Batch proccessing, that converts PiqoQuant PTU into fluorescent image

This script can generate multicolour images in an automized way. The PTU conversion is done by

https://github.com/RobertMolenaar-UT/readPTU_FLIM install wx python for file selector app.

Main purpose of the script is one can proccess a batch or folder of PTU files and get a series fluorescent multicolor images with minimal user input. Tested system : Picoquant MT200 with FLIMBEE with 4x SPAD and a multiharp 150.

Each PTU file is checked for

• if it is a 2D image.
• Autodetect # APD channels
• Suports 'PIE' or 'normal' excitation
• Features Zstack image projection and FRET efficiency
• Common errors are catched and listed in the end.

You need to define the channels in the initial part of the code: Ch, NameLabel, Coloring, Gain, PIE TimeGate, FRET

• Config1 = Set_Channel_Info(1, 'Alexa647' , 'Red' ,2 ,1 , 'donor')
• Config2 = Set_Channel_Info(2, 'Alexa488' , 'Green' ,2 ,2 , 'acceptor')
• Config3 = Set_Channel_Info(3, 'DAPI-1 , 'Blue' ,2 ,3 , '-')
• Config4 = Set_Channel_Info(4, 'DAPI-2' , 'Blue' ,2 ,3 , '-')

1. Each channel is normalized from 0:1 and added into a RGB color image.
2. Availabe colors: 'Red', 'Green', 'YGreen' , 'Blue', 'Magenta', 'Cyan', 'Orange','Yellow'
3. Picoquant PIE TimeGates LASER fireing order is from the longst wavelenght to the shortest.
4. Optionan assign FRET 'donor', and 'acceptor' channels Set FRET =True
5. Zstack image projection can be made of the selected Z stack files. Set Zstack=True and Plot_OrthogonalProjections=True
6. Image Intensity [counts] data is saved in .dat (comma seperated file) Set *Save data files = True

SETUP configurations.

1. Change laser lines here in order of SEPIAII rackposition *SEPIA_laser_lines=[638,560,488,405]
2. Set Full objective in Symphotimetime64 or in the function *Read_objective()
3. PIE TAC ranges are auomatically calculated

Known limitiation: For bi-directional scanning, the readPTU_flim needs to be modified, code upon request.