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  1. CODEX® Processor
  2. Technical Notes

Background Subtraction

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Last updated 5 years ago

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Formalin-fixed paraffin-embedded (FFPE) samples and certain tissue types such as liver or brain may have high auto-fluorescence that significantly impact signal-to-noise ratio (SNR). In turn, low SNR may reduce the accuracy of gating, clustering and downstream spatial analysis. To improve SNR, blank cycles corresponding to the estimated auto-fluorescence can be acquired and subtracted from raw marker intensities.

Successful background subtraction requires 1) a start blank cycle, 2) an end blank cycle and 3) channel exposure times.

Cycle

Channel 1

Channel 2

Channel 3

Channel 4

1

DAPI-1

Blank

Blank

Blank

2

DAPI-2

Pan-cytokeratin

Ki67

CD68

3

DAPI-3

CD4

CD21

SMA

4

DAPI-4

Blank

Blank

Blank

Biomarkers must not contain 'Blank' in its name. 'Empty' channels will not be used for background subtraction. If variable exposure times are used, use the shortest exposure time of the corresponding channel to reduce over-subtraction.

Cycle

Channel 1

Channel 2

Channel 3

Channel 4

1

10 ms

500 ms

350 ms

500 ms

2

10 ms

500 ms

350 ms

600 ms

3

10 ms

500 ms

500 ms

500 ms

4

10 ms

500 ms

350 ms

500 ms

As a reference, default exposure times for Akoya-validated CODEX® conjugates are listed .

Background signal is determined from the blank cycle. A multitude of CODEX experiments have demonstrated that over time (i.e., as cycle number increases) background can either increase or decrease, depending on tissue type and tissue preparation conditions. Thus, we subtract background from signal channels using both start and end blank channels to account for changes in autofluorescence during a CODEX experiment (Figure 1).

Additionally, we recognize that differences in exposure times between start blank, end blank, and signal channel may affect background subtraction. To account for differences in exposure time we use a ratio of signal exposure time to blank exposure time (Figure 2).

here
Figure 1. Background subtraction as a function of cycle number. Prior to subtraction, the intensity of start and end blank channels (i.e., autofluorescence at the beginning and end of the experiment) is scaled linearly by cycle number to account for changes in background intensity during the CODEX experiment.
Figure 2. Background subtraction as a function of cycle number and exposure time. To account for differences in exposure time between blank channels and signal channels background subtraction is applied using a ratio of signal exposure time to blank exposure time. We recommend setting blank exposure to the minimum signal channel exposure time.