Imaging Mass Cytometry

[ssivajothi]

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

[mleipold]

Hi Santhosh,

My first guess would be that the energetics of intercalation wouldn't be favorable in frozen tissue. There's a certain amount of energy needed to get the intercalator to *insert* between the DNA base-pairs (helix distortion, etc).

Could you instead use an anti-histone antibody to specifically stain your nuclei?


Mike

[cguidos]

Hi Santosh - I will back-up Mike's guess with some data from our experience, which is that the optimal Ir concentration to use needs to be empircally determined for each tissue and fixation method. So far we have generally found that we need to use more than the 100nM we use for suspension staining.

Also - anti-histone 3 is a good idea for human tissue but does not work well on mouse tissue. The clone sold by Fluidigm is listed as cross-reactive by the original supplier. Although we can get it stain mouse cells in suspension, the intensity is notably lower than when staining human cells in suspension. When we used this Ab for IMC of mouse frozen sections we saw no staining at all.

Hope this helps
Cindy

[vitozan]

Hi there,
I am successfully using the standard Iridium intercallator we use for suspenion MC in IMC both for FFPE as well as frozen sections and never really had a problem with it. Usually 5 min incubation at a concentration of ca 10x of the sMC concentration is usually enough to get a good nuclear signal, well suited for cell area segmentation.
Maybe also try a Hoechst/Dapi staining on the same tissue (it is compatible with IMC including Iridium staining) to check if the tissue integrity is not a problem.

Also for staining I frozen sections I have been successful a standard FFPE staining protocol, just skipping the heat retrieval steps. No real optimization was needed.

[tomash]

Hi all,

The intercalator issue is a little irritating, but FYI, the effect will be different for different tissues. In the bone marrow and spleen we get broad smearing of theintercalator, but in brain sections we get pretty decent staining.

Finding a more specific anti-nuclear antibody (if you can find a decent HH3 clone for the mouse) is definitely worthwhile.

Tom

[ssivajothi]

I recently tested different concentrations of Ir incubating for different time points on frozen sections of mouse bladder tissue.

The best result was 0.3uM - 0.5uM final Ir concentration for 5 minutes. Staining of nuclei was clean and similar to FFPE sections. The longer the incubations time, the more the background/smearing effect became.

This should serve as a good starting point and may have to be optimized for different tissues

Best,
Santhosh