The FACS machine at our core facility has a 670 LP (long pass) filter to detect PerCP and PerCP-Cy5.5. Yet, unlike the band pass (BP) filters these seem to accept a large chunk of the emission spectra (from 670 nm onwards).
Looking at the attached spectral schematic it seems this layout can interfere with detection of PE-Cy7 with the 780/60 BP filter. Is this true? How does this affect bleeding between these fluorochromes? And most importantly: how much hair does an average human being loose whilst trying to get to grips with FACS?
Hi Pedro,
before PerCP / PerCP-Cy5.5 detector should still be BP filter but it works even without but you have to be more careful on the correct compensations. The wavelength range of light incident on PerCP/PerCP-Cy5.5 detector is in this case determined by LP mirror for PE-Cy7 and LP mirror for PerCP/PerCP-Cy5.5
Are you using FACSCanto? Those machines often have such configuration. Anyway, if on a typical BD digital instrument (Canto/LSRII/Fortessa/Aria/etc), usually a first mirror/filter that receives the light from the blue laser is 700-something mirror (reflecting everything below 700-smth) stacked with a 780/60 filter for PE-Cy7. The reflected light goes to the next PMT that has 670LP filter, but the higher end of the spectrum is cut off already, so no problem with PE-Cy7.
If you are using a FACSCalibur etc with FL3 channel equipped with 670LP, then all wavelenghts longer than 670 nm would reach the PMT, and there will be no way to distinguish PE-Cy7 and PerCP5.5 fluorescence. If that's the case, don't use them simultaneously.
Hi, you are right, with a 670LP filter you would detect both signals - the PerCP/PerCP-Cy5.5. and the PE-Cy7. You also mention that there is a filter for the PE-Cy7 - a 780/60 filter. So I would assume that the layout of the system you have, meaning the order and arrangment of the filters in the light path, makes sure that the PE-Cy7 signal is not registred by the LP filter, e.g. by placing the 780/60 before the LP filter. You will seem some of the PerCP in this detector (long tail to the right) but the PE-Cy7 should not be in the PerCP one.
Anna and Christa: I checked and you are absolutely right. There is a 735LP mirror in position A (where the PE-Cy7 filter is) meaning little PE-Cy7 signal reaches the 670LP :)
Thanks people!!!
Hi Pedro,
The setup can work because the usual order of detection is first Cy7 and then PerCP-Cy5.5. Meaning the 780/60 Cy7 fluorescence is taken away before the laser hits the LP670 for the PerCP-Cy5.5. However, as nothing is perfect I think it is prone for bleed through. You should be careful with the compensation and use good controls. Or, you change the filters. I saw you're in CMB. Which machine are you using? The FACS Aria has a LP735 780/60 for Cy7 and a LP655 LP670 for PerCP-Cy5.5, but there is also a 710/50 filter which you could use additionally. Maybe we could even have a look together.
Concerning your last question, I still have lots of hair ;)
Dear Pedro,
In general LP670nm is for Cy5.5 and LP780 - for Cy 7, but better 630nm laser should be activated first. 488 excitation is worse for Far Red, though you can see signal with He-Ne and without filters at all - only its specificity from autofluorescecence will be several times lower.
Also in general fluorescent and confocal microscopy is better for handling to understand what you are doing. It has the advantage of cellular visualisation and 3D imaging. But in reality all microscopies are slower than Flow methods, Flow cytometry has the same functional measuring but it is much faster and has statistical-significant value.
My hair are grey but still many and even longer than Erik's in spite of many years with confocal and Flow...
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