Hello Jose, I have to disagree with the suggestion of aligning the FACScan yourself. Moving the steering plate will change lots more than light scatter noise- it will also have an effect on fluorescence channels. I would suggest that you ask your service engineer to align the light scatter channels using log amplification, and minimize the noise that way. I believe light scatter is typically aligned by the service engineer using linear amplification. The only adjustment needed would be to the forward scatter obscuration bar. The fact that you're seeing similar PMT voltage requirements for your unstained material on the FACScalibur means that it is probably true- the intensity is low. Peter > ---------- > From: > Ann.Atzberger@EMBL-Heidelberg.de[SMTP:Ann.Atzberger@EMBL-Heidelberg.de > ] > Sent: Thursday, December 17, 1998 9:05 AM > To: Cytometry Mailing List > Subject: Re: FACscan Problems > > > >Dear all, I would like to get some advice on whether my > >FACscan needs further fine tuning. I recently called the BD > >engineer to come and recheck the alignment and sensitivity > >of our machine. He says there is no problem with it but I am > >not satisfied and would like to get a second opinion. Our > >main application is flowcytometry of red blood cells and I > >see two problems: > > > >1. When we acquire using Log amplification for FSC and SSC > >we see a lot of background and have to increase the > >threshold on FSC to about 300. I understand that some > >background using these settings is normal to some extent but > >there was a time when my machine had no background at all > >using the same settings. This background is not due to > >impurities in our buffers because it is present even after > >filtering through 0.22 um filters. Also, it became worse > >after the engineer adjusted the machine. > > > >2. The other problem is that the PMT gain that I have to use > >to have unstained cells in the first decade of FL1 seems to > >be too high, around 700. We found the same when we used the > >FACscalibur across the hallway. The engineer said that he > >had checked everything. > > > >I would appreciate any suggestions as to how to deal with > >the above problems. > > > >-- > >Jose A. Stoute, MD > >Unit 64109, Box 401 > >USAMRU-Kenya > >APO AE 09831-4109 > >e-mail:stoutej@net2000ke.com > > stoutej@workmail.com > >Nairobi Tel 254-2-729303, Fax 254-2-714592 > >Kisumu Tel 254-35-22942/22903, Fax 254-35-22903 > > > > > >Content-Type: text/x-vcard; charset=us-ascii; name="vcard.vcf" > >Content-Transfer-Encoding: 7bit > >Content-Description: Card for Jose A. Stoute > >Content-Disposition: attachment; filename="vcard.vcf" > > > >Attachment converted: MACINTOSH:vcard.vcf 2 (TEXT/ttxt) (0000A111) > > > Hallo Jose, > > some tips that might help > > 1. Try aligning it yourself: if you look at the optical configuration > for > the FSC;there's a lens mounted on a square holder, the top part of the > holder is round with a hole in it. If you put a screwdriver (or > anything > that will fit) in the hole > you can adjust it by moving it carefully, at the same time watch the > FSC,SSC and FL signals. You need to run a sample and deactivate the > laser > shutter. Don't try unscrewing anything though, it can be difficult > getting > things back the way they were. > If your machine still has a warrantee, don't do this! > > 2. Your flow cuvette needs cleaning, the company has a special cleaner > for > this. If the machine has been in use for several years it should be > done. > Makes a huge difference. > > 3.Most operators are inclined to put the threshold on SSC when working > with > LOG amplification. > > 4.Unstained red cells may well have such a high PMT setting. Being > non-nucleated they might have less autofluorescence. I'm assuming we > are > talking about peripheral blood cells, non-chicken, non-fish, > non-fixed. > > 5. Is your laser working properly. How old is it? > > > good luck > > Ann > > > >
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