TITLE: Multi-parameter confocal TCSPC spectroscopy analysis
AUTHORS: Amber Yanas

[DESCRIPTION]
The following outlines the analysis of data generated by multi-parameter confocal time-correlated single photon counting (TCSPC) microscopy and spectroscopy (MicroTime-200; PicoQuant, GmbH).

[STEPS]
SECTION: Conversion of .ptu to .hdf5
1.    

SECTION: Conversion of .ptu to .hdf5
1.1. Use program: PQ_To_hdf5_All.ipynb 
SECTION: Conversion of .ptu to .hdf5
1.2. Enter pathname and run all to convert files from .ptu to .hdf5 
SECTION: Conversion of .ptu to .hdf5
1.3. A new folder will be created with these hdf5 files 
a.     Hint – convert to hdf5 in bulk to save time 
SECTION: Processing .hdf5 files for burst analysis and FRET
2.   

SECTION: Processing .hdf5 files for burst analysis and FRET
2.1. Use program: FRET_Anal_Y6_Vb.ipynb
SECTION: Processing .hdf5 files for burst analysis and FRET
2.2. Enter pathname and run RNA only sample 
SECTION: Processing .hdf5 files for burst analysis and FRET
2.3. Check you are using the appropriate correction factors, usually beta is the only factor that varies from experiment to experiment. Make sure beta is set so that the double labeled population median is at 0.5.  
SECTION: Processing .hdf5 files for burst analysis and FRET
2.4. 
 

SECTION: FRET Efficiency Proportion Analysis
3. FRET Efficiency Proportion Analysis
SECTION: Processing .hdf5 files for burst analysis and FRET
2.5. Make sure the time ranges are appropriate for your experiment and cover the decay fully. 

 

SECTION: Processing .hdf5 files for burst analysis and FRET
2.7. Separate files into individual folders: 

 

SECTION: Processing .hdf5 files for burst analysis and FRET
2.6. Enter pathname and run +protein sample
       Make sure beta is set so that the double labeled population median is at 0.5.
SECTION: FRET Efficiency Proportion Analysis
3.1. In the “FRET Efficiency” folder you created, go to the individual FRET Efficiency .csv file 
SECTION: FRET Efficiency Proportion Analysis
3.2. Add up the number of bursts that had a FRET Efficiency above and below 0.5 from the “E” column in the .csv file
a.     =IF(F5454>0.5,1,0)
b.     This will assign 0 below 0.5 and 1 above 0.5 
SECTION: FRET Efficiency Proportion Analysis
3.3. Sum the number of bursts that had a FRET efficiency above 0.5 
a.     =SUM(N2:N5454)
b.     This will be the numerator for your high FRET efficiency bursts 
SECTION: FRET Efficiency Proportion Analysis
3.4. Determine the number of bursts (sum the number of rows of data) 
a.     =ROWS(N2:N5454) 
b.     This is your denominator 
SECTION: FRET Efficiency Proportion Analysis
3.5. Determine the proportion of bursts above 0.5 – this is proportion high FRET
a.     Sum of bursts above 0.5 / sum of total bursts 
SECTION: FRET Efficiency Proportion Analysis
3.6. Determine the proportion of bursts below 0.5 – this is proportion low FRET 
a.     Sum of bursts below 0.5 / sum of total bursts 
SECTION: Burst Analysis
4. Burst Analysis
SECTION: Burst Analysis
4.1. The output will be called “BurstSearch.txt.”
SECTION: Burst Analysis
4.2. Copy the entire text file contents to an excel sheet 
SECTION: Burst Analysis
4.3. Copy the bursts for acceptor only, double labeled, and donor only 
SECTION: Burst Analysis
4.4. Determine the proportion of these bursts by taking the quantity of these individual burst populations over the quantity of bursts in the RNA only sample 
SECTION: Burst Analysis
4.5. Normalize the RNA only sample to the maximum annealing proportion of 0.75 (for the RNA used in Yanas PNAS 2014 paper). 
SECTION: Processing files for Anisotropy
5.  
SECTION: Processing files for Anisotropy
5.1. In Spyder - Use program: Anisotr_DDX2b_fig.py
SECTION: Processing files for Anisotropy
5.2. Enter pathname (make sure you separate the files into AO, DO, DL) 
SECTION: Processing files for Anisotropy
5.3. Determine the t shift, G, and time period that fits the decay curve of the RNA only sample:
a.     The G value should bring the decay curve to zero 
b.     The t shift should align the beginning of the horizontal and perpendicular anisotropy decays.
c.     The time period should begin when the data is less sparse in the decay curve and end when the curve has flatlined  

SECTION: Processing files for Anisotropy
5.4. Use the “G” “e” “T” commands to set these parameters.
a.     For example: type “G”, enter, then “1” (as your G value), enter. You have now set the G factor. Repeat for the other parameters.
SECTION: Processing files for Anisotropy
5.5. 
 

SECTION: Processing files for Anisotropy
5.6. Apply these values to the +protein and +protein/ATP samples
SECTION: Processing files for Anisotropy
5.7. Stop program and enter pathname for +protein samples
a.     Use the “G” “e” “T” commands to set these parameters.
SECTION: Processing files for Anisotropy
5.8. The values will be saved to SavedData.csv file 
SECTION: Processing files for Anisotropy
5.9. Determine B/B0 by using the anisotropy of the tightly bound protein only sample as B0 and using the B value column as B. Plot these values over time to observe anisotropy changes.
SECTION: Processing .hdf5 files for FCCS
6.   
 
  
SECTION: Processing .hdf5 files for FCCS
6.1. Use program: FCS_AnalY4_All_B.ipynb
SECTION: Processing .hdf5 files for FCCS
6.2. Enter pathname and run all 
SECTION: Processing .hdf5 files for FCCS
6.3. Use beta value determined in the burst analysis program 
SECTION: Processing .hdf5 files for FCCS
6.4. Check each file has a good fit 
SECTION: Processing .hdf5 files for FCCS
6.5. In Spyder use program: PlotResults1.py
SECTION: Processing .hdf5 files for FCCS
6.6. Enter pathname and run files 
SECTION: Processing .hdf5 files for FCCS
6.7. Extract file names, tau values, and N values  
SECTION: Processing .hdf5 files for FCCS
6.8. Copy and paste this output to excel 
SECTION: Processing .hdf5 files for FCCS
6.9. Split these cells into individual cells 
SECTION: Processing .hdf5 files for FCCS
6.10. Copy and paste transpose to sort data into vertical columns 
SECTION: Processing .hdf5 files for FCCS
6.11. Convert tau values to diffusion coefficients with the following equations: