Advantages of the Alexa Fluor dyes in Molecular Imaging

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Advantages of the Alexa Fluor dyes in Molecular
Imaging
New Secondary Detection Tools for Flow Cytometry
and IHC, Small Animal In Vivo Imaging Reagents
Dana Brozban Technical Sales Specialist Imaging
and Microscopy dana.brozban_at_invitrogen.com
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Todays Presentation

• Focus on Key Molecular Probes Products and
  Applications
• Alexa Fluor Dyes
• Zenon Technology for secondary detection
• Small Animal In Vivo Imaging (SAIVI Reagents)

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Desirable Characteristics of Fluorophores

• 1) Emission at wavelengths that minimize
  problems with autofluorescence
• 2) Minimal spectral overlap with other fluors
  (Minimal Compensation)
• 3) Bright (high QY and Extinction Coefficient)
• 4) Minimal photo bleaching for sorting and
  microscopy
• 5) Minimal non-specific cell binding

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What Does a Fluorescent Dye Look Like?

• Rigid conjugated ?-electron system is generally
  required for fluorescence

Fluorescein Rigid, fluorescent
Phenolphthalein Flexible, non-fluorescent
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Efficiency or Total Fluorescence
? Absorbance x Path Length
M

• ? Extinction Coefficient, Path Length is
  typically 1 cm for most cuvettes, M the molar
  concentration

• More highly conjugated (ring structures)
  compounds give higher extinction coefficient as
  they absorb more light.
• - Benzene has an extinction coefficient of
  1000
• - Ethidium Bromide has an extinction
  coefficient of 6000
• Quantum Yield Number of Photons Emitted by the
  Fluorochrome divided by the number of photons
  absorbed.
• Another way to look at the quality of a
  fluorochrome is to use
• e x Q Yield Efficiency or Total Fluorescence.

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Protecting the fluorescence signal
Q What are the problem faced with standard
fluorescent dyes?
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Protecting the fluorescence signal

• Problem
• Low signal intensity
• Photobleaching
• Sensitivity to environmental conditions

Solution The Alexa Fluor dyes brighter, more
photostable, pH insensitive
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Immuno Histochemistry Workflow
Workflow Problems
Culture Cells
Fix cells
Permeabilise
Blocking Buffer
Primary Antibody
Low Signal Intensity
Photobleaching
Secondary Antibody
Non-specific Binding
Nuclear Stain
Mount
Filter Quality
Microscopy
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Multiplexing
Alexa Fluor Dyes across the spectrum
Colour Selection ? Brightness ?
Photostability
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Multiplexing-The Four Main Colours
Emission wavelengths
DAPI/UV
FITC
TRITC
FAR RED
Alexa Fluor 488 Fluorescein (FITC) Cy2
Alexa Fluor 555 Rhodamine, TAMRA, TRITC Cy3
Alexa Fluor 647 Cy5, APC
Alexa Fluor 350 Coumarin, AMCA
Alexa Fluor 594 Texas Red, Cy3.5
Colour Selection ? Brightness ?
Photostability
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Alexa Fluor 488 and fluorescein
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Alexa Fluor 488 vs Fluorescein Bleaching
2x Real Time
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Alexa Fluor Dyes pH Insensitive
Alexa Fluor488
FITC
Oregon Green
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Alexa Fluor Dyes Photo Stability
Laser-scanning cytometry EL4 cells
biotin-anti-CD44 streptavidin conjugates
Photobleaching dye solutions in capillary tubes
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Better Performance
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Alexa Fluor 647 vs. Cy5
Compare Alexa Fluor conjugates to any other
fluorescent conjugates
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Alexa Fluor 405 / Pacific Blue

• Great match for the 407 nm line of the krypton
  laser and the 405 and 408 violet diode lasers.

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Alexa Fluor 405 / Pacific Blue

• Alexa Fluor 405 Brightness- on the order of
  magnitude of FITC

• Alexa Fluor 405 is purported to be less prone to
  problems with non- specific binding.

• Photo-stability 24 hour exposure, no reduction
  in signal

• Pacific Blue is brighter than Alexa Fluor 405.

• Pacific Blue has no compensation issues with UV
  excitable dyes

• Studies have shown a decrease in signal of
  Pacific Blue following fixation.
• Therefore if you need to fix, consider using
  Alexa Fluor 405

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Alexa Fluor 488 succinimidyl ester
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Single Conjugates
Single
Single Conjugates are made by attaching small
fluorescent molecules (fluorophores) to the
antibody at optimal F to P ratios.
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Types of Fluors
Tandem Fluorescence Resonance Energy Transfer
(FRET) Dyes

• Emission of one dye overlaps the excitation of
  a second.
• When close, energy is transferred to the
  second.
• The second emits a longer wavelength of light
  than the first.

