Claude, Some weeks ago I did a preliminary test of PharRed on our FACStar Plus using the same filters, and DID see a signal with useful +/- separation. The PMT in this case is Hamamatsu R1477-04, I don't know how that compares with the 957-08. (the search function on the Hamamatsu website won't work for me..) According to the website, the R1477 is a high sensitivity variant of the R928, and the R3896 is an even higher sensitivity variant of the R1477. ;~} From your description below, the PharRed signal is missing when both dichroic and LP filters are in, also with the dichroic removed; what happens with just the dichroic in, and the 740EFLP removed? I suggest you compare the PMTs you have, and select the one with the best APC sensitivity for the PharRed channel. Most of us are not aware that there is a wide variation in sensitivity between tubes of the same model! (Comparing the three R1477 tubes supplied with our instrument, there is about 150 volts difference between the best and worst to achieve the same signal level!) It may also be worth checking your filters in a scanning spectrophotometer to verify they are as labelled. Flow Well, Joseph. At 08:06 24/5/2001, Claude Cantin wrote: >Hi All >I have been unable to detect a PharRed signal from samples I have been >given, and don't know if the problem is instrument or labeling error. > >MoFlo set up is as follows: >Hammatsu 957-08 PMT all around >Omega Optical Filter Set XF501 (consists of 710 DRLP, 670DF40, and 740EFLP) >Melles Griot 35mW HeNe > >Red laser alignment seems fine: "Molecular Probes AlignPlus beads for >633" are almost off scale if no ND filter, at 399V, linear gain 1. BD's >APC like Calibrites show excellent separation + / - ( >2 logs) >580V. Whether I measure APC in FL5 (remove 710LP dichroic, use 670/40BP) >or FL6 (710LP plus 670BP), I obtain good APC signal. But no PharRed with >the 710LP dichroic / 740EFLP in FL5, and no PharRed signal even if I >remove the 710LP dichroic. > >BD Pharmingen Technical data sheet recommends a Hammatsu R3896 PMT for >improved red sensitivity. Is the Hammatsu 957-08 deficient in the >740 range? > >If not, what's up? > >Thanks, >Claude
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