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change some names in the resulting data frame and add cytosol image
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@buddekai
buddekai committed Jan 26, 2023
1 parent 0e043c9 commit 37bc12e
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Showing 2 changed files with 95 additions and 50 deletions.
118 changes: 76 additions & 42 deletions R/cellPixels.R
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Original file line number Diff line number Diff line change
Expand Up @@ -133,6 +133,7 @@ cellPixels <- function(input_dir = NULL,
dir.create(output_dir, showWarnings = FALSE)

# Create empty data fram -------------------------------------------------

df_results <- data.frame(
"fileName" = file_names,
"manual_quality_check" = rep(NA, number_of_images),
Expand All @@ -143,24 +144,27 @@ cellPixels <- function(input_dir = NULL,
"number_of_nuclei_with_second_protein" = rep(NA, number_of_images),
"color_of_third_protein_in_cytosol" = rep(NA, number_of_images),
"number_of_cells_with_third_protein" = rep(NA, number_of_images),
"intensity_sum_red_full" = rep(NA, number_of_images),
"intensity_sum_green_full" = rep(NA, number_of_images),
"intensity_sum_blue_full" = rep(NA, number_of_images),
"intensity_sum_red_nucleus_region" = rep(NA, number_of_images),
"intensity_sum_green_nucleus_region" = rep(NA, number_of_images),
"intensity_sum_blue_nucleus_region" = rep(NA, number_of_images),
"intensity_sum_red_without_nucleus_region" = rep(NA, number_of_images),
"intensity_sum_green_without_nucleus_region" = rep(NA, number_of_images),
"intensity_sum_blue_without_nucleus_region" = rep(NA, number_of_images),
"intensity_sum_red_foreground" = rep(NA, number_of_images),
"intensity_sum_green_foreground" = rep(NA, number_of_images),
"intensity_sum_blue_foreground" = rep(NA, number_of_images),
"intensity_sum_red_foreground_without_nucleus_region" = rep(NA, number_of_images),
"intensity_sum_green_foreground_without_nucleus_region" = rep(NA, number_of_images),
"intensity_sum_blue_foreground_without_nucleus_region" = rep(NA, number_of_images),
"intensity_mean_red_background" = rep(NA, number_of_images),
"intensity_mean_green_background" = rep(NA, number_of_images),
"intensity_mean_blue_background" = rep(NA, number_of_images),
"intensity_sum_full_red" = rep(NA, number_of_images),
"intensity_sum_full_green" = rep(NA, number_of_images),
"intensity_sum_full_blue" = rep(NA, number_of_images),
"intensity_sum_nucleus_region_red" = rep(NA, number_of_images),
"intensity_sum_nucleus_region_green" = rep(NA, number_of_images),
"intensity_sum_nucleus_region_blue" = rep(NA, number_of_images),
"intensity_sum_full_without_nucleus_region_red" = rep(NA, number_of_images),
"intensity_sum_full_without_nucleus_region_green" = rep(NA, number_of_images),
"intensity_sum_full_without_nucleus_region_blue" = rep(NA, number_of_images),
"intensity_sum_cytosol_region_red" = rep(NA, number_of_images),
"intensity_sum_cytosol_region_green" = rep(NA, number_of_images),
"intensity_sum_cytosol_region_blue" = rep(NA, number_of_images),
"intensity_sum_foreground_red" = rep(NA, number_of_images),
"intensity_sum_foreground_green" = rep(NA, number_of_images),
"intensity_sum_foreground_blue" = rep(NA, number_of_images),
"intensity_sum_foreground_without_nucleus_region_red" = rep(NA, number_of_images),
"intensity_sum_foreground_without_nucleus_region_green" = rep(NA, number_of_images),
"intensity_sum_foreground_without_nucleus_region_blue" = rep(NA, number_of_images),
"intensity_mean_background_red" = rep(NA, number_of_images),
"intensity_mean_background_green" = rep(NA, number_of_images),
"intensity_mean_background_blue" = rep(NA, number_of_images),
"number_of_total_pixels" = rep(NA, number_of_images),
"number_of_pixels_nucleus_region" = rep(NA, number_of_images),
"number_of_pixels_foreground" = rep(NA, number_of_images),
Expand Down Expand Up @@ -683,14 +687,15 @@ cellPixels <- function(input_dir = NULL,
# Fill holes
nmask <- EBImage::fillHull(nmask)

