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rename zstack projections and add parameter to function to export nor…
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…malized versions of images
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@buddekai
buddekai committed Jan 16, 2024
1 parent 06199bb commit bb7cbc4
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Showing 4 changed files with 189 additions and 17 deletions.
1 change: 1 addition & 0 deletions .gitignore
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Original file line number Diff line number Diff line change
Expand Up @@ -4,4 +4,5 @@
.Ruserdata
detectCilia.Rproj
inst/testscriptDevelop.R
inst/extdate/testImageCzi/test.czi
output_compare/
2 changes: 1 addition & 1 deletion DESCRIPTION
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Original file line number Diff line number Diff line change
@@ -1,7 +1,7 @@
Type: Package
Package: detectCilia
Title: Detect and measure the lengths of primary cilia in microsopy images
Version: 0.7.5
Version: 0.7.6
Authors@R:
person("Kai", "Budde-Sagert", , "[email protected]", role = c("aut", "cre"),
comment = c(ORCID = "0000-0001-6836-9865"))
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182 changes: 170 additions & 12 deletions R/detectCilia.R
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Original file line number Diff line number Diff line change
Expand Up @@ -26,7 +26,7 @@
#' threshold to find cilia positions (either "mean" or "max").
#' @param use_histogram_equalization_for_threshold_calculation A Boolean
#' stating whether to use a histogram equalization algorithm for determining
#' threshold values.
#' threshold values and finding cilia in projection.
#' @param threshold_by_density_of_cilium_pixels A Boolean.
#' * `TRUE` (the default): Disregard the custom threshold values (if given)
#' and, instead, use a function to calculate the binarization thresholds by
Expand Down Expand Up @@ -55,6 +55,10 @@
#' @param slice_distance A number showing the distance of two consecutive
#' z-stack layers in micrometers.
#' @param nuc_mask_width_height Number of pixels (integer) for nuclei detection.
#' @param export_normalized_images A Boolean.
#' * `TRUE`: Also export the images with a normalized version of the original
#' z-stack projection/z-stack layer.
#' * `FALSE` (Default): Do not export normalized images.
#'
#' @importFrom magrittr %>%
#' @importFrom rlang .data
Expand Down Expand Up @@ -90,7 +94,8 @@ detectCilia <- function(
number_size_factor = NULL,
pixel_size = NULL,
slice_distance = NULL,
nuc_mask_width_height = NULL) {
nuc_mask_width_height = NULL,
export_normalized_images = FALSE) {


# 0. Basics --------------------------------------------------------------
Expand Down Expand Up @@ -468,6 +473,15 @@ detectCilia <- function(
# Count number of cells
nucNo <- max(nmask_watershed)

if(export_normalized_images){
EBImage::writeImage(x = nmask_watershed,
files = file.path(output_dir,
paste(input_file_name,
"_nucleimask.tif",
sep = "")),
bits.per.sample = 8,
type = "tiff")
}


# ---------------------------------------------------------------------- #
Expand Down Expand Up @@ -573,7 +587,7 @@ detectCilia <- function(
EBImage::writeImage(x = Image_projection_histogram_equalization_normalized,
files = file.path(output_dir,
paste(input_file_name,
"_stack_cilia_all_histogram_equalized_normalized.tif",
"_projection_cilia_all_histogram_equalized_normalized.tif",
sep = "")),
bits.per.sample = 8,
type = "tiff")
Expand Down Expand Up @@ -1053,7 +1067,7 @@ detectCilia <- function(
EBImage::writeImage(x = Image_projection_histogram_equalization_normalized,
files = file.path(output_dir,
paste(input_file_name,
"_stack_cilia_all_histogram_equalized_normalized.tif",
"_projection_cilia_all_histogram_equalized_normalized.tif",
sep = "")),
bits.per.sample = 8,
type = "tiff")
Expand Down Expand Up @@ -1453,6 +1467,24 @@ detectCilia <- function(

