It’s a terrific series of video to introduce something detail in flow cytometry. Here is my problem about how instrument know to excite the dyes by different laser. Let me make my question more specifically. In the spectrum viewer, I found laser 355 and laser 488 could both excite the fluorochromes, such as FITC, PE, APC, although laser 488 could create higher emission spectrum. Could you explain this for me? thanks!!!
Hi Fei Tu, thank you for your kind comments and great question. Understanding your fluorochromes and their excitation and emission profiles is critical to any fluorescence-based experiment. If we take FTTC as an example and we look at its excitation spectrum you can see that it will be excited to varying degrees from 350nm up to about 510nm but as this curve is normalised we know that we should use the 488nm laser to excite as close as possible to the excitation maximum. However as you can see a 355nm laser or a 405nm laser will also excite FITC just with less efficiency. What this means in practise is that if we have an antigen that is highly expressed and labelled with FITC we will see some excitation with the Violet laser and some emission at around about 530nm which would be in the same filter on the Violet laser as something like BV510. We can account for this overlap by compensation, or unmixing if we're using a full spectrum approach, but it will impact on the resolution so it's important to know that this might happen. No matter which laser wavelength is used to excite FITC the emission profile will always be the same. Remember the emission profile is a probability curve so we know that most of the time FITC will emit photons in the green region of the spectrum. We are always trying to maximise our signal to noise ratio and to maximise our resolution in our flow cytometry experiments so understanding the spectra is crucial.
Using Beads instead of CS&T, are there specific values for Area and Height measurement provided by the company? or we can use our own reference point on FSC-A and FSC-H Histograms and adjust Laser delay. My Facility Assistant said we can set any measurement as a reference point, that sounded confusing.
The area and height will be altered by changing the voltage so there are no specific measurements provided. Adjust the voltage so that the height signal is where you want it and then adjust the ASF so that area is at the same value. Laser delay will be adjusted by looking at fluorescence not scatter (which will only come from one laser on most cytometers).
@@DerekDaviesCytometry I truly understand Laser delay comes later for individual lasers but before that we adjust voltage for FSC to get to some desired values on individual FSC-A and FSC-H histograms. As you said, "we adjust voltage so that height signal is where I want it". My question is how would a person know where my height signal should be on FSC-H histogram? e,g. My Bead population should be at a value of say 100 on FSC-H Histogram, why not 250.
@@junaidrehmani9829 If you are looking at cells, you typically set the forward scatter so that you are able to see your cells and they resolve well and can pull out from debris. You would not set your cells (or beads) so high that they are off scale. You may be working with a heterogenous population in terms of scatter (PBMC, or even beads that have different sizes) and you need to set the FSC and SSC so all cells are able to be seen on your plot. This might mean that lymphocytes are at ~50k on FSC whereas your granulocytes may be around 200k. If you have one population and you're setting it so they are at 250k, you leave yourself little room to see anything that may have a high forward scatter. When I'm looking at rainbow beads, I typically set the FSC to be around 100k for these beads which have a single population by scatter. Then I use this as a reference point for every day when I run QC to make sure there are no differences. Some labs may use 150k or 50k. There's no rule for where to set it. Also, I'd recommend looking at your scatter not by histogram, but by dot plot. Make sure your desired events on are on scale, then from there check your area scaling factor and make any fine tune adjustments to voltage after that. - Kathy
@@OpenFlowCytometry Thank you so much Kathy and Derek. This was useful, appreciate your time and patience. We run Histograms in addition to conventional dot plot.
