Don't FRET

Don't FRET! is a python package featuring a web application for performing burst search on confocal smFRET data.

pip install dont-fret

Process photon files

dont-fret process filename.ptu

Will process the file. It will perform burst search as specified in the configuration file. Output (default) are burst photons and bursts as .pq files.

Run the web application

To launch with the default configuration file:

dont-fret serve

To create a local default configuration file:

dont-fret config

Then you can edit the created config file. To launch the web application with a specific config file:

dont-fret serve --config config.yaml

Configuration

Configuration for channels, photon streams and (default) burst search settings is done from the config .yaml file.

First, define your channels:

channels: # refactor channels in code to channel_identifiers
  laser_D:
    target: nanotimes
    value: [ 0, 1000 ]
  laser_A:
    target: nanotimes
    value: [ 1000, 2000 ] # intervals are inclusive, exclusive
  det_D:
    target: detectors
    value: 1
  det_A:
    target: detectors
    value: 0

Currently supported targets are nanotimes, detectors and timestamps. These are as read from the file and not converted to seconds. Modulo is supported (untested) for us-ALEX:

channels: # refactor channels in code to channel_identifiers
  laser_D:
    target: timestamps
    value: [ 0, 100 ]
    modulo: 200

This will assign photons with a timestamp modulo 200 in the range from 0 up to 100 to "laser_D".

Next, define your photon streams. Photons streams are combinations of channels ("AND"):

streams:
  DD: [laser_D, det_D]
  DA: [laser_D, det_A]
  AA: [laser_A, det_A]
  AD: [laser_A, det_D]

!IMPORTANT The notation used here is excitation then emission, thus the FRET stream is 'DA' while in literature the FRET photon stream is often written as A|D (Acceptor emission during donor excitation).

!IMPORTANT
At the moment apparent FRET and stoichiometry are calculated from the defined photon streams and it is required the following streams are defined: 'AA', 'DD', 'DA' (=FRET). This is expected to be changed in future updates.

Development

Download a test file:

wget https://kuleuven-my.sharepoint.com/:u:/g/personal/jochem_smit_kuleuven_be/Efy7ur779ARNiBlP05Ki7NMBabKX3auswj30xmpRLaIfPg?e=E6wWoZ&download=1

If autoreload (refresh web application upon code changes) doesnt work, run from: solara run dont_fret\tmp.py -- --config default_testing.yaml

Create a new release

• Create a new release on github. Create a new tag with the version (format: v0.1.0)
• github actions creates release on pypi
• done!