# Save black-and-white-image as nucleus mask
nucleus_mask <- nmask
# # Save black-and-white-image as nucleus mask -> don't use this and
# # use the resultnig mask instead
# nucleus_mask <- nmask

# Number of pixels of the nucleus mask
number_of_pixels_nucleus_region <- sum(nucleus_mask)
number_of_pixels_nucleus_region <- sum(nmask)

# Number of pixels of the foreground without the nucleus part
mask_foreground_wo_nucleus <- mask_foreground - nucleus_mask
mask_foreground_wo_nucleus <- mask_foreground - nmask
mask_foreground_wo_nucleus[mask_foreground_wo_nucleus < 0] <- 0

number_of_pixels_foreground_without_nucleus_region <- sum(mask_foreground_wo_nucleus)
Expand Down Expand Up @@ -937,12 +942,16 @@ cellPixels <- function(input_dir = NULL,
print(paste("Number of nuclei: ", nucNo, sep=""))


# Save a (0/1 = black and white) mask of nuclei
nmask_bw <- nmask
nmask_bw[nmask_bw != 0] <- 1

# Use the nucleus mask to leave only the nuclei part of the images
Image_loaded <- EBImage::Image(data = image_loaded)
Image_nucleus_part <- Image_loaded * EBImage::toRGB(nucleus_mask)
Image_nucleus_part <- Image_loaded * EBImage::toRGB(nmask_bw)

# Use the nucleus mask to cut out nuclei of the images
non_nucleus_mask <- 1-nucleus_mask
non_nucleus_mask <- 1-nmask_bw
Image_non_nucleus_part <- Image_loaded * EBImage::toRGB(non_nucleus_mask)


Expand Down Expand Up @@ -1171,6 +1180,13 @@ cellPixels <- function(input_dir = NULL,
}
cytosolmask[cytosolmask %in% to_be_removed] <- 0

# Save a (0/1 = black and white) mask of cytosol
cytosolmask_bw <- cytosolmask
cytosolmask_bw[cytosolmask_bw != 0] <- 1

# Use the cytosolmask to leave only the cytosol part of the images
Image_loaded <- EBImage::Image(data = image_loaded)
Image_cytosol_part <- Image_loaded * EBImage::toRGB(cytosolmask_bw)

# Go through every stained cytosol and keep those that contain a nucleus
n_c_mask <- cytosolmask * nmask_watershed
Expand Down Expand Up @@ -1409,29 +1425,33 @@ cellPixels <- function(input_dir = NULL,
df_results[i,"number_of_cells_with_third_protein"] <- cell_with_proteins_No
}

df_results[i,"intensity_sum_red_full"] <- sum(image_loaded[,,1])
df_results[i,"intensity_sum_green_full"] <- sum(image_loaded[,,2])
df_results[i,"intensity_sum_blue_full"] <- sum(image_loaded[,,3])
df_results[i,"intensity_sum_full_red"] <- sum(image_loaded[,,1])
df_results[i,"intensity_sum_full_green"] <- sum(image_loaded[,,2])
df_results[i,"intensity_sum_full_blue"] <- sum(image_loaded[,,3])

df_results[i,"intensity_sum_nucleus_region_red"] <- sum(Image_nucleus_part[,,1])
df_results[i,"intensity_sum_nucleus_region_green"] <- sum(Image_nucleus_part[,,2])
df_results[i,"intensity_sum_nucleus_region_blue"] <- sum(Image_nucleus_part[,,3])

df_results[i,"intensity_sum_red_nucleus_region"] <- sum(Image_nucleus_part[,,1])
df_results[i,"intensity_sum_green_nucleus_region"] <- sum(Image_nucleus_part[,,2])
df_results[i,"intensity_sum_blue_nucleus_region"] <- sum(Image_nucleus_part[,,3])
df_results[i,"intensity_sum_full_without_nucleus_region_red"] <- sum(Image_non_nucleus_part[,,1])
df_results[i,"intensity_sum_full_without_nucleus_region_green"] <- sum(Image_non_nucleus_part[,,2])
df_results[i,"intensity_sum_full_without_nucleus_region_blue"] <- sum(Image_non_nucleus_part[,,3])

