compensation the process of correcting for flourescence crosstalk
DESCRIPTION
Compensation The process of correcting for flourescence crosstalk. History of compensation by traditional flow cytometry Compensating Image Stream Data Troubleshooting compensation in IDEAS. Why is compensation necessary?. Broad emmision spectra - PowerPoint PPT PresentationTRANSCRIPT
CompensationThe process of correcting for flourescence crosstalk
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• History of compensation by traditional flow cytometry
• Compensating Image Stream Data
• Troubleshooting compensation in IDEAS
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Why is compensation necessary?
• Broad emmision spectra
• Imperfections in fluorescence filtering cause leakage into other channels
• Tandem conjugates
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The electromagnetic spectrum
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Resonance Energy Transfer
Resonance energy transfer is distance dependent and occurs without radiant release from the donor molecule
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Spectral overlap
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Band Pass Filters
Filters with transmission that is high for a particular band of frequencies, but that falls to low values above and/or below this band
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Beam splitter• A beam splitter is an optical device that splits a beam of
light in two.
• A dichroic mirror is a type of beam splitter that is able to split light of different wavelengths.
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Traditional flow cytometer flow cell
BD FACSCalibur Optics
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The ImageStream® SystemCh1470-
500nm
Ch2400-
470nm
Ch3500-
560nm
Ch4560-
595nm
Ch5595-
660nm
Ch6660-
730nmtdiSynchCW2.mov Frame.mov
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Post Mag Lens
Deep Blue Corrector
Spectral Decomposition
Stack
Petzval Lens Sets
Field Lens
Objective &
Quartz Cuvette
Spectral Decomposition6 Spectral Channels 488-505nm Scatter 560-595nm PE 400-470nm DAPI 595-660nm PI, 7-AAD 505-560nm FITC 660-730nm Cy5, DRAQ5
0.3 degree separation per channel
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Flourescence Microscopy•The fluorescent microscope utilizes filter cubes that narrow the wavelenth of excitation and emission designed specifically for each fluorochrome.
•Fluorescence microscopes typically do not apply compensation because each color is taken with a different set of optimized filters.
From SemRock
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The crosstalk can be quantified and corrected
Givans
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Compensation of flow data during acquisition
Figures from BD FACS Academy
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Formula for traditional compensation
D1 = 1F1+ 2F1 +3F1 +…nF1
D2 = 1F2+ 2F2 +3F2 +…nF2
Dn = 1Fn+ 2Fn +3Fn +…nFn
xFn = the amount of signal in detector n that originates from fluorophore x
Dn = the measured signal in detector n
For each detector D=the sum of fluorescence from each fluorophore
A matrix is defined such that each detector measured value is the sum of the peak fluorescence plus the spillover amount from every other fluorochrome.The matrix is used to remove the contribution of the non-peak fluorochrome. This gives you the ‘true’ value for the peak fluorochrome.
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Factors affecting matrixes
• Crosstalk is fluorochrome specific
• Tandem conjugates can have dual band fluorescence due to the inefficiency of RET to the acceptor or degradation of the conjugate
• Bright vs Dim fluorescence
• Autofluorescence is cell specific
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Compensation of dim to bright cells
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BD Fluorescence Spectrum Viewer
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BD Fluorescence Spectrum Viewer
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BD Fluorescence Spectrum Viewer
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Single color control samples used to calculate a 6x6 matrix.Post-acquisition compensation is applied to images on a pixel by pixel basis in IDEAS.
Spectral Compensation
3_Intensity1000 1e4 1e5
1000
1e4
1e5
1e6
3_Intensity
4_In
tens
ity
Un-compensated
5_Intensity1000 1e4 1e5 1e6
01000
1e4
1e5
1e6
5_Intensity
6_In
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Un-compensated
3_Intensity-1000 0 1000 1e4 1e5
-10000
1000
1e4
1e5
1e6
3_Intensity
4_In
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Compensated
5_Intensity-1000 0 1000 1e4 1e5
-10000
1000
1e4
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1e6
5_Intensity
6_In
tens
ity
Compensated
SSC Brightfield FITC PE PE-Alexa610 Draq-5
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Compensating ImageStream data pixel by pixel• Corrections are applied before spectral compensation.
• ASSIST values are used for:• 1.Darkcurrent correction• 2.Brightfield gain correction• 3.Spatial registration
Brightfield compensation is done using the background around the objects and is automatically computed and applied when the data file is loaded in IDEAS.
Single fluorochrome compensation control files are used for fluorescence crosstalk compensation.
