fluorescencecompensation - Discoveries Need Dollarsfluorescencecompensation -发现需要美元.ppt

Fluorescence Compensation In Flow Cytometry Frank Battye WEHI Cytometry Lab Flow Cytometry: Multi-colour Detection Fluorescein / PE spillover 2-Colour Fluorescence Compensation 2-Colour Fluorescence Compensation 2-Colour Fluorescence Compensation 2-Colour Fluorescence Compensation Suitable Single-Colour Controls Tricky Issues: Effects of High Compensation Tricky Issues: Unequal Intensities Tricky Issues: Tandem Conjugates Tricky Issues: “Auto”fluorescence Equations: 3-colour Equations: Multi-Colour Matrix Fluorescence Compensation Multi-Colour Compensation Solution.. Effect of Pairwise Compensation of 3 Colours Single Colour Controls: Spillover Practical Issues: Mechanisms Used in Cytometers Compensation Mechanisms: Scans, ‘Calibur, LSR, Star Compensation Mechanisms: MoFlo Compensation Mechanisms: MoFlo Compensation Mechanisms: DiVa Setting Compensation on DiVa Data * THE WALTER ELIZA HALL INSTITUTE OF MEDICAL RESEARCH .au/cytometry/presentations.html 0 20 40 60 80 100 480 500 520 540 560 580 600 620 emission l (nm) FL1 Fluorescein FL2 PE The 2-colour compensation equation (background fluorescence ignored): FL1 = FluorA + f * FluorB FL2 = FluorB + g * FluorA (where f and g are the spillovers) These can be solved exactly to give: FluorA = (FL1 - f * FL2)/(1 - f * g) FluorB = (FL2 - g * FL1)/(1 - f * g) i.e. in 2-colour fluorescence compensation, the compensation factor = - the spillover The 2-colour spillover equation (background fluorescence ignored): FL1 = 1.0 f f1 FL2 g 1.0 f2 (where f and g are the spillovers) Ignoring the normalisation, these give: f1 = 1.0 -f FL1 f2 -g 1.0 FL2 i.e. in 2-colour fluorescence compensation, the compensation factor = - the spillover ( ( ( ) ) ) ( ( ( ) ) ) The 2-colour spillover equation (background fluorescence ignored): FL1 = 1.0 .01 f1 FL2 .25 1.0 f2 (where f and g are the spillovers) Ignoring the normalisation, these give: