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Fluorescence Compensation In Flow Cytometry

Fluorescence Compensation In Flow Cytometry. Frank Battye WEHI Cytometry Lab. http://www.wehi.edu.au/cytometry/presentations.html. Flow Cytometry: Multi-colour Detection. FL2. PE. Fluorescein / PE spillover. FL1. 100. 80. 60. Fluorescein. 40. 20. 0. 480. 500. 520. 540. 560.

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Fluorescence Compensation In Flow Cytometry

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  1. Fluorescence Compensation In Flow Cytometry Frank Battye WEHI Cytometry Lab http://www.wehi.edu.au/cytometry/presentations.html

  2. Flow Cytometry: Multi-colour Detection

  3. FL2 PE Fluorescein / PE spillover FL1 100 80 60 Fluorescein 40 20 0 480 500 520 540 560 580 600 620 emission (nm) l

  4. 2-Colour Fluorescence Compensation 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

  5. ) ( ) ( ) ( 2-Colour Fluorescence Compensation 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 ) ) ( ) ( (

  6. 2-Colour Fluorescence Compensation 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: f1 = 1.0 -.01 FL1 f2 -.25 1.0 FL2 i.e. in 2-colour fluorescence compensation, the compensation factor = - the spillover ) ( ) ( ) ( ) ) ( ) ( (

  7. 4 10 3 10 FL2 - 0 % FL1 FL2 - 30 % FL1 FL2 - 36 % FL1 FL2 - 20 % FL1 2 FL2-H 10 1 10 0 10 0 1 2 3 4 10 10 10 10 10 FL1-H 2-Colour Fluorescence Compensation Mean FL2: 7.9 units

  8. Suitable Single-Colour Controls Mouse Spleen Cell Type: “Positives” and “negatives” should differ only in the presence of the specific fluorochrome. Fluorochromes: While the emission spectra of fluorescein and PE don’t vary significantly from batch to batch, that is not the case for all fluorochromes (e.g. Tandem conjugates vary considerably). fluor2 T-cells Non-T Thy-1.fluor1

  9. RAW DATA COMPENSATED DATA 4 4 10 10 PE spill = 420 + 50 Tricolor = 1200 + 144 PE = PE spill - 35% Tricolor = background + 100 3 3 10 10 2 2 FL2 (PE) FL2 (PE) 10 10 1 1 10 10 0 0 10 10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 FL3 (Tricolor) FL3 (Tricolor) Tricky Issues: Effects of High Compensation Tip: Don’t try to detect low level staining against a high level of spillover.

  10. 4 4 10 10 3 3 10 10 2 2 PE (immunofluorescence) PE (immunofluorescence) 10 10 1 1 10 10 0 0 10 0 1 2 3 4 10 10 10 10 10 10 PI (non-viable) PI (DNA) 0 0 200 400 600 800 1k Tricky Issues: Unequal Intensities Viable cells Tip: Combine PE immunoflu. with PI for viability but not with PI for cell cycle. Alternatives: FITC for immunoflu. or 7AAD for cell cycle

  11. Cy5 Cy5 Cy5 PE PE.Cy5 Cy5 400 450 500 550 600 650 700 750 800 850 Tricky Issues: Tandem Conjugates • Problems: • Emission at the PE emission peak (570nm) • Excitation at the Cy5 excitation peak (600-650nm) Tip: Whether these spillovers are manageable depends on the cytometer and the PE.Cy5 batch. Alternatives are perCP, perCP.Cy5.5 or PE.Cy7

  12. 5 10 4 10 PE-A 3 10 650 0 -650 3 4 5 -650 0 650 10 10 10 FITC-A Tricky Issues: “Auto”fluorescence Tip: Once compensations have been set using valid control samples, don’t be tempted to “fiddle” with them to make the data look “better”.

  13. Equations: 3-colour • The SPILLOVER equations: • FL1 detector = FITC + 1% PE + 0.5% PI (1) • FL2 detector = PE + 25% FITC + 5% PI (2) • FL3 detector = PI + 34% PE + 0.5% FITC (3) • These can be solved (with greater difficulty) but bear in mind: • Remark: We have ignored the background level of electronic and optical noise in the instrument and autofluorescence on the cells. • Remark: These equations are linear and so are the detected signals. • Question: How now will the required compensations relate to the spillover percentages?

