TITLE: Formalin Fixed Paraffin Embedded (FFPE) Tissue Preparation for Digital Pathology Quantification Using QuPath
AUTHORS: Toby J Curless, Yau Lim, Zane Jaunmuktane

[DESCRIPTION]
QuPath is an open-source digital pathology analysis software. Here, it is used to quantify various misfolded proteins across anatomical regions. 

This protocol is appropriate for both human and mouse tissue, and will outline the methods of tissue preparation, digitisation, segmentation and importing images into QuPath. 


[STEPS]
SECTION: Tissue Preparation
1. Generate tissue sections using standard microtome sectioning protocols. 
SECTION: Tissue Preparation
2. Perform immunohistochemistry staining as per standard protocols.
SECTION: Tissue Preparation
3. Coverslip stained slides as per standard protocols.
SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
6. Insert slide rack into a NanoZoomer S360 slide scanner (Hamamatsu – model: C13220).

 
SECTION: Tissue Preparation
4. Following cover slipping, wipe slides with 100% ethanol to remove ink/debris, then wipe slides with a dry microfiber cloth to remove any residual dust/debris.
SECTION: Tissue Preparation
5. Insert slides facing upwards into the NanoZoomer slide rack.
SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
7. Select the scan profile in “Scan Settings” – full scan profile can be seen in the table below


 

SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
8. Select the output profile in “Scan Settings” – full output profile can be seen in the table below – asterisks (*) are in place of file names – choose file as appropriate.


 

SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
9. Additional settings: Image compression set to JPEG (quality 70), resolution: 230 nm/pixel (110198 DPI), colour profile: NanoZoomer S360 (C13220_MI_00) default.
SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
10. Scan slides by selecting "Start Batch".
SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
11. Manually select focus points and areas to be scanned for each section.
SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
12. Following slide scanning, remove slides from the scanner and store appropriately.
SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
13. Access the scanned slides via NZConnect 1.0.36 (IVD) – the images will appear in the folder in which they were saved in the “output path settings” – note: if a temporary export path was chosen, files should be manually selected and moved (cut and paste) to the desired folders.
SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
14. Select the desired anatomical regions to annotate, for example the hippocampus. Select the “Freehand” tool on NZConnect.
SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
15. Either left-click across the perimeter of the region of interest, or click and drag around the perimeter. Once the perimeter is fully covered, double click to finish the annotation.
SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
16. Label the annotation appropriately – here you can choose to display the area and perimeter of the region, in addition to changing the colour of the perimeter. It is recommended to standardise the colour used for different anatomical regions for easy identification.

  

SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
18. Download and install the most recent version of QuPath on your device (https://qupath.github.io/).
SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
17. Once all annotations are completed, the annotations must be published – this can be done for each image individually, or whole folders can be published at once. When hovering over the image or folder with the mouse, 3 vertical dots will appear in the top right corner of the selection box. Left click the dots and select “Publish annotations” – publishing options can be modified to allow access to specific users, and to automatically share annotations when added. Choose the desired settings and select “Publish”
SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
19. Open QuPath and select “create project” on the home page. Alternatively, select “file” → “Create New Project”. 
SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
21. Once a new project is created, select “Add Images” – select all of the scanned images (.ndpi files) to be imported, In the drop-down boxes select: “Default (let QuPath Decide)” for the image provider, and “Brightfield (H-DAB)” for the set image type. 
SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
20. Create a new folder and save to the desired location.
SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
22. Annotations will need to be separated from the original ndpi file – this will require the use of a custom script. Once separated, 2 files for each scanned slide should be available – 1 .ndpi file (this is the original, non-segmented file), and 1 .ndpa file – this is the separated annotations.
SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
23. The separated annotations will need to be imported to QuPath. This will require the use of another custom script – select “Automate”→ “Show script editor” → “File” → “Open” 

SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
24. Select the script from the file browser – here we used a script saved as a “GROOVY FILE”.
SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
25. Select “Run”  →  “Run for project” – Using the arrows in the pop-up box (Select project image), select all of the desired images and transfer across to the “Selected” box.
SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
26. Once all images are selected, click “Ok”.
SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
27. All annotations should now be imported into QuPath and analysis can be conducted.

 

SECTION: Digitisation and Segmentation of Tissue Sections, and Importing Images to QuPath
28.