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Tandem Conjugates - Energy Transfer Dyes
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Tandem Conjugates - Energy Transfer Dyes
R-PE
APC
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R-Phycoerythrin (PE-Alexa Fluor 610)

• Large multi-subunit, globular (240 kDa)
  protein derived from algae.

• gt20 chromophores per molecule

• High quantum yield (bright)

• Excitation 488 nm and 532 nm

• Emission Maximum 575 nm

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Alexa Fluor 610 Carboxylic Acid, Succinimidyl
Ester
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PE-Alexa Fluor 610
R-Phycoerythrin, Chains A, B, K, and L
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PE-Alexa Fluor 610 an alternative to PE-Texas Red

• Energy transfer dye excited at 488, with maximal
  emission at 628 nm.

• Higher quantum yield than PE-Texas Red with lower
  compensation in PE channel

• Ideally suited for multicolor applications with
  the superior alternative for PE-Cy5
  PE-Alexa Fluor 647

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APC-Alexa Fluor 750
Allophycocyanin, Chains A and B
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APC-Alexa Fluor 750 Analysis

• Instrument Tritech1 modified BD FACScan 2

• Human peripheral blood lymphocytes

• APC-Cy7 and APC-AF750 detected through 765/20
  filter, split from APC with 675 SP

1 Tritech, Edgewater, MD
2 BD, Becton Dickinson Biosciences
Immunocytometry Systems, San Jose, CA
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APC-Alexa Fluor 750 vs. APC-Cy7

• APC-Alexa Fluor 750 is perfectly interchangeable
  with APC-Cy7.
• APC-Alexa Fluor 750 is brighter than APC-Cy7 by
  as much as 2-fold.
• APC-Alexa Fluor 750 exhibits roughly half the
  compensation of APC-Cy7.
• APC-Alexa Fluor 750 is significantly more
  photostable than APC-Cy7.

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Antibodies and ImmunoFluorescence
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Fluorescence reagents-Labeling

• Direct-Labeled Primary Antibody
• Lowest Background
• Potentially low signal due to abundance of
  target or dye
• Dye could affect antigen recognition site

• Zenon Technology
• Brighter Signal
• Dye does NOT affect antigen recognition site

• Indirect-Labeled Secondary Antibody
• Higher Background
• Brighter Signal

• Tyramide Signal Amplification (TSATM)
• Higher Background
• Brightest Signal

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Fluorescence reagents-Direct

• Simple and easy to follow protocols
• Reactive dye is pre-measured no need to weigh
  out small quantities of dye
• Procedure (including purification) takes 2
  hours, with little hands on time
• Three kit sizes

Alexa Fluor Protein Labeling Kit (1 mg)
Alexa Fluor Monoclonal Labeling Kit (100 ug)
New - Alexa Fluor Microscale Protein Labeling
Kit (20-100 ug)
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Fluorescence reagents-Zenon Labeling
1 µg primary antibody
Block with non-specific IgG Use directly
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Zenon labeling technology- Stability
Kinetically trapped complex formation key to
making the system work.
Fast complex formation
Slow complex dissociation
Blocked mouse anti-biotin IgG1 Alexa Fluor 488
Zenon Capture by BSA-biotin in microtiteer plate
Mouse anti-biotin IgG1
Alexa Fluor 488 Zenon Capture by BSA-biotin in
microtiteer plate
Control IgG1
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WHY use ZENON?

• Simple
• No need to use secondary antibodies anymore
• Speed
• Zenon labeling complexes are ready to use for
  cell staining within 5 minutes
• Quantitative Labeling
• 100 of the primary antibody sample is labeled.
• No Preparation
• Removal of exogenous proteins such as serum
  albumin
• from primary antibody samples is unnecessary.
• Compatibility
• Multiple Zenon Onelabeled mouse antibodies can
  be used
• in the same immunolabeling protocol.
• Economy
• A standard Zenon labeling requires only 1 µg of
  primary antibody

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Example 1 Triple Immunofluorescence Staining of
Muntjak Cells with Zenon

• anti-nuclear mouse mAb Alexa Fluor 350 Zenon
  labeling reagent
• F-actin Alexa Fluor 488 phalloidin
• anti-tubulin mouse mAb Alexa Fluor 568 Zenon
  labeling reagent

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Example 2 Switching Fluorescent Colors Same
Cell Type, Same Targets

• F-actin Alexa Fluor 350 phalloidin
• anti-tubulin mouse mAb Alexa Fluor 488 Zenon
  labeling reagent
• anti-nuclear mouse mAb Alexa Fluor 647 Zenon
  labeling reagent

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Just in case you were wondering
Muntjak
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Fluorescence reagents - Indirect
Goat anti-Rabbit IgG
We Offer a Wide Selection of Secondary Antibodies
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Tyramide Signal Amplification (TSA)
TSA can provide high resolution and high S/N
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Chromosome FISH Labeled Streptavidin vs TSA
Streptavidin Alexa Fluor 546
Alexa Fluor 546 TSA
10-fold increase in sensitivity w/ TSA
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Why Use TSA?

