Correction factors

[smXplorer]

It would seem useful to offer the ability to save calculated correction factors for 2-color smFRET:

• donor leakage in acceptor channel
• direct acceptor excitation by the donor laser
• gamma and beta factor

[tritemio]

Maybe.

To think about:

• Single-spot vs multispot. Per-channel or global?
• It is a piece of information whose provenance cannot be explained.
• the definition of direct-excitation may change in ALEX non-ALEX
• donor leakage and A-direct excitation is a dyes/setup property, but gamma can change from sample to sample with same dyes.
• If we include this post-analysis data why not add more?

In general I see the Photon-HDF5 file as "immutable raw data". Any post-analysis data should go in a different file (every time we write the file there is a risk of corruption). But for these 3 parameters it may be ok to do an exception.

[tritemio]

@smXplorer, after more thoughts I think it would be useful to provide these 3 fields as a standard place where to store these parameters. Even with the limitation that the source of these parameters would be not specified, a standard place is useful. They would be optional.

These parameters depend on both sample and setup, but it is hard to choose between sample or setup group. I would to put them in /photon_data/measurement_specs/. For example:

• /photon_data/measurement_specs/donor_leakage
• /photon_data/measurement_specs/acceptor_direct_excitation
• /photon_data/measurement_specs/gamma

These fields are spot-specific and would also be compatible automatically compatible with the multispot layout. This fields would not work for 3 or more color ALEX, and I think it's ok to leave this case out for now.

[smXplorer]

You could add a field (URL, DOI) for each parameter to specify its origin (Notebook, publication, etc).

[tritemio]

Yes, we could add a new filed corrections_factors_provenance containing an URL. It can be a DOI-URL (http://doi.org/XXXX) or any other URL (e.g. a github repository).

[tritemio]

There is the issue of which direct excitation definition to use.

Using reference: https://doi.org/10.1101/083287, the direct excitation Dir term can be expressed as a function of n_aa as in ALEX:

Dir = d_aa * n_aa

or as a function of physical parameters

Dir = d_T (gamma nd + na)

The link between the two is:

d_T = beta d_aa

The advantage of the second definition is that the same coefficient is valid for all measurements using the same A fluorophore and D laser (regardless of A-laser). In particular it can be used for single-laser smFRET measurements.

We can use the definition d_T and also add another field for beta so that the common ALEX definition d_aa can be derived.