Compensation The process of correcting for flourescence crosstalk In-house customer training seminar on compensation Compensation The process of correcting for flourescence crosstalk

History of compensation by traditional flow cytometry Compensating Image Stream Data Troubleshooting compensation in IDEAS AMNIS CORPORATION-Compensation

Why is compensation necessary? Broad emmision spectra Imperfections in fluorescence filtering cause leakage into other channels Tandem conjugates Imperfections in brightfield, scatter and fluorescence filtering cause leakage into other channels AMNIS CORPORATION-Compensation

The electromagnetic spectrum Flourochromes have a specific absorption and emmision sprectra and although some report the peak absorption or emmission, the actual absorptions and emmisions can be broad depending on the flourochrome. The strategic use optical filters and mirrors dissect the wavelengths of light so that we can detect specific flourochromes. AMNIS CORPORATION-Compensation

Resonance Energy Transfer Resonance energy transfer is distance dependent and occurs without radiant release from the donor molecule AMNIS CORPORATION-Compensation

Spectral overlap Here is an example of 3 flourochromes with absorption in the 488 nm range with 3 different emmision peaks. Note that the peak emmisions are broad and do “crosstalk” into adjacent channels. AMNIS CORPORATION-Compensation

Band Pass Filters A filter with a transmission that is high for a particular band of frequencies, but that falls to low values above and/or below this band Filters with transmission that is high for a particular band of frequencies, but that falls to low values above and/or below this band AMNIS CORPORATION-Compensation

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. In its most common form, a beam splitter is a cube, made from two triangular glass prisms which are glued together at their base. The thickness of the resin layer is adjusted such that (for a certain wavelength) half of the light incident through one "port" (i.e. face of the cube) is reflected and the other half is transmitted. AMNIS CORPORATION-Compensation

Traditional flow cytometer flow cell BD FACSCalibur Optics Traditionally compensation was done pre-acquisition because flow cytometry was designed to sort cells while collecting data. AMNIS CORPORATION-Compensation

The ImageStream® System Ch1 470-500nm Ch2 400-470nm Ch3 500-560nm Ch4 560-595nm Ch5 595-660nm Ch6 660-730nm AMNIS CORPORATION-Compensation

Spectral DecompositionStack 6 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 Deep Blue Corrector Spectral DecompositionStack Petzval Lens Sets Field Lens Objective & Quartz Cuvette Post Mag Lens AMNIS CORPORATION-Compensation

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. Flourescence microscopes typically do not use compensation because each color is taken with a different set of optimized filters. Get transmission data to demo. From SemRock AMNIS CORPORATION-Compensation

The crosstalk can be quantified and corrected Fitc PMT 500-560 PE PMT 545-605 Givans AMNIS CORPORATION-Compensation

Compensation of flow data during acquisition Classic flow cytometry, compensation is done pre-acquisition for the purposes of sorting cells. Easy to compensate in real time on a flow cytometer because it is computationally faster Figures from BD FACS Academy AMNIS CORPORATION-Compensation

Formula for traditional compensation 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 D1 = 1F1+ 2F1 +3F1 +…nF1 D2 = 1F2+ 2F2 +3F2 +…nF2 Dn = 1Fn+ 2Fn +3Fn +…nFn 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. AMNIS CORPORATION-Compensation

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 Biexponential plots, properly compensated data may have negative intensities and it’s impossible to plot a negative value on a log plot. The linear region of the biexponential plot allows you to do this. AMNIS CORPORATION-Compensation

Compensation of dim to bright cells AMNIS CORPORATION-Compensation

BD Fluorescence Spectrum Viewer AMNIS CORPORATION-Compensation

BD Fluorescence Spectrum Viewer AMNIS CORPORATION-Compensation

BD Fluorescence Spectrum Viewer AMNIS CORPORATION-Compensation

Spectral Compensation Post-acquisition compensation is applied to images on a pixel by pixel basis in IDEAS. Single color control samples used to calculate a 6x6 matrix. SSC Brightfield FITC PE PE-Alexa610 Draq-5 Channel imagery consists of signal from the desired channel as well as leakage from other channels due to: Broad emission characteristics of probes Limitations in optical filtration Compensation removes the overlap such that channel imagery reflects only the appropriate probe AMNIS CORPORATION-Compensation

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. Each pixel on a CCD detector has a characteristic baseline output known as dark current offset and a responsivity to light exposure, known as the pixel gain. Correcting for these offsets although low allow for increased sensitivity of dim flourescence. These values are collected and saved when running ASSIST and automatically applied to the data in IDEAS. 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. AMNIS CORPORATION-Compensation

Spectral Compensation Dark Current Correction Pixelated Imagery Read Out 1023 500 800 700 1000 475 775 675 950 425 725 625 1023 Spectral Overlap Signal Signal Signal 40 Spectral Overlap 35 25 Dark Current Green Channel Orange Channel Red Channel AMNIS CORPORATION-Compensation

