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1、Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,23-13536-00 Rev.01,Fluorochromes,*,Fluorochrome Properties,Desirable properties for fluorochromes:,High relative brightness,Narrow emission spectrum(low spectral overlap in combination),Easily con
2、jugated(for immunophenotyping),Fluorochromes can be characterized by:,Type of molecule,Excitation and emission wavelengths,Relative brightness,1,Fluorochrome Molecule Types,Small organic molecules,examples:FITC,BD Horizon,TM,V450,Cy7,Fluorescent proteins,examples:PE,APC,PerCP,Tandem dyestypically,th
3、e coupling of a fluorescent protein donor with a small organic molecule acceptor,examples:PE-Cy7,PerCP-Cy,TM,5.5,Nanocrystals(Qdots)inorganic semiconductors,examples:Qdot 565,Qdot 605,2,Some Common Fluorochromes,Fluorochrome,Type of molecule,Fluorescein isothyocyanate(FITC),Small organic,Alexa Fluor
4、 488,Small organic,Phycoerythrin(PE),Protein,PE-Cy,TM,5,Protein tandem,Peridinin chlorophyll protein(PerCP),Protein,PerCP-Cy,TM,5.5,Protein tandem,PE-Cy,TM,7,Protein tandem,Allophycocyanin(APC),Protein,Alexa Fluor 647,Small organic,APC-Cy,TM,7,Protein tandem,BD,TM,APC-H7,Protein tandem,BD Horizon,TM
5、,V450,Small organic,Pacific Blue,TM,Small organic,BD Horizon,TM,V500,Small organic,AmCyan,Protein,3,Small Organic Fluorochromes,Advantages,Low molecular weight,Easy to conjugate,d,irect attachment to free amino groups on mAb,Excellent stability,Extremely consistent emission spectra,Disadvantages,Sma
6、ll Stokes Shift(50,100 nm),Tend to be less bright,4,Protein Fluorochromes,Advantages,Good stability,Consistent emission spectra,Medium Stokes Shift(75,200 nm),Tend to be more bright,Disadvantages,High molecular weight,More difficult to conjugate,intermediaries needed to attach to mAb,5,Tandem Dye Fl
7、uorochromes,Advantages,Very large Stokes Shift(150,300 nm),Tend to be very bright,often brighter,than the fluorescent protein donor,Disadvantages,High molecular weight(similar to fluorescent protein),Difficult to make consistently(lot-to-lot variation in emission properties),Harder to conjugate,(sam
8、e as,fluorescent protein),Some tandems have poor stability,6,Nanocrystal Fluorochromes,Advantages,Large Stokes Shift(100,500 nm),Tend to be very bright,Emission peaks are consistent and narrow,and do not change with variations in the excitation source,Highly resistant to photobleaching,Nanocrystals
9、share biophysical and conjugation properties,Disadvantages,Difficult to conjugate,Instability of bindings,Cytotoxicity,Wide excitation range produces cross-laser spillover,7,Excitation and Emission,Excitation wavelengths determine lasers that can excite the fluorochrome.,Emission wavelengths determi
10、ne filters and PMTs that can measure the emission signal.,8,Know Your Cytometer,Cytometer Configuration,BD FACSCanto,TM,II 4-2-2 configuration is shown below,BD,TM,LSR II 4-2-2 configuration is similar,4 detectors for blue laser,2 detectors for red laser,2 detectors for violet laser,9,Typical Excita
11、tion and Emission,BD FACSCanto II 4-2-2(BD LSR II 4-2-2 is similar),Detector Range,Violet Laser 405 nm,Blue Laser 488 nm,Red Laser 633 nm,410490 nm,BD Horizon V450 Pacific Blue,TM,500560 nm,BD Horizon V500 AmCyan,515545 nm,FITC,Alexa Fluor,488,564606 nm,PE,650670 nm,APCAlexa Fluor,647,670735 nm,PerC
12、P-Cy5.5PE-Cy5PerCP,750810 nm,PE-Cy7,BD APC-H7APC-Cy7,10,Fluorochrome Use Depends on the Cytometer Configuration,6-color,8-color,More than,8 colors,FITC,Alexa Fluor 488,FITC,Alexa Fluor 488,FITC,Alexa Fluor 488,PE,PE,PE,PE-Texas Red,PE-Alexa,Fluor 610,or PE-Alexa Fluor 594,PE-Cy5,PerCP,or PerCP-Cy5.5
13、,PE-Cy5,PerCP,or PerCP-Cy5.5,PE-Cy5,PerCP,or PerCP-Cy5.5,PE-Cy7,PE-Cy7,PE-Cy7,APC,Alexa Fluor 647,APC,Alexa Fluor 647,APC,Alexa Fluor 647,APC-Cy5.5,Alexa Fluor 680,Alexa Fluor 700,APC-H7,APC-Cy7,APC-H7,APC-Cy7,APC-H7,APC-Cy7,BDHorizon V500,AmCyan,BDHorizon V500,AmCyan,BDHorizon V450,Pacific Blue,BD
14、Horizon V450,Pacific Blue,Pacific Orange,Q-dots,11,Fluorochrome Brightness,The brightness of a fluorochrome depends on two factors:,Molar Extinction Coefficient(,),measures how well a fluorochrome,absorbs energy.,Quantum Yield(Qy),is the ratio of photons emitted to photons absorbed.,Brightness,=,x,Q
15、y,Relative Brightness,=,Brightness of PE,Brightness,12,Some Fluorochromes are MUCH Brighter,PE is 50 x brighter than FITC and 10 x brighter than APC.,APC is 5x brighter than Pacific Blue.,Extinction coefficient is more significant than quantum yield in determining brightness.,Fluorochrome,Molar Exti
16、nction Coefficient(mol,-1,x cm,-1,),Quantum Yield,Relative Brightness(to PE),PE,1960000,0.98,100.00%,PE-Cy5,1960000,0.8,81.63%,PerCP,320000,NA,16.66%,APC,232000,0.68,8.21%,FITC,67000,0.50,1.74%,Pacific Blue,36000,0.80,1.5%,13,D,D,=difference between the medians of the positive and negative populations,W,=spread(2 x rSD)of the negative population,Stain Index,Stain Index=,Stain index is a practical way to characterize the brightness of a marker with respect to a given optical configuration.,D,W,W,
流式細(xì)胞技術(shù)熒光抗體
