Flow Cytometry Seminar Series 2017 : Fundamental Principles Spillover correction in multicolor flow cytometry 28.02.2017 http://www.cytometry.uzh.ch
Contents Fluorescence and its detection Absorption and emission Detection and spillover of spillover Theory and calculation Practical example Limitations of compensation
Fluorophores Synthetic dyes and fluorescent proteins pdb.org Usually have conjugated systems of π electron that can absorb and release energy in the form of photons.
Fluorescence Absorption Absorption: a molecule absorbs a photon and enters a higher energy, excited state. Excited state Transition probability E = h λ Transition energy Ground state 1) Transitions to different vibrational levels possible, but only in defined energies. 2) Some transitions are more likely than others. -> Absorption spectrum
Fluorescence Stokes Shift 1) Fast relaxation to lowest energy excited state level -> Energy released as heat. Excited state Heat E = h λ Photons Ground state 2) Relaxation to an energy level of the ground state -> Energy released as a photon.
Fluorescence Summary Fluorophores absorb and emit light over a range of wavelengths. An emitted photon is of lower energy (higher wavelength) than the absorbed one.
Fluorescence Detection Normal detectors are blind to the wavelength -> optical filters AHF.de
Fluorescence Detection Light filtering: 1) Long pass (LP) filter Reflects everything below its designated wavelength.
Fluorescence Detection Light filtering: 1) Long pass (LP) filter Reflects everything below its designated wavelength. 2) Band pass (BP) Lets only a narrow band of wavelengths through.
Fluorescence Detection Detection window (DW): Combined effect of LP and BP filter. Detected are all photons passing both filters. -> Only a part of the total dye emission is detected in the channel.
Fluorescence Detection Multiple dyes need multiple detection windows (and detectors).
Fluorescence Detection DW2 Multiple dyes need multiple detection windows. Problem: Emission of one dye is detected in multiple DWs.
Signal Flow Cytometry Facility Fluorescence Detection DW2 DW2
Signal Flow Cytometry Facility Fluorescence Detection DW2 Spillover DW2
Detection Summary DW2 We have to use optical filters because our detectors are colorblind. Each filter window only detects a part of the total fluorophore emission. Because emission spectra are broad, a single detection window can receive light from multiple fluorophores. -> Spillover
Signal Flow Cytometry Facility Spillover 1 Signal arriving on the detector is the correct signal + spillover: 1 S = + 2 S DW2 = + 2 DW2
Signal Signal Signal Flow Cytometry Facility Spillover 1 Spillover is a constant quantity: Can be determined from single stain controls. 2 2 1 DW2 DW2 DW2 s FITC = S 2 FITC s PE = S 1 PE
Signal Flow Cytometry Facility Spillover 1 With spillover constants s: S 1 PE = s PE S = + S 1 PE = + s PE 2 = s FITC 2 DW2 2 S DW2 = + = s FITC + 2 equations for 2 unknowns.
Signal Flow Cytometry Facility Spillover 1 Using linear algebra to solve the system of equations (much more convenient for computers): S = + s PE S DW2 = s FITC + S S DW2 = 1 s PE s FITC 1 s = 1 s PE s FITC 1 2 s 1 S S DW2 = s 1 s DW2 s 1 S S DW2 =
Signal Signal Flow Cytometry Facility Spillover One simple multiplication yields the corrected signals: 1 s 1 S S DW2 = 2 DW2 For s, we only need the single stain controls. Extension to more channels/colors is straightforward. DW2 s 1 = 1/(1 s PE s FITC ) (s PE /(1 s PE s FITC )) (s FITC /(1 s PE s FITC )) 1/(1 s PE s FITC )
Signal Flow Cytometry Facility Spillover Background bg 1 bg 2 a 1 a 2 1 What if we also have other contributions to the signal? Auto-fluorescence (a) Other background (bg) S = + s PE + a 1 + bg 1 S DW2 = s FITC + + a 2 + bg 2 2 s 1 S a1 + bg 1 S DW2 a 2 + bg 2 = DW2 Background can be subtracted from signal before compensation to obtain the correct signal.
