Seitenhistorie
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- Get the real sample ready for mounting (eg. imaging chamber out of the incubator)
- Select pre-set settings that include 488/561, continuous, focus on the well, select a nice cell that has both constructs
- Zoom to fill the screen with the sample, instead of having a majority of black pixels
- Then select different pre-sets in ZEN, 485 only, 256x256 px, 40 frames time course
- SymPhoTime: confirm that the settings match the new pre-set settings in ZEN
- Start in SymPhoTime
- Start in ZEN (sends a trigger to SymPhoTime, to start the measurement)
- Evaluate the detection rate. Ideally, we count number of photons/second at 5% (160000/sec) of the repetition rate (32 MHz)
- At minimum, we pick up at least 1000/second.
Routine loop: go find another cell
- In ZEN, switch back to "standard" GFP/RFP settings, and find another cell that has comparable, good expression levels.
- Focus and zoom the cells, go back to pre-set measurement settings for flim (eg. 485 etc.)
- Start in SymPhoTime, start in ZEN, wait for acquisition.
- Interpret
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- , and evaluate data
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- . Besides judging expression level from images, count rate can also be used to compare between cells.
- Record data in existing template word file.
- Always include a donor-only control!
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