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Pixelated Imagery
0
25
35
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Dark Current
SpectralOverlap
Signal
Green Channel Orange Channel Red Channel
Read Out 1023 1023 1023500 500 800 700 800 500 800 5007001023
1000 1000 1000475 475 775 675 775 475 775 475675
SpectralOverlap
Signal
950 950 950425 425 725 625 725 425 725 425625
Signal
Dark Current Correction Spectral Compensation
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Darkcurrent correction
• Each pixel on a CCD detector has a characteristic baseline output known as dark current offset.
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Brightfield gain correction
• Each pixel on a CCD detector has a characteristic responsivity to light exposure, known as the pixel gain.
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Spatial registration
• Spatial registration errors between image channels is measured by imaging the same object (SpeedBead) in all 6 channels simultaneously and comparing the location of the images.
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IDEAS Tools
• The corrections are available to the user in IDEAS during data analysis.
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Spectral Compensation: Matrix Development Cross talk matrix is determined by calculating best fit linear regression for each dye into
each channel. Slope of linear regression is matrix coefficient. A 6x6 martix of linear equations is solved for each pixel in every image to remove cross talked light.
5_Intensity1000 1e4 1e5 1e6
01000
1e4
1e5
1e6
5_Intensity
6_In
tens
ity
Un-compensated
3_Intensity1000 1e4 1e5
1000
1e4
1e5
1e6
3_Intensity
4_In
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Un-compensated
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Potential pitfalls to watch for
• Saturation• Mis-alignment• Coefficients calculated on poorly fit lines• Autoflourescence• Camera staging
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Misalignment
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Saturation
• Saturation occurs when the pixel can no longer quantify the available light.
• The 10 bit detector provides 1024 bins and once 1023 is reached the pixel can no longer quantify the signal and compensation becomes impossible.
• • It is therefore critical that events with saturated pixels be eliminated
• During data acquisition the laser power, camera sensitivity and cell classifiers are used to reduce saturation
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Saturation
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Undercompensation due to saturated pixels…
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Staging the camera
z
128 FWD Stage Selection
32 FWD Stage Selection8 Stage Selection
Active Channel96 pixels wide by
512 tallX6
Inactive Region12 pixels wide by
512 tallX6
Single or multitap readout register suitable for 50KHz line rate18 tap register suitable for 400KHz line rate
Active Channel96 pixels wide
by512 pixels tall
X6
Inactive area12 pixels wide
byX6
256 FWD Stage Selection
• Charged Coupled Device
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Properly compensated single color controls
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Mis-compensated single color controls
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Compensation variation with laser power
• Jurkat cells stained with NFkB FITC were run in the absence of brightfield and a 1000 images were collected with 488 laser excitation ranging from 20mw to 200mw at 20mw increments. Compensation values were calculated in IDEAS graphed over changing laser power. Compensation variation with increasing laser power shows little to no correlation to changing laser power.
Compensation Variation with Laser power
-0.05
0.25
0.55
0.85
20 40 60 80 100 120 140 160 180 200
488 excitation in mw
% C
ompe
nsat
ion FITC into Channel 1
FITC into Channel 2
FITC into Channel 3
FITC into Channel 4
FITC into Channel 5
FITC into Channel 6
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20 mw positive 20 mw Blank
100 mw positive 100 mw Blank
200 mw positive 200 mw Blank
Compensation variation with laser power
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Two methods for calculating the matrix
• The Means method is used for uniform objects like beads.
• The Best Fit is used for objects that have a varied level of fluorescence.
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Coefficient error
• Check the matrix
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IDEAS Compensation
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Workflow• Collect files of single color fluorescent controls with brightfield turned off
• Open IDEAS and start a New Matrix under compensation
• Add the single color control files to the analysis (open and load files)
• Select the single cells using the scatter area vs. aspect ratio dot plot to use as the compensation population
• Assign the positive populations to the appropriate channels
• Create the compensation matrix
• Validate the matrix
• Save the matrix
• Use the matrix to open data files
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Go forth and compensate!
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• Two channels of interest X and Y• Compute the variance of X (VarX), variance of Y (VarY) and the
covariance of X and Y (CovXY). CovXY measures the degree to which 2 values vary together
• Define a 2 x 2 matrix:
• Eigen Value =
• The major Eigen value is the positive value• Slope =
Calculating the compensation matrix
VarYCovXYCovXYVarX
Eigen values can be found for square symmetric matrices. There are as many eigen values as there rows (or columns) in the matrix. Conceptually they can be considered to measure the strength (relative length) of an axis (dervied from the square symmetric matrix). Each eigen value has an associated eigen vector. An eigen value is the length of an axis, the eigen vector determines its orientation in space.
4
)(*2
22 VarYVarXCovXYVarYVarXVarYVarX
CovXYVarXvalueeigenmajor )(
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Pixelated Imagery
1023
0
25
35
40
Dark Current
SpectralOverlap
Signal
Green Channel Orange Channel Red Channel
Read Out 1023 1023 1023500 500 800 700 800 500 800 500700