Multiplexed Protein Map analysis

This section explains step-by-step the image analysis protocol from the generation of cell segmentations in Cell Profiler 1 to the identification of Multiplexed Cell Units and finally to the creation of Multiplexed Protein Maps.
Protocol
  1. Structure you project directory containing the images of the different cycles as suggested in the pdf document "Explanations_Generation_MultiplexedProteinMaps.pdf". Cell Profiler 1 and the Scripts for the generation of Multiplexed Protein Maps (MMP) expect the specified layout.
  2. Generate object segmentations such as Cells, Nuclei, and Cytoplasms using Cell Profiler 1 of one of the 4i cycles. Ideally, the one were you have a signal to identify the cell outlines. An example pipeline called "4iPipeline_1_SegmentNucleiAndCells.mat" is found in "CPPipelines.zip".
  3. Align and shift the segmentations previously generated (step 2) so that the match up with 4i images from other cycles. Then save the shifted segmentations. We usually use DAPI as a reference signal (C01) to align images of different cycles. An example pipeline called "4iPipeline_2_LoadSegmentationAndMeasure.mat" is found in "CPPipelines.zip". Repeat step 3 until all 4i cycle directories contain segmentation files.
  4. Generate a CSV file which contains the metadata information of the cells from which you want to extract the Multiplexed Protein Profiles (MMP). Read "Explenations_Generation_MultiplexedProteinMaps.pdf" on the specific vlaues required to be included in the CSV.
  5. Execute Script_01_GenerateMPPMatrix,m, Script_02_ProcessMPPMatrix.m, Script03_ClusterMPPMatrix_with_SOM.R, Script_04_ClusterSOMNodes_with_PG.m, Script_05_Proximity_Size_Calculation.m, Script_06_MPMGeneration.m sequentially to generate a Multiplexed Protein Map of your 4i images.
    The script Script1to6_MPMGeneration_FullWorkflow.m is a concatination of all MatLab script previously mentioned and simplifies the MPM generation, since some inputs are already set. We suggest to use it to make your life easier.

Quick description of the 6 scripts:
  • Script_01_GenerateMPPMatrix.m alignes images of different 4i cycles and extracts from each 2D position within the cell segmentation of a cell of interest the MPP. It also generated MPP metadata, which will be necessary to measure Spatial Proximity Scores (SPS).
  • Script_02_ProcessMPPMatrix.m processes the MPM matrix and prepares it for clustering via Self-Organizing Maps (SOMs).
  • Script03_ClusterMPPMatrix_with_SOM.R clusters the MPM matrix with a SOM. You will need R for this step, and FlowSOM.
  • Script_04_ClusterSOMNodes_with_PG.m further clusters the the SOM with Phenograph to identify Multiplexed Cell Units (MCUs). You will need Phenograph.
  • Script_05_Proximity_Size_Calculation.m calculates the pairwise SPS for each MCU in each cell, as well as the area of MCUs relative to the cell areas.
  • Script_06_MPMGeneration.m uses SPSs anf MCU areas to generate a Multiplexed Protein Map and an overview of the 4i channel loadings of each MCU.

Spatial projection of Multiplexed Cell Units on to a group of cells

Materials needed:
CellProfiler1
cellatlas.org or
cellatlas.org/downloads version 1.0.5811Bugfix

Custom 4i modules
cellatlas.org/downloads

Scripts for generating Multiplexed Protein Maps
cellatlas.org/downloads

R and FlowSOM
https://www.r-project.org/​
https://bioconductor.org/packages/release/bioc/html/FlowSOM.html

Phenograph and tSNE (cyt implementation)

1x computer 
(Ideally 1x high-performance cluster for the image analysis)
Download
Explanations_Generation_MultiplexedProteinMaps.pdf
File Size: 94 kb
File Type: pdf
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4i_CPModules.zip
File Size: 59 kb
File Type: zip
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4i_CPPipelines.zip
File Size: 7 kb
File Type: zip
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MPMGeneration.zip
File Size: 119 kb
File Type: zip
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