6. Vignettes on t-CyCIF data¶

We provide example vignettes to help users run Mistic. Check out our Mistic experiments page for examples Mistic has been tested on and Functions for a full list of functions Mistic uses.

6.1. t-CyCIF image on Lung adenocarcinoma metastasis to lymph node¶

This t-CyCIF image is in OME-TIFF format, 13GB in size with dimensions 10101 x 9666, and has 44 marker channels. To simultaneously test Mistic for scalability, we created duplicates of this image. In Mistic experiments 5.3, we use 70 duplicates of this image and render it on Mistic for 6 markers: CD45, Keratin, aSMA, FoXP3, PD-1, PD-L1.

Download Mistic as described here Code Installation
- Navigate to Mistic_code/code/user_inputs/
Download data from link.

Go to Files -> DATASET-1 -> LUNG-1-LN -> 40X-> ometiff.

Download lung-1-ln_40x.ome.tif and place it in the /user_inputs/figures/ folder

Create say 3 copies of this .tif file in the /figures folder

Navigate to the /user_inputs/metadata folder
- Upload the imaging markers of interest as Markers_ids.csv
  A sample .csv is provided in the /metadata/t-CyCIF folder
  
  If using the sample .csv, move this file from /metadata/t-CyCIF to /metadata folder
- Upload the markers.csv provided by t-CyCIF
  Go to link.
  
  Go to Files -> DATASET-1 -> markers.csv
- Optional uploads:
  Image tSNE co-ordinates as X_imagetSNE.csv
  
  If no user-generated tSNE co-ordinates are provided, Mistic will generate a set of t-SNE coordinates to render the images
  
  Cluster labels as Cluster_categories.csv
  
  If cluster labels are not provided, Mistic will cluster the images using a Bayesian mixture model.
  
  Patient_ids as Patient_ids.csv
  
  Treatments as Treatment_catgories.csv
  
  Patient response as Response_categories.csv
  
  If any of these are unavailable, Mistic will use either the randomly-generated or user-provided tSNE points without any color coding i.e. dots are colored in gray, for the live panels.
  
  Sample metadata files are provided for reference in the /metadata/t-CyCIF folder
  
  If using the sample metadata, move the files from /metadata/t-CyCIF/ into the /metadata folder
Open a command prompt (or the Terminal application), change to the directory containing /code and type
- bash mistic.sh
- This runs a bokeh server locally and will automatically open the interactive dashboard in your browser at http://localhost:5098/image_tSNE_GUI
From the GUI, choose ‘t-CyCIF’ from the dropdown menu, check the boxes for markers of interest and select other options based on image metadata (for example, border, image layout) and click ‘Run’

6.2. t-CyCIF image on Primary lung squamous cell carcinoma¶

This is an example where Mistic can be used to view a stack montage made up of the individual markers for a single multiplexed image (see Mistic experiments 5.4). We show this option on the t-CyCIF image on Primary lung squamous cell carcinoma. This is in OME-TIFF format for all 44 marker channels.

Download Mistic as described here Code Installation
- Navigate to Mistic_code/code/user_inputs/
Download data from link.
- Go to Files -> DATASET-1 -> LUNG-3-PR -> 40X-> singletiff.
- Download the 44 single channel tifs (lung-3-pr_40x_x.tif) and place it in the /user_inputs/figures/ folder
Navigate to the /user_inputs/metadata folder
- Upload the markers.csv provided by t-CyCIF
  Go to link.
  
  Go to Files -> DATASET-1 -> markers.csv
Open a command prompt (or the Terminal application), change to the directory containing /code and type
- bash mistic.sh
- This runs a bokeh server locally and will automatically open the interactive dashboard in your browser at http://localhost:5098/image_tSNE_GUI
From the GUI, choose ‘t-CyCIF’ from the dropdown menu, select the ‘Stack montage’ option and click ‘Run’