Last updated: 2023-05-01

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Aim

Quickly check the cell number in GFP and RFP channels over 3 days of imaging in BioSpa + Cytation

Researcher

ML

# load the required packages if (!require('pacman')) install.packages('pacman')
# pacman::p_load(package1, package2, package_n) if (!require('BiocManager',
# quietly = TRUE)) install.packages('BiocManager')

library(data.table)
library(DT)
library(here)
library(dplyr)
library(ggplot2)
library(plotly)
library(tidyverse)
library(readxl)
# check if here() correctly identified the directory
here::here()
[1] "/home/mli/ML_Rhi/Rhi_test_analysis"
# set the file prefix

prefix <- "ML_Rhi_test"

Known technical issues

The Edge wells (2 outer rows, A/B/O/P 1-24, and Col 1/2/23/24) are in focus, but inner wells are not.

That has made object segmentation difficult with weak signals using CellProfiler’s filter based object identification.

I have trialed CellPose’s Deep Learning Model Cyto and Nuclei and unfortunately without user training the default model performed worse than Cell Profiler.

Data Preprocessing and heat map

We can see there are “inner rings” with very high object numbers, likely the cells are out of focus here.

Find values to filter numbers

Incorrectly segmented objects usually have abnormal mask size.

Use filtered data tp see object counts

This is not perfect method but we can see edge wells that are in focus the cell count make sense and steady increasing. The inner wells whose are out of focus still have abnormal cell counts (one can see almost 2 populations by day 3)


 

Analysed by Mark Li

Victorian Centre for Functional Genomics

 


sessionInfo()
R version 4.2.0 (2022-04-22)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: CentOS Linux 7 (Core)

Matrix products: default
BLAS:   /usr/lib64/libblas.so.3.4.2
LAPACK: /usr/lib64/liblapack.so.3.4.2

locale:
 [1] LC_CTYPE=en_AU.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=en_AU.UTF-8        LC_COLLATE=en_AU.UTF-8    
 [5] LC_MONETARY=en_AU.UTF-8    LC_MESSAGES=en_AU.UTF-8   
 [7] LC_PAPER=en_AU.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=en_AU.UTF-8 LC_IDENTIFICATION=C       

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] readxl_1.4.2      forcats_1.0.0     stringr_1.5.0     purrr_1.0.1      
 [5] readr_2.1.4       tidyr_1.3.0       tibble_3.1.8      tidyverse_1.3.2  
 [9] plotly_4.10.1     ggplot2_3.4.1     dplyr_1.1.0       here_1.0.1       
[13] DT_0.27           data.table_1.14.8 workflowr_1.7.0  

loaded via a namespace (and not attached):
  [1] googledrive_2.0.0   colorspace_2.1-0    deldir_1.0-6       
  [4] ellipsis_0.3.2      rprojroot_2.0.3     htmlTable_2.4.1    
  [7] base64enc_0.1-3     fs_1.6.1            rstudioapi_0.14    
 [10] farver_2.1.1        bit64_4.0.5         fansi_1.0.4        
 [13] lubridate_1.9.2     xml2_1.3.3          splines_4.2.0      
 [16] cachem_1.0.6        knitr_1.42          Formula_1.2-4      
 [19] jsonlite_1.8.4      broom_1.0.3         cluster_2.1.4      
 [22] dbplyr_2.3.0        png_0.1-8           compiler_4.2.0     
 [25] httr_1.4.4          backports_1.4.1     assertthat_0.2.1   
 [28] Matrix_1.5-3        fastmap_1.1.0       lazyeval_0.2.2     
 [31] gargle_1.3.0        cli_3.6.0           later_1.3.0        
 [34] formatR_1.14        htmltools_0.5.4     tools_4.2.0        
 [37] gtable_0.3.1        glue_1.6.2          Rcpp_1.0.10        
 [40] cellranger_1.1.0    jquerylib_0.1.4     vctrs_0.5.2        
 [43] xfun_0.37           ps_1.7.2            rvest_1.0.3        
 [46] timechange_0.2.0    mime_0.12           lifecycle_1.0.3    
 [49] googlesheets4_1.0.1 getPass_0.2-2       scales_1.2.1       
 [52] vroom_1.6.1         hms_1.1.2           promises_1.2.0.1   
 [55] parallel_4.2.0      RColorBrewer_1.1-3  yaml_2.3.7         
 [58] gridExtra_2.3       sass_0.4.5          rpart_4.1.19       
 [61] latticeExtra_0.6-30 stringi_1.7.12      highr_0.10         
 [64] checkmate_2.1.0     rlang_1.0.6         pkgconfig_2.0.3    
 [67] evaluate_0.20       lattice_0.20-45     htmlwidgets_1.6.1  
 [70] labeling_0.4.2      platetools_0.1.5    bit_4.0.5          
 [73] processx_3.8.0      tidyselect_1.2.0    magrittr_2.0.3     
 [76] R6_2.5.1            generics_0.1.3      Hmisc_4.8-0        
 [79] DBI_1.1.3           pillar_1.8.1        haven_2.5.1        
 [82] whisker_0.4.1       foreign_0.8-84      withr_2.5.0        
 [85] survival_3.5-3      nnet_7.3-18         modelr_0.1.10      
 [88] crayon_1.5.2        interp_1.1-3        utf8_1.2.3         
 [91] tzdb_0.3.0          rmarkdown_2.20      jpeg_0.1-10        
 [94] grid_4.2.0          callr_3.7.3         git2r_0.31.0       
 [97] reprex_2.0.2        digest_0.6.31       httpuv_1.6.9       
[100] munsell_0.5.0       viridisLite_0.4.1   bslib_0.4.2