Summarizes and visualizes the pixel intensities per spot and channel

Helper function for estimating the spillover matrix. This function visualizes the median pixel intensities per spot (rows) and per channel (columns) in form of a heatmap.

plotSpotHeatmap(
  object,
  spot_id = "sample_id",
  channel_id = "channel_name",
  assay_type = "counts",
  statistic = "median",
  log = TRUE,
  threshold = NULL,
  order_metals = TRUE,
  color = viridis(100),
  breaks = NA,
  legend_breaks = NA,
  cluster_cols = FALSE,
  cluster_rows = FALSE,
  ...
)

Arguments

object

a SingleCellExperiment object containing pixel intensities per channel. Individual pixels are stored as columns and channels are stored as rows.

spot_id

character string indicating which colData(object) entry stores the isotope names of the spotted metal. Entries should be of the form (mt)(mass) (e.g. Sm152 for Samarium isotope with the atomic mass 152).

channel_id

character string indicating which rowData(object) entry contains the isotope names of the acquired channels.

assay_type

character string indicating which assay to use (default counts).

statistic

the statistic to use when aggregating channels per spot (default median)

log

should the aggregated pixel intensities be log10(x + 1) transformed?

threshold

single numeric indicating a threshold after pixel aggregation. All aggregated values larger than threshold will be labeled as 1.

order_metals

should the metals be ordered based on spotted mass?

color

see parameter in pheatmap

breaks

see parameter in pheatmap

legend_breaks

see parameter in pheatmap

cluster_cols

see parameter in pheatmap

cluster_rows

see parameter in pheatmap

...

other arguments passed to pheatmap.

Value

a pheatmap object

Quality control for spillover estimation

Visualizing the aggregated pixel intensities serves two purposes:

1. Small median pixel intensities (< 200 counts) might hinder the robust estimation of the channel spillover. In that case, consecutive pixels can be summed (see binAcrossPixels).

2. Each spotted metal (row) should show the highest median pixel intensity in its corresponding channel (column). If this is not the case, either the naming of the .txt files was incorrect or the incorrect metal was spotted.

By setting the threshold parameter, the user can easily identify spots where pixel intensities are too low for robust spillover estimation.

See also

pheatmap for visual modifications

aggregateAcrossCells for the aggregation function

Author

Nils Eling (nils.eling@dqbm.uzh.ch)

Examples

path <- system.file("extdata/spillover", package = "imcRtools")
# Read in .txt files
sce <- readSCEfromTXT(path)
#> Spotted channels:  Dy161, Dy162, Dy163, Dy164
#> Acquired channels:  Dy161, Dy162, Dy163, Dy164
#> Channels spotted but not acquired:  
#> Channels acquired but not spotted:  

# Visualizes heatmap
plotSpotHeatmap(sce)


# Visualizes thresholding results
plotSpotHeatmap(sce, log = FALSE, threshold = 200)