Signal Signal Flow Cytometry Facility Spillover Background 1 bg 2 a 1 subtracted before bg 1 a 2 What if background is not compensation, like its usually the case with autofluorescence? The displayed absolute values will be wrong. 2 DW2 bg 1 bg 2 a 1 a 2 DW2 However, the difference between the compensated stained and compensated unstained population still yields the correct absolute signal.
Signal Flow Cytometry Facility Spillover Summary 1 Spillover can be completely compensated for. The only thing we need for compensation are single stain controls for each fluorophore used. Autofluorescence and background can be corrected for after compensation. 2 DW2 s 1 S S DW2 =
Practical Example (FlowJo) The difference between the positive and negative population medians is our fluorescent signal. Gates are only defined in main channel.
Practical Example (FlowJo) The ratios of the fluorescent signals in all channels for each fluorophore are then used to calculate the spillover matrix (s): Dyes Channels The compensation matrix (S 1 ) is the inverse of the spillover matrix (not shown by FlowJo).
A700 CD3 A488 CD32 A700 CD3 Flow Cytometry Facility Practical Example (FlowJo) - comp. + comp. - comp. + comp. PE CD86 APC-H7 CD19 01.03. 2017 - comp. + comp. - comp. + comp. PE-Cy5.5 CD209 A647 2H2
Spillover Limitations 1) can only be as good as the single stain measurements. -> any error in the single stain measurements will propagate to your compensated data. 2) introduces artifacts in populations that would not need full compensation (e.g. unstained and single stained populations). 3) Mathematically, any amount of spillover can be compensated. In practice, the total signal level and the relative signal contributions in one channel become limiting: 1) Linear range of the detector 2) Signal broadening (spillover spreading)
1) Single Stain Measurements The ideal single stain control is: As bright as possible -> High signal to noise ratio reduces measurement error. The same thing you measure in your sample (minus all the other dyes) -> Dyes can change their emission properties depending on their environment. Potential problems with single stain cell controls: Sample is precious and you cannot afford to use any for single stain controls. Low antigen expression -> low signal Cells with your marker are rare -> low signal
1) Single Stain Measurements Beads as substitute for cells: Bright and low background. Does not consume actual sample. Different environment than cell. Other substitutions: Less expensive antibody with same fluorophore Test first! Less expensive antibody with different fluorophore for same channel Don t do that.
2) Artifacts of Without compensation With compensation Green: 50% -> Red Red: 10% -> Green Extreme example By applying compensation to signals that result not from the fluorophores (background, instrument noise, autofluorescence), some amount of distortion is introduced.
Signal Measured signal Flow Cytometry Facility 3) Linear Range of the Detectors Oversaturation bg 1 a 1 1 and background correction are linear operations. -> only works within the linear range. Dark counts Linear Range Actual signal -> Adjust gain voltages to have brightest signal within the linear range. -> For correct compensation, gain voltages must not be changed during the experiment.
3) Signal Broadening Error in the measurement Photon emission (our signal) is a stochastic process that is described by a Poisson distribution. P k, λ = λk e λ k! Mean: λ Standard deviation: λ Even under perfect conditions, that is the variation in the measurement. The relative variation ( λ/λ) decreases with higher signals, but the absolute variation ( λ) increases.
3) Signal Broadening Spillover Spreading Mean: 10 3 SD: 32 Mean: 10 5 SD: 315 Mean: 10 3 SD: 315 Populations shifted (compensated) from high to low intensities take their variation with them. -> Compensated populations have larger variations than an original population at that signal level would have.
Limitations Summary Single stain measurements are a compromise between best signal and a control as similar to your sample as possible. Non-fluorophore signals (e.g. autofluorescence) are distorted by compensation. Keep signal within linear range of detectors or compensation cannot be applied. Spillover leads to signal spreading that cannot be corrected for.
Summary The main points: Fluorophores have broad emission spectra and are detected in multiple channels (spillover). Spillover can be compensated for with single stain controls. quality depends directly on the quality and suitability of the controls. Spillover leads to signal spreading that cannot be corrected for.
Some Reading Material An Introduction to for Multicolor Assays on Digital Flow Cytometers. BD Technical Bulletin Bagwell CB, Adams EG. Fluorescence spectral overlap compensation for any number of flow cytometer parameters. Ann NY Acad Sci. 1993 in Flow Cytometry. Roederer M. Current Protocols in Cytometry. 2002