[SCIF Flow Cytometry] Change in FACS Diva software

[David Gravano]

Hi Flow Users,

There's a minor change for you to note in the Diva software. Each time you log in you should now see a window pop up that says "CS&T Mismatch" and it will ask you if you want to "Use CST settings" or "Keep BD FACSDiva settings". You want to select "Use CST setting". I've posed this on each instrument's monitor to remind you. This ensures you're using the laser delay values that were calculated during the most recent instrument calibration run, which is done daily before each user comes on. Due to a reported Diva version 8 software glitch the most optimal settings may not have been applied when duplicating old experiments. This may have had a (likely minor and not noticeable) impact in the brightness of your fluorescent stains if the optimal settings were not being applied. Below is a more technical discussion of the problem if you're curious.

Thanks,
David






What's the glitch in Diva?
When I set up accounts for people, Diva version 8 gives the option to "always use the current CST settings". In past versions of Diva you couldn't do this. I had been selecting this for people when setting up new accounts and it was nice because you don't get that annoying window popping up that you should always check "Use CST settings" on. However, on the Purdue Cytometry Forum there has been a discussion that there is a glitch in Diva where sometimes it uses old CST settings. This happens when users duplicate past experiments. It will copy their laser delay values that were determined when the original experiment was set up. So I've unselected that option for everybody's account and now that's why the window pops up. Selecting "Use CST settings" should now give you the optimal laser delays every time. If you have any questions or concerns please let me know.

What is laser delay?
The lasers on our instruments are not co-linear. This means they are not aligned to all travel in the same optical path. Each laser hits the cells traveling through at a different point in space in the flow cell. This allows us to distinguish dyes from each other like PE-Cy7 and APC-Cy7 because while they have the same emission properties, they are excited by different lasers. APC-Cy7 is only excited by the red laser and PE-Cy7 only excited by the blue (on the LSR). Since each laser hits the sample in a spatially different location we can tell these dyes apart. But we need to know the amount of time it takes for the cell to travel between each laser so we can determine the fluorescent properties of the same cell across multiple lasers. If this time delay is off, the blue laser channels might be reading the cell properly, but the red laser channels may be taking their readings at the wrong time when the cell is not passing through the laser. This would look like the cell has no red signal. Or if it only captures data when the cell is already half way past, you'd get less signal than optimal. The delay values are time values that are determined with beads every day. They don't fluctuate wildly on a day to day basis but are an important value that's determined daily to ensure you're seeing the maximal signal.

What can affect laser delay?
When the service technicians come to align our lasers they may affect the laser delay values. This would be a minor change. Also any difference in system pressure would affect the laser delay because cells would travel at a different velocity. System pressure is tightly regulated and would only have a large change if there were a fluidic issue with the system. I've looked back and the delay values have been pretty constant over time, so if this has affected the quality of your data it is likely very minor.


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