2022年医学专题—流式细胞技术荧光抗体.ppt

Fluorochrome Properties,Desirable properties for fluorochromes:High relative brightnessNarrow emission spectrum(low spectral overlap in combination)Easily conjugated(for immunophenotyping)Fluorochromes can be characterized by:Type of moleculeExcitation and emission wavelengthsRelative brightness,1,23-13536-00 Rev.01,第一页，共三十五页。,Fluorochrome Molecule Types,Small organic moleculesexamples:FITC,BD HorizonTM V450,Cy7Fluorescent proteinsexamples:PE,APC,PerCPTandem dyestypically,the coupling of a fluorescent protein donor with a small organic molecule acceptorexamples:PE-Cy7,PerCP-CyTM5.5Nanocrystals(Qdots)inorganic semiconductorsexamples:Qdot 565,Qdot 605,2,23-13536-00 Rev.01,第二页，共三十五页。,Some Common Fluorochromes,3,23-13536-00 Rev.01,第三页，共三十五页。,Small Organic Fluorochromes,AdvantagesLow molecular weightEasy to conjugatedirect attachment to free amino groups on mAbExcellent stabilityExtremely consistent emission spectraDisadvantagesSmall Stokes Shift(50100 nm)Tend to be less bright,4,23-13536-00 Rev.01,第四页，共三十五页。,Protein Fluorochromes,AdvantagesGood stabilityConsistent emission spectraMedium Stokes Shift(75200 nm)Tend to be more brightDisadvantagesHigh molecular weightMore difficult to conjugateintermediaries needed to attach to mAb,5,23-13536-00 Rev.01,第五页，共三十五页。,Tandem Dye Fluorochromes,AdvantagesVery large Stokes Shift(150300 nm)Tend to be very brightoften brighter than the fluorescent protein donor DisadvantagesHigh molecular weight(similar to fluorescent protein)Difficult to make consistently(lot-to-lot variation in emission properties)Harder to conjugate(same as fluorescent protein)Some tandems have poor stability,6,23-13536-00 Rev.01,第六页，共三十五页。,Nanocrystal Fluorochromes,AdvantagesLarge Stokes Shift(100500 nm)Tend to be very brightEmission peaks are consistent and narrow,and do not change with variations in the excitation sourceHighly resistant to photobleachingNanocrystals share biophysical and conjugation properties DisadvantagesDifficult to conjugateInstability of bindingsCytotoxicityWide excitation range produces cross-laser spillover,7,23-13536-00 Rev.01,第七页，共三十五页。,Excitation and Emission,Excitation wavelengths determine lasers that can excite the fluorochrome.Emission wavelengths determine filters and PMTs that can measure the emission signal.,8,23-13536-00 Rev.01,第八页，共三十五页。,Know Your Cytometer,Cytometer ConfigurationBD FACSCantoTM II 4-2-2 configuration is shown below BDTM LSR II 4-2-2 configuration is similar,4 detectors for blue laser,2 detectors for red laser,2 detectors for violet laser,9,23-13536-00 Rev.01,第九页，共三十五页。,Typical Excitation and EmissionBD FACSCanto II 4-2-2(BD LSR II 4-2-2 is similar),10,23-13536-00 Rev.01,第十页，共三十五页。,Fluorochrome Use Depends on the Cytometer Configuration,11,23-13536-00 Rev.01,第十一页，共三十五页。,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 QyRelative Brightness=,Brightness of PE,Brightness,12,23-13536-00 Rev.01,第十二页，共三十五页。,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.,13,23-13536-00 Rev.01,第十三页，共三十五页。,D,D=difference between the medians of the positive and negative populationsW=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.,DW,W,Stain index can also be used to characterize the sensitivity of a fluoresence parameter.,14,23-13536-00 Rev.01,第十四页，共三十五页。,Typical CD4 Stain IndexesBD LSR II,APC has a higher CD4 stain index than PE-Cy5,but approximately 1/10th the relative brightness.,15,23-13536-00 Rev.01,第十五页，共三十五页。,Typical CD4 Stain IndexesBD FACSCanto II,FITC has approximately the same CD4 stain index as PerCP,but approximately 1/10th the relative brightness.,16,23-13536-00 Rev.01,第十六页，共三十五页。,Stain Index Factors,Stain index is dependent on the optical configuration and additional performance factors.Factors that can affect stain index include:Laser wavelength and powerDetector rangeDetector efficiencyBackground signalDont depend on published valuesmeasure stain index on your own system.,17,23-13536-00 Rev.01,第十七页，共三十五页。,CD4 Stain Indexes Across Cytometers,Stain Index Exercise,18,23-13536-00 Rev.01,第十八页，共三十五页。,Fluorescence Spillover,Emission of FITC in PE channel,19,23-13536-00 Rev.01,第十九页，共三十五页。,Significant Spillovers on 4-2-2 Configuration,20,23-13536-00 Rev.01,第二十页，共三十五页。,Spillover Decreases Sensitivity,Without CD45 AmCyan,With CD45 AmCyan,CD19 FITC,Spillover can significantly increase the variability of negative and dim populations,even after compensation is applied.,21,23-13536-00 Rev.01,第二十一页，共三十五页。,Lost Population due to Spillover,Lymphocytes stained with CD45 FITC and CD4 PE