Seitenhistorie

...

• 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

...

• 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) of the repetition rate (32 MHz)

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, record, and evaluate data, move on to next cell.