Preprocessing and normalization in histoCAT

erikg · Post by **erikg** » Wed Mar 14, 2018 3:48 pm

What do pre-processing step would you recommend prior to importing the uint16 images into HistoCat (hotspot removal etc)? How do you best normalize your images when combining larger image sets(ROIs) that you want to analyse with tSNE/Phenograph clustering.

DenisSchapiro · Post by **DenisSchapiro** » Wed Mar 14, 2018 4:57 pm

What do pre-processing step would you recommend prior to importing the uint16 images into HistoCat (hotspot removal etc)?

FYI - The newest histoCAT versions (>1.72) can load 32-bit tiff images, so no need to convert the images to 16-bit anymore.
1) I do not modify the raw images before loading into histoCAT. However, we use 99th percentile normalised data for t-SNE and PhenoGraph as suggested by the corresponding publications.
2) Some people in our lab prefer to apply noise filters or log-transformation as a pre-processing step, but this is decided based on the individual image quality. @Votti: Can you comment further on that?

How do you best normalize your images when combining larger image sets(ROIs) that you want to analyse with tSNE/Phenograph clustering.

This is a tough question. Ideally, we would like to use something as an internal control in each sample. One possibility is to use Histone-H3 for normalisation, since this marker is present in each segmented cell. Currently, I don't have a clear answer here, but we are working on it.

ElisaRasca · Post by **ElisaRasca** » Mon Oct 08, 2018 3:15 pm

When you say you use 99th percentile normalised data for t-SNE and PhenoGraph, do you mean that this normalization step is already present in the implementation of these algorithms in histoCAT, or that you perform the normalization BEFORE importing the data?

DenisSchapiro · Post by **DenisSchapiro** » Mon Oct 08, 2018 9:05 pm

The normalization is included in the implementation. It can be commented out in the source version of histoCAT if needed.

ElisaRasca · Post by **ElisaRasca** » Tue Oct 09, 2018 7:56 am

Thank you very much!

Post by **jonas** » Tue Aug 27, 2019 8:58 am

FYI, there is a related question in the following topic: viewtopic.php?f=4&t=1501&p=1808

JaJeago · Post by **JaJeago** » Mon Nov 02, 2020 3:37 pm

Dear Steve,

This is a pretty unique and tailored question. I will respond via email with your data example.

For the Clustergram, use the command: viewDelta

Best

Denis