• Increased sensitivity
• 100-fold compared to avidinbiotinylated enzyme
  complex (ABC)
• Generates multiple copies of Alexa Fluor
  dyes at target site
• Gives high-resolution signal amplification
• When normal amplification methods fail
• Low abundance targets

3 color sequential TSA
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Immuno Histochemistry Workflow
Workflow Problems
Culture Cells
Fix cells
Permeabilise
Blocking Buffer
Primary Antibody
Low Signal Intensity
Photobleaching
Secondary Antibody
Non-specific Binding
Nuclear Stain
Mount
Filter Quality
Microscopy
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Protecting the fluorescence signal-Antifade
Reagents

• Prolong Gold
• Optimized formulation-Superior antifade with
  little or no impact on initial fluorescence
  intensity
• Ready to use - dropper bottle
• Cures to a firm gel no more nail polish

NEW SlowFade Gold - antifade mounting media
which doesnt polymerize
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Immuno Histochemistry Workflow
Workflow Problems
Culture Cells
Fix cells
Permeabilise
Blocking Buffer
Primary Antibody
Low Signal Intensity
Photobleaching
Secondary Antibody
Non-specific Binding
Nuclear Stain
Mount
Filter Quality
Microscopy
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Increase S/N ratio Reducing Non-specific
background
Non-specific binding - any signal not related to
the target of interest, therefore increasing the
background.

• Problem
• Non-specific binding of dye conjugates
• Standard blocking agents dont work

• Solution
• Image-iTTM FX Signal Enhancer non-serum based
  blocking reagent

Decrease background - Increase Specific Signal
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Increase S/N Ratio - Image-iTTM FX signal
enhancer
BPAE Cells Stained with TMR Streptavidin
Not blocked
Blocked
Image-iTTM FX signal enhancer decreases
background fluorescence!
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Alexa Fluor Image-iT FX Image-iT SFX Kits

• The Alexa Fluor Image-iT FX Kits provide
  all of the dyes and reagents you need for optimal
  imaging of fixed cells and tissue sections for a
  more convenient price
• Alexa Fluor Dye conjugates for superior
  photostability and brightness
• ProLong Gold antifade reagent for reduced
  photobleaching independently of the color of your
  fluorochrome
• Image-iT FX Signal Enhancer for improved
  signal-to-noise ratio
• CultureWell chambered coverslips to make sample
  processing more convenient.

• NEW Alexa Fluor Image-iT SFX Kits Start-Up
  Kits
• Alexa Fluor Dye conjugates in small packaging
  combined with.
• Image-iT FX Signal Enhancer for superior,
  photostable, bright and highly specific staining !

NEW Easier to Use Kits
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Small Animal In Vivo Imaging (SAIVI)

• DRIVERS for INVITROGEN
• Bring our expertise in molecular biology to
  develop solutions in molecular imaging
• Develop tools to enable the acceleration of drug
  discovery
• Continue to support our customers in the
  development of useful tools as they use more
  in-vivo assays

• COMPELLING REASONS for Optical
• Low cost Compared to all
• Ease of use Compared to all
• High sensitivity
• superior to anatomical MR CT
• competitive with functional PET
• Optical can enable benchtop imaging for the PI
• Quantitative Optical can displace some PET

COMPELLING REASONS for NIR Injectables

• The VISION for INVITROGEN
• Leverage our Molecular Probes Labeling
  Detection technology in the Optical imaging space
  with injectable NIR reagents and Qdot technology
• LD
• Cell Biology
• In vivo Imaging
• Leverage our other appropriate technologies (gene
  regulation and delivery) into the in vivo imaging
  space

NIR injectables can simplify and improve optical
SAIVI
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Dye Technologies
Alexa Fluor Dyes

• Fluorescent Nanocrystals.

