5-CHANNELFLUORESCENCE IMAGING SIMPLIFIED
Reliable Multiplexing for the Non-specialist

CHROMA TECHNOLOGY CORP®

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Introducing simpler and more reliable5-channel fluorescence imaging
New, brighter fluorochromes from BD Biosciences and optimized filter sets from Chroma
Technology make multi-color imaging simpler and more accessible.

BV421

AlexaFluor 555

BV480

FITC

AlexaFluor488

BV480

AlexaFluor555

BV421

Draq5

Alexa Fluor 647

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Novel polymer chemistry led to the discovery and development of conductive
organic polymers.*These polymers led to the development of a new class of extremely bright fluorescentmolecules which have now become the BD Horizon Brilliant™ family of fluorochromes.

Polymers harvest light,
acting like a molecularantenna, due to a very large
Extinction Coefficienthυhυhυhυhυhυhυhυhυhυhυ(capacity to absorb photons)= Optical Segment* Nobel Prize in Chemistry, 2000 (Alan J. Heeger, Alan G. MacDiarmid and Hideki Shirakawa)

CHROMA TECHNOLOGY CORP®

in collaboration withHigh Quantum
Yield
results in anabundance ofphotons emitted
as fluorescenceBD Biosciences, a respected leader in the field of cellular analysis, acquired this technology.
Brilliant Violet 421 (BV421) was the first of these commercially available polymers.
Recognizing how useful these fluorochromes could be in imaging applications, BD developed
BV480. These exceptionally bright, photostable violet and blue fluorochromes are now
available for use in microscopy.

BV421

BV480

ɛ (λex) = 2,534,400 ; Φf = 0.70;

ɛ (λex) = 1,450,000 ; Φf = 0.69;>50-fold brighter thanCascade Blue or Pacific Blue>20-fold brighter than
DEAC or ECFPCHROMA TECHNOLOGY CORP®

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Brightness and spectral properties of BD Horizon Brilliant™ Violet
Fluorochromes enable:• Lower levels of excitation energy,
minimizing photodamage• Fewer numbers of molecules(increased sensitivity)• Shorter exposures, increasing speed• Increased parameters per sample

BV480

Alexa Fluor® 488

15ms Exposure

140ms Exposure

Draq5

Draq5

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BD Biosciences and Chroma Technology have collaborated to introduce thesefluorochromes to help advance fluorescence microscopy.
Chroma Technology recognized the opportunity to make 5-color imaging easily accessible
to anyone working with a basic fluorescence microscopy stand. BV421 and BV480 fill agap in the visible spectrum:

BV421
BV480
Widely availablefluors

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5 reliable fluorescence channels:
Reliable and popular fluors from respected suppliers provide the longer wavelengths.
5 colors, with excitation wavelengths from approx. 390-650nm, and emission wavelengths
from approx. 420-700nm allow for the use of standard light sources and cameras:

BV421
BV480
*488
*568

*647

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The equipment you need:+Standard Microscope

What You
Don’t Need+

Standard Light Source}

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Standard Camera

Advanced imaging expertise

Sophisticated technologies or equipment

in collaboration with

The filters you need:
Because these
are all very brightfluors, we are able
to use narrow-band
excitation andemission filters tomaximize spectral
separation.
Shown here areseparate excitationand emission filter
graphs for ease ofillustration.

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With Excitation Filters

BV421

in collaboration with

With Emission Filters

BV480

*488

*568

*647

Alternative filter options:
Many workers prefer to use multiband filter sets for multi-channel imaging. Chroma offers
5-band (or more) filter sets for use with external filter wheels to house multiple excitationand emission filters with one multiband polychroic mirror in a cube. This allows for evenmore fluorochromes and fast, efficient image acquisition, including z-stacks.

