail
Lysed/Washed 2x
488 ex FACScan
530/30 FL1
585/42 FL2
620 LP FL3
UNCOMPENSATED
Stock 650 LP would havesubstantially lower
FL2/3 cross-talk
CD3
CD8
CD8
CD4
Flow Summary
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y
Current Qdot materials are comparable to best dyes in intensity.
The narrow emission spectra of the Qdot materials result in verylow cross-talk.
The Qdot channels off the violet laser are free of compensation.
There are significant benefits to using Qdot materials inmulticolor single-laser analysis.
Current solutions available: Conjugation Kits
Streptavidin Conjugates
Reactive Qdot Nanocrystals Custom Conjugation Services
In vivo Imaging
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g g
Qdot nanocrystal materials can be imaged at scales from centimeters to nanometers.
Ballou, B. et al., Bioconjugate Chemistry, 15(1)2004.
Real-time Receptor Ligand Tracking
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p g g
EGF-Qdot Conjugate co-internalizes with ErbB2
Novel retrograde transport
mechanism via filopodia EGFR homodimerizes with erbB2
but erbB3
Lidke, D. et. al. Nature Biotechnology 22 (20), 2004
4.5 sec/frame; 100 framesSequential confocal scans
Qdot 605-EGF conjugate (erbB1)erbB3-Citrine
Photostability allows unprecedentedreal-time continuous monitoring of
receptor-molecule (ligand, drug)dynamics
EGF Receptor Internalization
Multiphoton Imaging Vascular Imaging in Ovary
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Noninvasive- Imaging through skin- High contrast angiography
Stable- No toxicity observed after
venous injection
Bright- Very high S/B- Excellent discrimination
from auto-fluorescence- Fine structural and dynamic
information can be obtained
FITC - Dextran Qdot ITK Carboxyl QDsSingle MP slice
Qdot ITK Carboxyl QDs250 m image stack
Larson, et.al., Science 300(5624) 2003.
In-vivo Vascular Imaging
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Long circulation time allows detailed vascular imaging. Also useful for marking vascular structures in tissue sections.
Chick embryo venous injection at increasing resolution
Bright signal allows highly detailed vascular analysis
Red colors allow deeper, higher resolution imaging than dyes
Courtesy of Greg Fisher, Byron Ballou and Alan Waggoner, Carnegie Mellon University
Zebrafish with Qdot Conjugates
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Rieger, et al. Dev Dyn 18, 2005.
Whole MountIHC from in-vivo
Acetylated Tubulin
axonsQdot 605 SAv Conj.
vascular
Tracking cells by fluorescence and luminescence
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Xenogen IVISExpt Courtesy
Steve Smith
Results like bioluminescence without transformed cells
C57B1/6 Mice
B16F10 luc cellsLoaded with Qtracker705 Cell Labeling Kit
2x106cells injectedvia tail vein
Cell fate monitoredfor 24 hours.
Fluorescence tracksbioluminescence
results until colonyformation.
In-vivo imaging across the continuum
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Molecular Targeting
Non-targeted Intravascular Cell Surface Interstitial Intracellular Metabolic
Cellular Tracking
Passive Proliferation Uptake Metabolism Reporter Systems
Post-mortem analysisPhysiology Molecular
Quantum Dots
Alexa Fluors
Quantum Dots
Alexa Fluors
Quantum Dots
Alexa Fluors
Quantum Dots
Alexa Fluors
Live Cell and Animal Imaging
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Image noninvasively, then follow up at higher resolution Compatible with GFP imaging
Imaging at greater depth and resolution
Infrared materials can be imaged through skin and other
tissues effectively
Repeated imaging without repeated dosing.
Longitudinal imaging from in-vivo to intravital to post-mortem
to electron microscopy.
Qdot Western Blotting products
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ActinVimentinGAPDH
Rat TissueLysate Blot
Western Analysis with Qdot Conjugates
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Sensitivities comparable or better than reported chemiluminescence levels.
NO FILM, NO DARKROOM, NO RUSH.
Simple multicolor detection without dedicated instrument
Chemiluminescence imaging systems
Gel Documentation systems
Trans-illuminator and color camera with anti-haze filter
No stripping and reprobing required
Faster experiments More reliable data
More reliable quantitative analysis
2-3 orders of magnitude linear dynamic range with single exposure
Extended exposures extend dynamic range to 4-5 orders of magnitude
More Sensitive than the Gold Standard ECL
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12.5 6.25 3.1 1.6 0.8 0.4 0.2 0.1 0.05 (ng)
Qdot 655 ConjugateKodak Imager
ECL DetectionKodak Imager
ECL DetectionHyperfilmPlus 5 min
100 pg
1600 pg
400 pg
Purified protein dilution series with identical antibodies Qdot Conjugates deliver sensitivity dramatically higher than ECL Reagents even
under the optimal film-based detection. Combination with Millipore Immobilon-FL Transfer Membrane provides highest
sensitivity.
