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- Prepare a coverslip with a droplet of rhodamine, and find focus using the edge of the drop (that is a sharp line, then go into the drop a bit and start measuring)
- Perform a continuous FCS measurement existing pre-set settings: 488 nm, point-scan, cw laser (argon)
- this happens at 500 KHz repetition (of detector), but is with CW laser so you can't draw lifetime conclusions
- Get diffusion time. During the course, we saw 32 µs for rhodamine.
- Reveal count rate: how many events per molecule do we detect in the setup?
- Correction collar: Maximize this number by adjusting the objective correction collar. That is the optimal correction.
- Pinhole alignment: Can be simply used here to automatically optimize X and Y position of pinhole based on intensity.
SymPhoTime first, start observing photons
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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, and evaluate data. 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!