BV421
BV480
*488
*568

*647

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in collaboration withBD Horizon Brilliant™ fluorochromes excel in fluorescence imaging applications:
Immunocytochemistry

Immunohistochemistry

BV421
Draq5

BV480
Draq5

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Addition of BV421 and BV480 enables more effective multiplexing and flexibility:

5-ChannelOptions

BV421

BA480

488 fluor
OR
Various Green FPs

568/555/546 fluor
OR
Various Red FPs

Draq5 (DNA)

BV421

BA480

488 fluor
OR
Various Green FPs

568/555/546 fluor
OR
Various Red FPs

647 fluor

DAPI (DNA)BA480

488 fluor
OR
Various Green FPs

568/555/546 fluor
ORVarious Red FPs

647 fluor

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Robust 5-color imaging:
DAPI, BV480, Alexa Fluor® 488, Alexa Fluor® 555, Alexa Fluor® 647

DAPI

E Cadherin BV480

Tubulin Alexa Fluor® 488

Ki-67 Alexa Fluor® 555

GM130 Alexa Fluor® 647

Merge

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Robust 5-color imaging:
BV480, Alexa Fluor® 488, Alexa Fluor® 555, BV421, DRAQ5

Cytochrome C BV480

Histone H3 Alexa Fluor® 488

Ki67 Alexa Fluor® 555

LAMP1 BV421

DRAQ5Merge

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Robust 5-color imaging:
GFP, RFP, Alexa Fluor® 647, BV480, BV421

CX3CR1 eGFP

CCR2 RFP

B220 Alexa Fluor® 647

CD3 BV480

CD4 BV421

Merge

Thank you to collaborators in the Catherine Hedrick lab at La Jolla Institute of Allergy and Immunology for data.

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Chroma Filter Set Recommendations for 5-channel imaging:
Recommended

Alternate

Fluor 1:

BV421 (or DAPI)

49027

Fluor 2:

BV480

49032

49001

Fluor 3:*488 (or various GFPs)

49303

49003

Fluor 4a:*568 (or mCherry)

49031

49306

Fluor 4b:*546, 555 (or TagRFP, mApple, mRuby)

49305

49004

Fluor 5:*647 (or Draq5)

49009= Best Compatibility

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= Good Compatibility

in collaboration with= Not Compatible

For Best Results
We’re providing new tools to workers who use fluorescence microscopy in order to simplify 5color imaging.
However, as always when immunolabeling, care must be taken to control for variables like
non-specific labeling, cross-reactivity and autofluorescence.
BD Horizon Brilliant™ fluorochromes are bright enough to be effectively used as direct
immunofluorescence probes, for one-step immunolabeling. This greatly simplifies
immunolabeling and greatly reduces non-specific fluorescence and background noise.
When expressing and imaging fluorescent proteins, careful technique and expertise andknowledge of potential problems is required. BV480 provides an extremely bright cyanchannel. To optimize the use of fluorescent proteins, use GFP-like or YFP-like proteins alongwith Red proteins similar to mApple, mRuby, TagRFP.

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in collaboration withSummary
Two new bright fluorochromes fromBD Biosciences plus Chroma’s optimized
filter sets enable simplified 5-color imaging.
BV421 and BV480 fill a gap in the visible spectrum.
With specific fluorescence filter sets, reliable tools now
enable imaging with up to 5 colors using basic tools
available to the non-Specialist.
Visit the Chroma website for details about filter sets:www.chroma.com/products/single-band-sets
Please visit the BD Bioscience website for details about
BD Horizon Brilliant™ fluorochromes:
http://www.bdbiosciences.com/research/cellularimaging

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With Emission Filters

THANK YOU
Published in Bellows Falls, Vermont, USChroma optical filters and related products may be purchased online or via phone. For
more information, please e-mail sales@chroma.com, visit www.chroma.com or call
800.824.7662.

BD Biosciences provides flow cytometers, reagents, tools and a wide range of services.
For more information, please visit www.bdbiosciences.com or call 877.232.8995.

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