DetectionLimit
Qdot Western Blots Can be Stored for Months
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Day 2: 40 pg detection
3 months later: 150 pg detectionStored in TBS
Stored in TBS, the Qdot Western Blots retain signal for months.
Stored dry, they may retain signal even longer.Plenty of time to get imaging conditions optimized for publication.
Sensitive Quantitative Western Analysis
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1n
g
500p
g
250p
g
125p
g
62p
g
31p
g
16p
g
8p
g
4p
g
Pure GSTGoat anti-GSTQdot 655 anti-Goat Conjugate
Secondary Detection of GST
R2 = 0.9951
0
200
400
600
800
1000
0 200 400 600
pg / lane GST
Mean
Intensity
Detection limit 4-8 pg GST
Fluorescence detection with
chemiluminescent sensitivity
Stability for repeated analysis
Broad linear range
Progressive exposures extend range 100 fold linear range for each exposure
Images acquired with a KODAK Image Station 2000MM Multimodal Imaging System
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Simple, Fast and Economical
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ECL on Film
Transfer (1 hr)|
Block (1 hr)
|Primary (1 hr)
|Rinse (0.25 hr)
|
Secondary (1 hr)|
Rinse (0.25 hr)|
Substrate, Film, Develop (1 hr)
|Substrate, Film, Develop (1 hr)
|Substrate, Film, Develop (1 hr)
Qdot Conjugate
Transfer (1 hr)|
Block (1 hr)
|Primary (1 hr)
|Rinse (0.25 hr)
|
Secondary (1 hr)|
Rinse and Image 3x(0.25 hr)
Save hours in the darkroomwith Qdot Western Blotting detection
Simple, Fast and Cost Effective
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$19.281:20001100-2Qdot Conjugate
$28.031:10001100-2Qdot Conjugate
$31.051:5000RPN2108(GE-Amersham)
ECL
$37.051:5000RPN2132
(GE-Amersham)
ECL Plus
$35.451:25000RPN2138(GE-Amersham)
ECL Advance
Cost per blotAntibody DilutionProduct NumberDetectionMethod
Useful in Cell Signaling Research
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0 10 20 40 60 (min)
phospho Akt
phospho ERK 1/2
pan Akt
pan ERK 1/2
Phosphoryation of Akt and Erk in A431 Cells Stimulated by EGF (25ng/mL)This is a typical cell-signalling model system.
} Qdot 705 } Qdot 605
Color value indicates ratio of modified to total protein This is a typical class of experiment in cell-signalling research.
Analysis of multiple bands allows single experiment with rich content.
The alternative experiment would take several days with standard methods.
Akt
ERK1/2
Ornberg, et al., Nature Methods 2(1) 2005, 79-81
Qdot Western Blotting Benefits
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Simple
Fluorescence imaged directly, no substrate addition No dependence on time (CL) or photostability issues (dyes)
Robust, stable signals allow repeated imaging
Imaging can be done on gel imagers, CCD imagers, and laser
scannersvery flexible Quantitative
Linear quantitative range over 2.5 orders of magnitude
Stable signal ensures reliable measurements
Sensitivity as good as best reported chemiluminescence methods Multiplexed
No stripping and reprobing to detect multiple bands
Single source excitation with emission filters ensures reliable
signal ratios
Conclusions
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Qdot Conjugates provide substantial benefits in detection
Ultimate in photostability Sensitivity rivals or exceeds the best methods Multiplexing capability dramatically simplified Wide variety of available products ensures application needs are
met
Distinct product lines appropriate for many applications Microscopy Flow Cytometry
Live cell/live animal imaging Western Blotting Immunoassays
Reactive Qdot nanocrystal materials available for your chemistry(Innovators Tool Kit Quantum Dots)
Organic Carboxyl Amino(PEG)
Independent Accolades
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Science magazines Top 10 Scientific Breakthroughs of 2003.[Quantum dot bio-imaging is]the most exciting newtechnique to emerge from the collaboration of physicistsand biologists.
Forbes/Wolfe Nanotech Reports Top 5 Breakthroughs of
2003. Number 1: In vivo labeling with quantum dots
LARTA Nano Republic Conference 2003. Most promisinginnovation award.
Small Times magazines 2003 Researcher of the Year.Quantum Dot founder Paul Alivisatos.
2004 Fortune Cool Companies winner.
PublicationsInvitrogen Materials Are the Standard
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~230 peer reviewed publications (Biological Apps since 1998)
92 used QDC Materials (2003-2005) PathologyFluorescence/EM/FISH Live Cell Microscopy (dynamics) Single Molecule Analysis FRET
Arrays Microfluidics/Patterning Pathogen Detection And many more
2 used competitor materials (reporting quenching) Others used self-fabricated materials (great ideas) Large number of review articles
Quantum DotInvitrogen Nanocrystal Technologies materials ensure
consistency, support, and optimal performance every time.
Collaborators
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Mark Ellisman NCMIR-UCSD
Alan Waggoner Carnegie Mellon University
Paul Wylie TTP Labtech Watt Webb Cornell University
Mario Roederer NIH-Vaccine Research Center