Imaging Mass Cytometry
Posted: Tue Jan 09, 2018 5:20 pm
Hi all,
I am sure many of you are aware of the Hyperion Imaging System (IMC) which was announced as the latest addition upstream of the CyTOF and performs multiplexed metal-tagged antibody based imaging on frozen and paraffin embedded sections. There is not a lot of information available for users of this technology since it is very new. I was hoping there can either be a thread or sub-section on this forum where we can discuss all things IMC related- experiences, issues/troubleshooting, tips, wish lists, antibodies etc. I think this will be tremendously useful for the early adopters who are trying to figure things out, as this forum has been for the CyTOF community.
To get started, I can share my (limited) experience here:
- Amazing technology which is going to enable key discoveries to be made. Really exciting space to work in. The images come out really well, no autofluoresence background (there is some background noise which usually disappears upon normalization)
- It is SLOW! The acquisition time for 1 sq.mm of tissue is ~50 minutes. You will really need to account for this when planning your experiments or advising clients/collaborators.
- The most convenient aspect is that it can run completely unattended once you start acquisition. No worries about clogs or errors. The longest acquisition I have performed is ~4 days of continuous run. You just have to make sure you have enough gases to last the run.
- The most time consuming part is building and testing your antibody panel, especially for FFPE tissue. I test my antibodies by regular IHC prior to conjugation. The pre-conjugated Fluidigm antibodies work very well too.
- Issues: The one problem I have right now is that the staining protocol that was recently published does not work well for me on frozen tissue. The issue here is the intercalator does not work well, it is not marking the nucleus distinctly, which means I cannot do single cell segmentation downstream. On FFPE tissue this works perfectly. I have not identified the cause yet, if anyone can point to a solution that would he helpful.
I hope this is useful for somebody and I encourage others to share their experiences with the IMC. I will also continue to do so.
Santhosh
I am sure many of you are aware of the Hyperion Imaging System (IMC) which was announced as the latest addition upstream of the CyTOF and performs multiplexed metal-tagged antibody based imaging on frozen and paraffin embedded sections. There is not a lot of information available for users of this technology since it is very new. I was hoping there can either be a thread or sub-section on this forum where we can discuss all things IMC related- experiences, issues/troubleshooting, tips, wish lists, antibodies etc. I think this will be tremendously useful for the early adopters who are trying to figure things out, as this forum has been for the CyTOF community.
To get started, I can share my (limited) experience here:
- Amazing technology which is going to enable key discoveries to be made. Really exciting space to work in. The images come out really well, no autofluoresence background (there is some background noise which usually disappears upon normalization)
- It is SLOW! The acquisition time for 1 sq.mm of tissue is ~50 minutes. You will really need to account for this when planning your experiments or advising clients/collaborators.
- The most convenient aspect is that it can run completely unattended once you start acquisition. No worries about clogs or errors. The longest acquisition I have performed is ~4 days of continuous run. You just have to make sure you have enough gases to last the run.
- The most time consuming part is building and testing your antibody panel, especially for FFPE tissue. I test my antibodies by regular IHC prior to conjugation. The pre-conjugated Fluidigm antibodies work very well too.
- Issues: The one problem I have right now is that the staining protocol that was recently published does not work well for me on frozen tissue. The issue here is the intercalator does not work well, it is not marking the nucleus distinctly, which means I cannot do single cell segmentation downstream. On FFPE tissue this works perfectly. I have not identified the cause yet, if anyone can point to a solution that would he helpful.
I hope this is useful for somebody and I encourage others to share their experiences with the IMC. I will also continue to do so.
Santhosh