Image <- image_data[,,,i]

if(export_normalized_images){
Image_normalized <- image_data[,,,i]
Image_normalized <- EBImage::normalize(Image_normalized)

Image_normalized[coordinates_layer1] <- 1
Image_normalized[coordinates_layer2] <- 1

Image_normalized <- EBImage::Image(data = Image_normalized, colormode = "color")
EBImage::writeImage(x = Image_normalized,
files = file.path(output_dir,
paste(input_file_name,
"_cilia_layer_",
i, "_normalized.tif",
sep = "")),
bits.per.sample = 8,
type = "tiff")
}

Image[coordinates_layer1] <- 1
Image[coordinates_layer2] <- 1

Expand Down Expand Up @@ -1489,6 +1521,24 @@ detectCilia <- function(
df_cilium_information$pos_y[df_cilium_information$layer == -1],
rep(2, length(df_cilium_information$pos_x[df_cilium_information$layer == -1]))), ncol = 3)


if(export_normalized_images){
Image_projection_cilia_normalized <- Image_projection_cilia
Image_projection_cilia_normalized <- EBImage::normalize(Image_projection_cilia_normalized)

Image_projection_cilia_normalized[coordinates_layer1] <- 1
Image_projection_cilia_normalized[coordinates_layer2] <- 1

EBImage::writeImage(x = Image_projection_cilia_normalized,
files = file.path(output_dir,
paste(input_file_name,
"_projection_cilia_unconnected_normalized.tif",
sep = "")),
bits.per.sample = 8,
type = "tiff")
}


Image_projection_cilia[coordinates_layer1] <- 1
Image_projection_cilia[coordinates_layer2] <- 1

Expand All @@ -1497,7 +1547,7 @@ detectCilia <- function(
EBImage::writeImage(x = Image_projection_cilia,
files = file.path(output_dir,
paste(input_file_name,
"_stack_cilia_unconnected.tif",
"_projection_cilia_unconnected.tif",
sep = "")),
bits.per.sample = 8,
type = "tiff")
Expand Down Expand Up @@ -1572,12 +1622,12 @@ detectCilia <- function(
# }
# rm(k)

# TRUE ciliary objects
# Cleaned ciliary objects
Image_projection_cilia_connected_copy <- Image_projection_cilia_connected
df_cilium_all_cilia <- df_cilium_information[df_cilium_information$layer == -99 & df_cilium_information$cilium_shape,]
coordinates_layer1 <- matrix(data = c(df_cilium_all_cilia$pos_x, df_cilium_all_cilia$pos_y, rep(1, length(df_cilium_all_cilia$pos_x))), ncol = 3)
coordinates_layer2 <- matrix(data = c(df_cilium_all_cilia$pos_x, df_cilium_all_cilia$pos_y, rep(2, length(df_cilium_all_cilia$pos_x))), ncol = 3)

Image_projection_cilia_connected[coordinates_layer1] <- 1
Image_projection_cilia_connected[coordinates_layer2] <- 1

Expand All @@ -1586,7 +1636,7 @@ detectCilia <- function(
EBImage::writeImage(x = Image_projection_cilia_connected,
files = file.path(output_dir,
paste(input_file_name,
"_stack_cilia_connected_cleaned.tif",
"_projection_cilia_connected_cleaned.tif",
sep = "")),
bits.per.sample = 8,
type = "tiff")
Expand All @@ -1597,6 +1647,23 @@ detectCilia <- function(
coordinates_layer1 <- matrix(data = c(df_cilium_all$pos_x, df_cilium_all$pos_y, rep(1, length(df_cilium_all$pos_x))), ncol = 3)
coordinates_layer2 <- matrix(data = c(df_cilium_all$pos_x, df_cilium_all$pos_y, rep(2, length(df_cilium_all$pos_x))), ncol = 3)

if(export_normalized_images){
Image_projection_cilia_connected_normalized <- Image_projection_cilia_connected
Image_projection_cilia_connected_normalized <- EBImage::normalize(Image_projection_cilia_connected_normalized)

