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5-CHANNELFLUORESCENCE IMAGING SIMPLIFIEDReliable Multiplexing for the Non-specialistCHROMA TECHNOLOGY CORP in collaboration with
Introducing simpler and more reliable5-channel fluorescence imagingNew, brighter fluorochromes from BD Biosciences and optimized filter sets from ChromaTechnology make multi-color imaging simpler and more accessible.BV421AlexaFluor aq5Alexa Fluor 647CHROMA TECHNOLOGY CORP in collaboration with
Novel polymer chemistry led to the discovery and development of conductiveorganic 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 largeExtinction ity 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 QuantumYieldresults in anabundance ofphotons emittedas 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 developedBV480. These exceptionally bright, photostable violet and blue fluorochromes are nowavailable for use in microscopy.BV421BV480ɛ (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 thanDEAC or ECFPCHROMA TECHNOLOGY CORP in collaboration with
Brightness and spectral properties of BD Horizon Brilliant VioletFluorochromes enable: Lower levels of excitation energy,minimizing photodamage Fewer numbers of molecules(increased sensitivity) Shorter exposures, increasing speed Increased parameters per sampleBV480Alexa Fluor 48815ms Exposure140ms ExposureDraq5Draq5CHROMA TECHNOLOGY CORP in collaboration with
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 accessibleto anyone working with a basic fluorescence microscopy stand. BV421 and BV480 fill agap in the visible spectrum:BV421BV480Widely availablefluorsCHROMA TECHNOLOGY CORP in collaboration with
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 wavelengthsfrom approx. 420-700nm allow for the use of standard light sources and cameras:BV421BV480*488*568*647CHROMA TECHNOLOGY CORP in collaboration with
The equipment you need: Standard MicroscopeWhat YouDon’t Need Standard Light Source}CHROMA TECHNOLOGY CORP Standard CameraAdvanced imaging expertiseSophisticated technologies or equipmentin collaboration with
The filters you need:Because theseare all very brightfluors, we are ableto use narrow-bandexcitation andemission filters tomaximize spectralseparation.Shown here areseparate excitationand emission filtergraphs for ease ofillustration.CHROMA TECHNOLOGY CORP With Excitation FiltersBV421in collaboration withWith Emission FiltersBV480*488*568*647
Alternative filter options:Many workers prefer to use multiband filter sets for multi-channel imaging. Chroma offers5-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.BV421BV480*488*568*647CHROMA TECHNOLOGY CORP in collaboration with
BD Horizon Brilliant fluorochromes excel in fluorescence imaging ryBV421Draq5BV480Draq5CHROMA TECHNOLOGY CORP in collaboration withFISH
Addition of BV421 and BV480 enables more effective multiplexing and PI (DNA)BA480CHROMA TECHNOLOGY CORP 488 fluorORVarious Green FPs488 fluorORVarious Green FPs488 fluorORVarious Green FPsin collaboration with568/555/546 fluorORVarious Red FPsDraq5 (DNA)568/555/546 fluorORVarious Red FPs647 fluor568/555/546 fluorORVarious Red FPs647 fluor
Robust 5-color imaging:DAPI, BV480, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647DAPIE Cadherin BV480Tubulin Alexa Fluor 488Ki-67 Alexa Fluor 555GM130 Alexa Fluor 647MergeCHROMA TECHNOLOGY CORP in collaboration with
Robust 5-color imaging:BV480, Alexa Fluor 488, Alexa Fluor 555, BV421, DRAQ5Cytochrome C BV480Histone H3 Alexa Fluor 488Ki67 Alexa Fluor 555LAMP1 BV421DRAQ5MergeCHROMA TECHNOLOGY CORP in collaboration with
Robust 5-color imaging:GFP, RFP, Alexa Fluor 647, BV480, BV421CX3CR1 eGFPCCR2 RFPB220 Alexa Fluor 647CD3 BV480CD4 BV421MergeThank you to collaborators in the Catherine Hedrick lab at La Jolla Institute of Allergy and Immunology for data.CHROMA TECHNOLOGY CORP in collaboration with
Chroma Filter Set Recommendations for 5-channel imaging:RecommendedAlternateFluor 1:BV421 (or DAPI)49027Fluor 2:BV4804903249001Fluor 3:*488 (or various GFPs)4930349003Fluor 4a:*568 (or mCherry)4903149306Fluor 4b:*546, 555 (or TagRFP, mApple, mRuby)4930549004Fluor 5:*647 (or Draq5)49009 Best CompatibilityCHROMA TECHNOLOGY CORP Good Compatibilityin collaboration with Not Compatible
For Best ResultsWe’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 likenon-specific labeling, cross-reactivity and autofluorescence.BD Horizon Brilliant fluorochromes are bright enough to be effectively used as directimmunofluorescence probes, for one-step immunolabeling. This greatly simplifiesimmunolabeling 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.CHROMA TECHNOLOGY CORP in collaboration with
SummaryTwo new bright fluorochromes fromBD Biosciences plus Chroma’s optimizedfilter sets enable simplified 5-color imaging.BV421 and BV480 fill a gap in the visible spectrum.With specific fluorescence filter sets, reliable tools nowenable imaging with up to 5 colors using basic toolsavailable to the non-Specialist.Visit the Chroma website for details about filter e visit the BD Bioscience website for details aboutBD Horizon Brilliant h/cellularimagingCHROMA TECHNOLOGY CORP in collaboration withWith Emission Filters
THANK YOUPublished in Bellows Falls, Vermont, USChroma optical filters and related products may be purchased online or via phone. Formore information, please e-mail sales@chroma.com, visit www.chroma.com or call800.824.7662.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.CHROMA TECHNOLOGY CORP in collaboration with
New, brighter fluorochromes from BD Biosciences and optimized filter sets from Chroma . filter sets enable simplified 5-color imaging. BV421 and BV480 fill a gap in the visible spectrum. . 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 .
