| 9 | | 1. Draw an ROI around region of interest (cell, part of cell, cell cycle) |
| 10 | | 1. Perform analysis in SPCImage |
| 11 | | 1. Select cut off for k1 |
| 12 | | 1. output results to excel where the k1, k2 values are transformed in to E'fret conditon, (1-k1/k2*100) |
| 13 | | 1. Generate heat map from E'fret, each map must have min, max set by hand |
| 14 | | 1. Repeat for each region of interest |
| 15 | | 1. Combine results to get averages for different treatments, cell cycle. |
| | 9 | Here are some more details on the workflow that I'm doing when I analyse FLIM FRET data: |
| | 10 | |
| | 11 | 1. Launching data corresponding to an acquisition of " No FRET conditions" |
| | 12 | 1. Draw an ROI around region of interest (cell, part of cell (nucleus) , different stage of mitosis...) |
| | 13 | 1. Determine background level |
| | 14 | 1. Determine if it is necessary to increase binning by looking how many photons count I have in average in my ROI. |
| | 15 | 1. Perform analysis in SPCImage by applying a monoexponential decay model and fixing the background value. |
| | 16 | 1. From the analysis of a single ROI, k no fret = k2 value is output in Excel. |
| | 17 | 1. Repeat this analysis of each ROI of a picture and then for n acquisitions of the " No FRET conditions" |
| | 18 | 1. Save intensity image as tiff; save k no FRET map as tiff |
| | 19 | 1. Calculate a k no fret= k2 average value from the n k2 values analysed. |
| | 20 | 1. Launching data corresponding to an acquisition of " FRET conditions" |
| | 21 | 1. Draw an ROI around region of interest (cell, part of cell (nucleus) , different stage of mitosis...) |
| | 22 | 1. Determine background level |
| | 23 | 1. Determine if it is necessary to increase binning by looking how many photons count I have in average in my ROI. |
| | 24 | 1. Perform analysis in SPCImage by applying a biexponential decay model and fixing the background value AND FIXING THE K2 PARAMETER FROM THE ANALYSIS (1-3). |
| | 25 | 1. From this analysis of a single ROI, k1=k FRET distribution is saved manually as a .csv to be latter open in Excel and displayed in publication. |
| | 26 | 1. In SPCimage, for a single ROI generate E 'fret map which is (1-k1/k2*100) ; each map must have min, max lifetime values range set by hand. Displaying E FRET map by using pseudo color continuous or discrete is set up by hand. Save this E FRET map manually as a tiff file. |
| | 27 | 1. Repeat this analysis of each ROI of a picture and then for n acquisitions of the " FRET conditions" |
| | 28 | 1. Perform calculations in Excel (normalisation FRET populations, etc..) |
| | 29 | 1. Combine results to get averages for different treatments, conditions or for different stage of mitosis or cell cycle. |
| | 30 | |