Image_projection_cilia_connected_normalized[coordinates_layer1] <- 1
Image_projection_cilia_connected_normalized[coordinates_layer2] <- 1

EBImage::writeImage(x = Image_projection_cilia_connected_normalized,
files = file.path(output_dir,
paste(input_file_name,
"_projection_cilia_connected_normalized.tif",
sep = "")),
bits.per.sample = 8,
type = "tiff")
}


Image_projection_cilia_connected[coordinates_layer1] <- 1
Image_projection_cilia_connected[coordinates_layer2] <- 1

Expand All @@ -1605,7 +1672,7 @@ detectCilia <- function(
EBImage::writeImage(x = Image_projection_cilia_connected,
files = file.path(output_dir,
paste(input_file_name,
"_stack_cilia_connected.tif",
"_projection_cilia_connected.tif",
sep = "")),
bits.per.sample = 8,
type = "tiff")
Expand All @@ -1615,6 +1682,11 @@ detectCilia <- function(
Image_projection_numbers <- Image_projection_cilia_connected
image_projection_numbers <- as.array(Image_projection_numbers)

if(export_normalized_images){
image_projection_numbers_normalized <- as.array(
Image_projection_cilia_connected_normalized )
}

for(i in unique(df_cilium_all$ciliumNumber) ){
ciliumNumber <- i
pos_x <- df_cilium_all$pos_x[df_cilium_all$ciliumNumber == i][
Expand All @@ -1628,6 +1700,18 @@ detectCilia <- function(
pos_y = pos_y,
number_size_factor = number_size_factor,
number_color = "red")

if(export_normalized_images){
image_projection_numbers_normalized <- addNumberToImage(
image = image_projection_numbers_normalized,
number = ciliumNumber,
pos_x = pos_x,
pos_y = pos_y,
number_size_factor = number_size_factor,
number_color = "red")
}


}
rm(i)

Expand All @@ -1636,12 +1720,24 @@ detectCilia <- function(
EBImage::writeImage(x = Image_projection_numbers,
files = file.path(output_dir,
paste(input_file_name,
"_stack_cilia_all_numbers.tif",
"_projection_cilia_all_numbers.tif",
sep = "")),
bits.per.sample = 8,
type = "tiff")


if(export_normalized_images){
Image_projection_numbers_normalized <- EBImage::Image(data = image_projection_numbers_normalized,
colormode = "color")
EBImage::writeImage(x = Image_projection_numbers_normalized,
files = file.path(output_dir,
paste(input_file_name,
"_projection_cilia_all_numbers_normalized.tif",
sep = "")),
bits.per.sample = 8,
type = "tiff")
}


# 5.4 Add nuclei and cilium numbers to image -----------------------------

Expand Down Expand Up @@ -1695,8 +1791,29 @@ detectCilia <- function(
number_size_factor = number_size_factor,
number_color = "blue")


if(export_normalized_images){
image_projection_numbers_normalized <- addNumberToImage(
image = image_projection_numbers_normalized,
number = i,
pos_x = df_nuclei_positions$pos_x[df_nuclei_positions$nuc_number_new == i],
pos_y = df_nuclei_positions$pos_y[df_nuclei_positions$nuc_number_new == i],
number_size_factor = number_size_factor,
number_color = "green")

image_projection_numbers_normalized <- addNumberToImage(
image = image_projection_numbers_normalized,
number = i,
pos_x = df_nuclei_positions$pos_x[df_nuclei_positions$nuc_number_new == i],
pos_y = df_nuclei_positions$pos_y[df_nuclei_positions$nuc_number_new == i],
number_size_factor = number_size_factor,
number_color = "blue")
}


}


rm(i)
}

Expand All @@ -1715,11 +1832,31 @@ detectCilia <- function(
EBImage::writeImage(x = Image_projection_numbers,
files = file.path(output_dir,
paste(input_file_name,
"_stack_cilia_all_numbers_nuclei.tif",
"_projection_cilia_all_numbers_nuclei.tif",
sep = "")),
bits.per.sample = 8,
type = "tiff")


if(export_normalized_images){
# Add border of nuclei and save file
Image_projection_numbers_normalized <- EBImage::Image(image_projection_numbers_normalized)
EBImage::colorMode(Image_projection_numbers_normalized) <- "color"
EBImage::colorMode(nmask_watershed) <- "gray"

Image_projection_numbers_normalized <- EBImage::paintObjects(x = nmask_watershed,
tgt = Image_projection_numbers_normalized,
col='#ff00ff')

EBImage::writeImage(x = Image_projection_numbers_normalized,
files = file.path(output_dir,
paste(input_file_name,
"_projection_cilia_all_numbers_nuclei_normalized.tif",
sep = "")),
bits.per.sample = 8,
type = "tiff")
}

# 5.6 Normalize and histogram equalized image ----------------------------

Image_projection_histogram_equalization_normalized <- EBImage::normalize(
Expand All @@ -1728,11 +1865,32 @@ detectCilia <- function(
EBImage::writeImage(x = Image_projection_histogram_equalization_normalized,
files = file.path(output_dir,
paste(input_file_name,
"_stack_cilia_all_histogram_equalized_normalized.tif",
"_projection_histogram_equalized_normalized.tif",
sep = "")),
bits.per.sample = 8,
type = "tiff")

EBImage::writeImage(x = Image_projection,
files = file.path(output_dir,
paste(input_file_name,
"_projection.tif",
sep = "")),
bits.per.sample = 8,
type = "tiff")

if(export_normalized_images){
Image_projection_normalized <- EBImage::normalize(
Image_projection)

EBImage::writeImage(x = Image_projection_normalized,
files = file.path(output_dir,
paste(input_file_name,
"_projection_normalized.tif",
sep = "")),
bits.per.sample = 8,
type = "tiff")
}


# 5.7 Save function call and used parameter values -----------------------

Expand Down
21 changes: 17 additions & 4 deletions inst/testscript.R
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Original file line number Diff line number Diff line change
Expand Up @@ -65,8 +65,6 @@ pixel_size <- 0.219645 # in \mu m
# Distance between two layers in micrometers
slice_distance <- 0.31607# in \mu m

cilium_color <- "red"

# Manually set mask width here because image is small
nuc_mask_width_height <- 100

Expand All @@ -79,6 +77,8 @@ output_dir <- file.path(getwd(), "output")
# Directory of the images
input_file <- system.file("extdata", "testImageCzi", "CiliaImage.czi",
package = "detectCilia", mustWork = TRUE)
input_file <- system.file("extdata", "testImageCzi", "test.czi",
package = "detectCilia", mustWork = TRUE)

# <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

Expand All @@ -88,13 +88,26 @@ input_file <- system.file("extdata", "testImageCzi", "CiliaImage.czi",
detectCilia_output_list <- detectCilia::detectCilia(
input_dir_tif = input_dir,
output_dir = output_dir,
cilium_color = cilium_color,
pixel_size = pixel_size,
slice_distance = slice_distance,
nuc_mask_width_height = nuc_mask_width_height)
nuc_mask_width_height = nuc_mask_width_height,
number_size_factor = 0.2)

## SECOND EXAMPLE CZI FILE -------------------------------------------------

# Obtain all positions of cilia in every z-layer
detectCilia_output_list2 <- detectCilia::detectCilia(input_file_czi = input_file,
output_dir = output_dir)





# Example for detectCilia paper --------------------------------------------
input_file <- file.path("C:", "Users", "Kai", "Documents", "git",
"gitlabElaine", "2022 DetectCilia", "workflow",
"190815_EV38_2_Collagen_ITSwithAsc+Dexa_63x_zstack_3.czi")
detectCilia_output_list3 <- detectCilia::detectCilia(input_file_czi = input_file,
export_normalized_images = TRUE)


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