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- Timestamp:
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03/15/10 09:33:28 (14 years ago)
- Author:
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dzmacdonald
- Comment:
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Legend:
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v5
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v6
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| 7 | 7 | Currently users using SPCImage have to go through a number of steps to generate their analysis results. |
| 8 | 8 | |
| 9 | | Here are some more details on the workflow that I'm doing when I analyse FLIM FRET data: |
| | 9 | Here are some more details on the workflow to analyse FLIM FRET data: |
| 10 | 10 | |
| 11 | 11 | 1. Launching data corresponding to an acquisition of " No FRET conditions" |
| 12 | 12 | 1. Draw an ROI around region of interest (cell, part of cell (nucleus) , different stage of mitosis...) |
| 13 | 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. |
| | 14 | 1. Determine if it is necessary to increase binning by looking at the average photon count in the ROI. |
| 15 | 15 | 1. Perform analysis in SPCImage by applying a monoexponential decay model and fixing the background value. |
| 16 | 16 | 1. From the analysis of a single ROI, k no fret = k2 value is output in Excel. |
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| 18 | 18 | 1. Save intensity image as tiff; save k no FRET map as tiff |
| 19 | 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" |
| | 20 | 1. Launch data corresponding to an acquisition of " FRET conditions" |
| 21 | 21 | 1. Draw an ROI around region of interest (cell, part of cell (nucleus) , different stage of mitosis...) |
| 22 | 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. |
| | 23 | 1. Determine if it is necessary to increase binning by looking at the average photon count in the ROI. |
| 24 | 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 | 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. |
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| 28 | 28 | 1. Perform calculations in Excel (normalisation FRET populations, etc..) |
| 29 | 29 | 1. Combine results to get averages for different treatments, conditions or for different stage of mitosis or cell cycle. |
| 30 | | |
| 31 | 30 | |
| 32 | 31 | Typically a user will generate 100 fret images per day, each days aquisiton can take up to 2 days to analyse. |
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