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SAIVI TM Imaging Reagents

• CONTRAST AGENTS
• To enable intravital imaging and
• blood flow imaging
• Alexa Fluor NIR Dyes
• Labelled colloidal agents
• Labelled proteins
  
• Quantum Dot
• Qtracker non-targeted solution for tail vein
  injection

• CORE TECHNOLOGY
• Dyes
• Multiple NIR wavelengths
• AF 647, 680, 750
• Multiplexing
• Spectral separation
• Multiple chemistries
• Better specificity
• Brighter
• Patented Bodipy dye portfolio
  
  
• Quantum Dots
• Expanded in-vivo utility

• CUSTOMER TOOLS
• Dye Labeling Kits
• 3 degrees of flexibility
• Multiple NIR wavelengths
• AF 647, 680, 750
• NIR Nanocrystals
• Multiplexing
• Spectral separation
• Scale (0.1mg, 1.0 mg)
• Degree of Labeling Control for brightness vs
  specificity vs pharmacokinetics
• Quantum Dots
• Conjugation Kits

• TARGETED PROBES
• Broad Applications (2006)
• To enable
• Customer Research in
• Apoptosis
• Angiogenesis
• Cancer
• Inflammation
• Cell Tracking (Now)
• Qtracker cell labeling kits
• Confidential Custom
• Services (Now)
• Aimed at Pharma/Biotech

Available Now Available
Now
Available Now
Unparalleled flexibility
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Alexa Fluor NIR Dye (SAIVI) Protein Labeling Kits

• Simple and easy to follow protocols
• Reactive dye is pre-measured no need to weigh
  out small quantities of dye
• Procedure (including purification) takes 2
  hours, with little hands on time
• Two kit sizes

Alexa Fluor Protein Labeling Kit (1 mg)
Alexa Fluor Monoclonal Labeling Kit (100 ug)
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SAIVI TM Labeling Kits Three degrees of
flexibility
Control of Degree of Labeling (DOL) of a typical
IgG anitbody with Alexa Fluor 680 NIR dye using a
SAIVI Alexa Fluor 680 0.1 mg Antibody/Protein
Labeling Kit. Alteration of DOL was achieved
without a significant change in protein volume or
concentration. Final yield of purified protein
remains constant across the DOL range.
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In-vivo imaging across the entire continuum
Molecular Targeting
Non-targeted Intravascular Cell Surface
Interstitial Intracellular
Metabolic
Quantum Dots
Quantum Dots
Alexa Fluors
Alexa Fluors
Cellular Tracking
Passive Proliferation Uptake
Metabolism Reporter Systems
Quantum Dots
Alexa Fluors
Post-mortem analysis
Physiology Molecular
Quantum Dots
Alexa Fluors
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Alexa Fluor Dyes Simply The Best

• Brightness
• Photostability
• Color Selection
• pH Insensitivity
• Water Solubility
• Ease of Use
• Wide Selection

• Broad Selection

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Immuno Histochemistry Workflow
Workflow Problems
Culture Cells
Fix cells
Permeabilise
Blocking Buffer
Primary Antibody
Low Signal Intensity
Photobleaching
Secondary Antibody
Non-specific Binding
Nuclear Stain
Mount
Filter Quality
Microscopy
58
Semrock BrightLine filters

• 2 to 4 times the brightness
• Twice the contrast
• Extremely low crosstalk
• Unparalleled performance
• brightest most discriminating filters for the
  fastest measurements
• Unique spectral capabilities
• unlock new capabilities that you once only
  dreamed about!
• Proven reliability for permanent performance
• work in hot, humid or corrosive environments
• Highest batch-to-batch reproducibility
• ensure the repeatable manufacturing of your
  product

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Independent images taken by experts in the field

• Differences in film density and resulting overall
  film quality are readily apparent

Semrock Filters
Ion-Beam Sputtered Hard Coating
Major Competitor
E-beam Evaporated Soft Coating
Identical conditions
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BrightLine spectra versus leading competitor
Exciter / emitter pairs for FITC
Competitors multiband set
Photographs of a Molecular Probes FluoCells 2
slide taken on an Olympus BX41 microscope using a
Spot Insight Color camera by Diagnostic
Instruments Inc. with competing DAPI/FITC/Texas
Red multiband filters sets
BrightLineTM multiband set
BrightLineTM Multiband visibly superior
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and, quantitatively superior!
Competitor
BrightLineTM
Color images were captured by a Spot Insight
color CCD camera by Diagnostic Instruments Inc.
Analysis based on monochrome images captured by a
QImaging Retiga cooled 12-bit CCD camera
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Compared to the competitors best filter set
Competitor
Competitor
Semrock BrightLine
Semrock BrightLine
Comparisons done under identical imaging
conditions using an Olympus BX61WI microscope
outfitted DSU spinning-disk confocal unit and a
Hamamatsu ORCA-ER monochrome CCD camera. RMK
sample courtesy of Mike Davidson, Molecular
Expressions.
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Semrock for the ultimate in
The Semrock BrightLine family When you want the
best images !
Brightness, Performance, Reliability !
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www.invitrogen.com/iProtocol
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www.invitrogen.com/iPath
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www.invitrogen.com/Antibodies
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Literature and brochures
Easy downloads from the web, or available on
request.
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You can have your own copy!
www.probes.invitrogen.com
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before
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Questions