compensation the process of correcting for flourescence crosstalk

CompensationThe process of correcting for flourescence crosstalk

AMNIS CORPORATION-Compensation

• History of compensation by traditional flow cytometry

• Compensating Image Stream Data

• Troubleshooting compensation in IDEAS


Why is compensation necessary?

• Broad emmision spectra

• Imperfections in fluorescence filtering cause leakage into other channels

• Tandem conjugates


The electromagnetic spectrum


Resonance Energy Transfer

Resonance energy transfer is distance dependent and occurs without radiant release from the donor molecule


Spectral overlap


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


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.


Traditional flow cytometer flow cell

BD FACSCalibur Optics


The ImageStream® SystemCh1470-

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730nmtdiSynchCW2.mov Frame.mov


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


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


The crosstalk can be quantified and corrected

Givans


Compensation of flow data during acquisition

Figures from BD FACS Academy


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.


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


Compensation of dim to bright cells


BD Fluorescence Spectrum Viewer


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

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SSC Brightfield FITC PE PE-Alexa610 Draq-5


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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Dark Current Correction Spectral Compensation


Darkcurrent correction

• Each pixel on a CCD detector has a characteristic baseline output known as dark current offset.


Brightfield gain correction

• Each pixel on a CCD detector has a characteristic responsivity to light exposure, known as the pixel gain.


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.


IDEAS Tools

• The corrections are available to the user in IDEAS during data analysis.


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.

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Potential pitfalls to watch for

• Saturation• Mis-alignment• Coefficients calculated on poorly fit lines• Autoflourescence• Camera staging


Misalignment


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


Saturation


Undercompensation due to saturated pixels…


Staging the camera

z

128 FWD Stage Selection

32 FWD Stage Selection8 Stage Selection

Active Channel96 pixels wide by

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Single or multitap readout register suitable for 50KHz line rate18 tap register suitable for 400KHz line rate

Active Channel96 pixels wide

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256 FWD Stage Selection

• Charged Coupled Device


Properly compensated single color controls


Mis-compensated single color controls


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

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20 mw positive 20 mw Blank



Compensation variation with laser power


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.


Coefficient error

• Check the matrix


IDEAS Compensation


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


Go forth and compensate!


• 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 )(


Pixelated Imagery

1023

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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 500700

compensation the process of correcting for flourescence crosstalk

Documents

compensation of dim

history of compensation

compensation necessary

compensation of flow

postacquisition compensation

peak fluorescence

sum of fluorescence

fluorescence microscopes