  14. Equations: Multi-Colour The SPILLOVER equations, including a“background” contribution : FL1 detector = S11 F1 + S12 F2 + S13 F3 .. + B1 (1) FL2 detector = S21 F1 + S22 F2 + S23 F3 .. + B2 (2) FL3 detector = S31 F1 + S32 F2 + S33 F3 .. + B3 (3) .. .. (..) These can be re-written in Matrix shorthand: d = Sf + b (Note: b = background for 1 cell) This cannot be solved because we now have too many unknowns! Our approximation is to use <b> = average background for the sample.

  15. Matrix Fluorescence Compensation The matrix equation d = Sf + <b> (S is the spillover matrix) Can be solved for f (the fluorochromes) if we can invert S: f = S-1(d - <b>) The inverted spillover matrix is called the compensation matrix. Thus: f = C(d - <b>) (C is the compensation matrix)

  16. f = C(d - <b>) (C is the compensation matrix; shown as %) X Multi-Colour Compensation Solution.. d = Sf + <b> (S is the spillover matrix; shown as %) X

  17. 4 4 4 10 10 10 3 3 3 10 10 10 Over-compensated FL2-H 2 FL1-H FL2-H 2 2 10 10 10 1 1 1 10 10 10 0 0 0 10 10 10 0 1 2 3 4 10 10 10 10 10 FL1-H 0 0 1 1 2 2 3 3 4 4 10 10 10 10 10 10 10 10 10 10 FL3-H FL3-H Effect of Pairwise Compensation of 3 Colours Tip: If a large overcompensation effect is seen, reduce the compensation setting causing it and correct the compensation afterwards using analysis software.

  18. Single Colour Controls: Spillover SSC-W

  19. Practical Issues: Mechanisms Used in Cytometers 'Scans, ‘Calibur, LSR, 'Star: Electronic subtraction of linear analog pulses. *Linear data for more accuracy *Susceptible to timing shifts *No inter-laser compensation MoFlo: Matrix computation from digitized electronic log amplified pulses. *Inter-laser compensation*Inaccurate log-amped data DiVa: Matrix computation from digitized linear pulses. *Inter-laser compensation *Linear data for greater accuracy *Digital calculation for greater accuracy

  20. FL1 1001011 Logamp 1-%2 ADC FL2 FACSCalibur FL3 1001011 Logamp 3-%4 ADC FL4 Compensation Mechanisms: 'Scans, ‘Calibur, LSR, 'Star

  21. logFL1 FL1 1001011 Logamp ADC *DSP (Digital Signal Processor) logFL2 FL2 Logamp ADC Compensation Mechanisms: MoFlo *Calculations: Linear FLn = 10logFLn/ChannelsPerDecade f = C.d where C is the compensation matrix, d is the detector vector {Linear FLn} and f is the fluorochrome vector {compFLn}

  22. 4 4 10 10 3 3 10 10 2 2 10 10 1 1 10 10 0 0 -10 -10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 Compensation Mechanisms: MoFlo Un-compensated Compensated FL2: PE FL2: PE FL1: FITC FL1: FITC

  23. FL1 1001011010110010 ADC *DSP (Digital Signal Processor) FL2 ADC Compensation Mechanisms: DiVa *Calculations: f = C.d where C is the compensation matrix, d is the detector vector {Linear FLn} and f is the fluorochrome vector {compFLn} Calculate Log scale values if required.

  24. 5 10 Digital System Data 4 10 4 10 PE-A 3 10 3 10 2 10 2 FL2-H 10 2 3 4 5 10 10 10 10 1 10 FITC-A 0 5 10 10 0 1 2 3 4 10 10 10 10 10 FL1-H 4 10 3 10 650 0 -650 3 4 5 -650 0 650 10 10 10 FITC-A Setting Compensation on DiVa Data Analog System Data

  25. http://www.wehi.edu.au/cytometry/presentations.html

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