Darkcurrent correction Each pixel on a CCD detector has a characteristic baseline output known as dark current offset. AMNIS CORPORATION-Compensation

Brightfield gain correction Each pixel on a CCD detector has a characteristic responsivity to light exposure, known as the pixel gain. AMNIS CORPORATION-Compensation

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. AMNIS CORPORATION-Compensation

IDEAS Tools The corrections are available to the user in IDEAS during data analysis. AMNIS CORPORATION-Compensation

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. AMNIS CORPORATION-Compensation

Potential pitfalls to watch for Saturation Mis-alignment Coefficients calculated on poorly fit lines Autoflourescence Camera staging AMNIS CORPORATION-Compensation

Misalignment AMNIS CORPORATION-Compensation

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 When a pixel can no longer quantify the available light. We have a 10 bit detector that gives us 1024 bins and once you reach 1024 the pixel is saturated. To apply compensation to an unknown quantity of light is impossible. Imagery with saturated pixels cannot be used to create a matrix nor be compensated. Figure with saturated image and a plot with saturation. Images with saturated pixels and point to crosstalk showing that the numbers do not change. AMNIS CORPORATION-Compensation

Saturation AMNIS CORPORATION-Compensation

Undercompensation due to saturated pixels… AMNIS CORPORATION-Compensation

Staging the camera Charged Coupled Device Active Channel 96 pixels wide by 512 pixels tall X6 Inactive area 12 pixels wide by 256 FWD Stage Selection Charged Coupled Device AMNIS CORPORATION-Compensation

Properly compensated single color controls AMNIS CORPORATION-Compensation

Mis-compensated single color controls AMNIS CORPORATION-Compensation

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. AMNIS CORPORATION-Compensation

Compensation variation with laser power 20 mw positive 20 mw Blank 100 mw positive 100 mw Blank 200 mw positive 200 mw Blank AMNIS CORPORATION-Compensation

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. AMNIS CORPORATION-Compensation

Coefficient error Check the matrix AMNIS CORPORATION-Compensation

IDEAS Compensation Open IDEAS for live demo of compensation from this slide. Return for discussion about saturation, problems AMNIS CORPORATION-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 A compensation matrix is calculated using single color fluorescent control files that are collected on the ImageStream in the absence of brightfield illumination. One file of between 500 and 1000 events should be collected for each fluorochrome in the experiment. Once the matrix is created it can then be applied to the experiment data when batch processing or opening a raw image file. 1. In the compensation drop down menu select New Matrix. 2. Click the Add button in the lower left portion of the window, and navigate to the single color no brightfield compensation control files. 3. Highlight each fluorescent control file using Ctrl-select and press open. A list of each compensation control file to be loaded appears in the directory. 4. When all control files have been added to the directory, click Load Files. 5. A prompt to combine the control files comes up. Select no to save on disk space or yes to combine each control sample into a single duplicate data file. 6. After the files open, a dot plot of scatter area vs. aspect ratio comes up. Using a region tool identify the single cells to use as your compensation population. 7. Select the newly created population from the compensation population drop down list, or use the “All” population if few outliers are present. 8. IDEAS will automatically identify the single color positive cells for each channel and present adjacent channel scatter plots with these events displayed in their respective colors. 9. Under “positive populations” is a list of each color channel. From the drop down list next to the channel names, select the appropriate single color positive population and do this for each fluorochrome used in the experiment. For example, if FITC was used, in channel 3 (green) select the “3_Positive” population. 10. Once the positive control populations are assigned to their corresponding channels, click the Create Compensation Matrix button. 11. To view the matrix, click on the Compensation Matrix tab in the upper right hand corner. 12. Validate the matrix by right-clicking each cell of the matrix. 13. The Matrix Coefficient Intensity Plot is displayed. If the green line of best fit does not exactly overlay the data points on the plot, click the Add Graph to Analysis Area button. 14. Using a region tool select a new positive population that excludes the outliers. 15. Assign the new population to the appropriate channel using its drop down list. 16. Click Create Compensation Matrix again to re-calculate the matrix using the new populations. 17. Select Save Compensation Matrix. AMNIS CORPORATION-Compensation

Go forth and compensate! AMNIS CORPORATION-Compensation

AMNIS CORPORATION-Compensation

AMNIS CORPORATION-Compensation

Calculating the compensation matrix 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 = 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. AMNIS CORPORATION-Compensation

Pixelated Imagery Green Channel Orange Channel Red Channel Read Out 1023 500 800 700 1023 Signal 40 Spectral Overlap 35 25 Dark Current Green Channel Orange Channel Red Channel AMNIS CORPORATION-Compensation