df_results[i,"intensity_sum_red_without_nucleus_region"] <- sum(Image_non_nucleus_part[,,1])
df_results[i,"intensity_sum_green_without_nucleus_region"] <- sum(Image_non_nucleus_part[,,2])
df_results[i,"intensity_sum_blue_without_nucleus_region"] <- sum(Image_non_nucleus_part[,,3])
df_results[i,"intensity_sum_cytosol_region_red"] <- sum(Image_cytosol_part[,,1])
df_results[i,"intensity_sum_cytosol_region_green"] <- sum(Image_cytosol_part[,,2])
df_results[i,"intensity_sum_cytosol_region_blue"] <- sum(Image_cytosol_part[,,3])

df_results[i,"intensity_sum_red_foreground"] <- sum((Image_loaded * EBImage::toRGB(mask_foreground))[,,1])
df_results[i,"intensity_sum_green_foreground"] <- sum((Image_loaded * EBImage::toRGB(mask_foreground))[,,2])
df_results[i,"intensity_sum_blue_foreground"] <- sum((Image_loaded * EBImage::toRGB(mask_foreground))[,,3])
df_results[i,"intensity_sum_foreground_red"] <- sum((Image_loaded * EBImage::toRGB(mask_foreground))[,,1])
df_results[i,"intensity_sum_foreground_green"] <- sum((Image_loaded * EBImage::toRGB(mask_foreground))[,,2])
df_results[i,"intensity_sum_foreground_blue"] <- sum((Image_loaded * EBImage::toRGB(mask_foreground))[,,3])

df_results[i,"intensity_sum_red_foreground_without_nucleus_region"] <- sum((Image_loaded * EBImage::toRGB(mask_foreground_wo_nucleus))[,,1])
df_results[i,"intensity_sum_green_foreground_without_nucleus_region"] <- sum((Image_loaded * EBImage::toRGB(mask_foreground_wo_nucleus))[,,2])
df_results[i,"intensity_sum_blue_foreground_without_nucleus_region"] <- sum((Image_loaded * EBImage::toRGB(mask_foreground_wo_nucleus))[,,3])
df_results[i,"intensity_sum_foreground_without_nucleus_region_red"] <- sum((Image_loaded * EBImage::toRGB(mask_foreground_wo_nucleus))[,,1])
df_results[i,"intensity_sum_foreground_without_nucleus_region_green"] <- sum((Image_loaded * EBImage::toRGB(mask_foreground_wo_nucleus))[,,2])
df_results[i,"intensity_sum_foreground_without_nucleus_region_blue"] <- sum((Image_loaded * EBImage::toRGB(mask_foreground_wo_nucleus))[,,3])

df_results[i,"intensity_mean_red_background"] <- mean(image_background[,,1])
df_results[i,"intensity_mean_green_background"] <- mean(image_background[,,2])
df_results[i,"intensity_mean_blue_background"] <- mean(image_background[,,3])
df_results[i,"intensity_mean_background_red"] <- mean(image_background[,,1])
df_results[i,"intensity_mean_background_green"] <- mean(image_background[,,2])
df_results[i,"intensity_mean_background_blue"] <- mean(image_background[,,3])

df_results[i,"number_of_total_pixels"] <- number_of_total_pixels
df_results[i,"number_of_pixels_nucleus_region"] <- number_of_pixels_nucleus_region
Expand Down Expand Up @@ -1591,6 +1611,20 @@ cellPixels <- function(input_dir = NULL,
type = "tiff",
bits.per.sample = 8)

# Images with marked cytosol
if(add_scale_bar){
Image_cytosol_part <- addScaleBar(image = Image_cytosol_part,
length_per_pixel = length_per_pixel_x_in_um)
}

Image_cytosol_part <- EBImage::Image(data = Image_cytosol_part, colormode = "Color")
EBImage::writeImage(x = Image_cytosol_part,
files = paste(output_dir, image_name_wo_czi, "_cytosol.tif", sep = ""),
type = "tiff",
bits.per.sample = 8)



# Images with marked nuclei and borders around the second protein in nuc
if(add_scale_bar){
Image_nuclei_numbers_proteins <- addScaleBar(
Expand Down
27 changes: 19 additions & 8 deletions man/cellPixels.Rd
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