Background Subtraction
Last updated
Last updated
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.
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.
As a reference, default exposure times for Akoya-validated CODEX® conjugates are listed here.
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).
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
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