23-13536-00 Rev. 01,Fluorochromes,23-13536-00 Rev. 01,2,Fluorochrome Properties,Desirable properties for fluorochromes: High relative brightness Narrow emission spectrum (low spectral overlap in combination) Easily conjugated (for immunophenotyping) Fluorochromes can be characterized by: Type of molecule Excitation and emission wavelengths Relative brightness,23-13536-00 Rev. 01,3,Fluorochrome Molecule Types,Small organic molecules examples: FITC, BD HorizonTM V450, Cy7 Fluorescent proteins examples: PE, APC, PerCP Tandem dyestypically, the coupling of a fluorescent protein donor with a small organic molecule acceptor examples: PE-Cy7, PerCP-CyTM5.5 Nanocrystals (Qdots)inorganic semiconductors examples: Qdot 565, Qdot 605,23-13536-00 Rev. 01,4,Some Common Fluorochromes,23-13536-00 Rev. 01,5,Small Organic Fluorochromes,Advantages Low molecular weight Easy to conjugatedirect attachment to free amino groups on mAb Excellent stability Extremely consistent emission spectra Disadvantages Small Stokes Shift (50100 nm) Tend to be less bright,23-13536-00 Rev. 01,6,Protein Fluorochromes,Advantages Good stability Consistent emission spectra Medium Stokes Shift (75200 nm) Tend to be more bright Disadvantages High molecular weight More difficult to conjugateintermediaries needed to attach to mAb,23-13536-00 Rev. 01,7,Tandem Dye Fluorochromes,Advantages Very large Stokes Shift (150300 nm) Tend to be very brightoften 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 (same as fluorescent protein) Some tandems have poor stability,23-13536-00 Rev. 01,8,Nanocrystal Fluorochromes,Advantages Large Stokes Shift (100500 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 share biophysical and conjugation properties Disadvantages Difficult to conjugate Instability of bindings Cytotoxicity Wide excitation range produces cross-laser spillover,23-13536-00 Rev. 01,9,Excitation and Emission,Excitation wavelengths determine lasers that can excite the fluorochrome. Emission wavelengths determine filters and PMTs that can measure the emission signal.,23-13536-00 Rev. 01,10,Know Your Cytometer,Cytometer Configuration BD 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,23-13536-00 Rev. 01,11,Typical Excitation and Emission BD FACSCanto II 4-2-2 (BD LSR II 4-2-2 is similar),23-13536-00 Rev. 01,12,Fluorochrome Use Depends on the Cytometer Configuration,23-13536-00 Rev. 01,13,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 Qy Relative Brightness =,Brightness of PE,Brightness,23-13536-00 Rev. 01,14,Some Fluorochromes are MUCH Brighter,PE is 50x brighter than FITC and 10x brighter than APC. APC is 5x brighter than Pacific Blue. Extinction coefficient is more significant than quantum yield in determining brightness.,23-13536-00 Rev. 01,15,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.,DW,W,Stain index can also be used to characterize the sensitivity of a fluoresence parameter.,23-13536-00 Rev. 01,16,Typical CD4 Stain IndexesBD LSR II,APC has a higher CD4 stain index than PE-Cy5, but approximately 1/10th the relative brightness.,23-13536-00 Rev. 01,17,Typical CD4 Stain IndexesBD FACSCanto II,FITC has approximately the same CD4 stain index as PerCP, but approximately 1/10th the relative brightness.,23-13536-00 Rev. 01,18,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 power Detector range Detector efficiency Background signal Dont depend on published valuesmeasure stain index on your own system.,23-13536-00 Rev. 01,19,CD4 Stain Indexes Across Cytometers,Stain Index Exercise,23-13536-00 Rev. 01,20,Fluorescence Spillover,Emission of FITC in PE channel,23-13536-00 Rev. 01,21,Significant Spillovers on 4-2-2 Configuration,23-13536-00 Rev. 01,22,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.,23-13536-00 Rev. 01,23,Lost Population due to Spillover,Lymphocytes stained with CD45 FITC and CD4 PE,CD45 FITC causes dim CD4+CD45+ to be difficult to distinguish due to significant FITC spillover into PE.,