From: Dave Coder (dave@nucleus.immunol.washington.edu)
Date: Thu Feb 03 1994 - 11:57:12 EST
Begin forwarded message: Date: 02 Feb 94 07:59:01 EST From: Marc.Langweiler@Dartmouth.EDU (Marc Langweiler) Subject: Sci Fi (ve) mystery To: cytometry@flowcyt.cyto.purdue.edu It came to pass that Curley found himself in northern New England, trying to do 3-color surface marker phenotyping. It so happens that he was using a cytometer from a company that Moe had gone to work for, based in south Florida. Amazingly, when he went looking for third color conjugates, he winds up talking to Larry, who had gone to work for a company in northern California specializing in PE-Cy5 conjugated antibodies! Curley bought several conjugates, and found that some of them, including CD3 and CD45 were bright as can be. He couldn't figure out why conjugates of CD33, CD10 and CD19 were so dim. He called Larry, who told him that his problem was probably due to a bum PMT in FL3, after making sure that he was using an appropriate filter, a 645 LPA. In fact, said Larry, I know of several other instances where users had to struggle with their cytometer manufacturers until they got a good PMT. One guy had to try 5 of them before he found one that worked! So Curley calls Moe, and tells him this, and Moe says bull#$%$! The problem is the conjugate. It's going to be dimmer than what you're used to seeing. For chrissakes, just look in their catalog...You excite the PE and it emits at a peak of 575. You now ask it to excite the Cy5, which is excited at 625-650. There ain't no way that you going to hit Cy5 with anything that's going to excite it much. So, bag that junk and use something that we make. By now Curley's really flustered. You know, starts that banging himself on the side of his head. But, since he's gotten a little smarter since his last gig, and has discovered cyberspace, he decides to ask the rest of the cytometry community for help. ........ The short answer is: These fluorochrome conjugates work great with 488nm excitation. (See below for details.) Detectors and filters are likely candidates. Moe should go back to throwing cream pies.... The long pass filter sounds fine, but what are the other dichroics? Also, the PMT could affect detection. Either it's bad, but more likely, is the location and/or alignment. That is, if it sits third in line after a series of dichroics each of which picks out a waveband, the light intensity drops by the time you get to PMT3. (For example, if each filter is 90% efficient in passing red light and there are 4 filters then, you have only about 66% of the light after the last filter.) Alignment of the PMT to properly illuminate the photocathode could also be a problem. Flurochrome conjugates work by resonance transfer not by fluorescence emission and re-absorption; PE-Cy5 is a stellar synthetic example. For energy transfer to work, you need a pair of fluorochromes that have absorbance and emission spectra that overlap, are located about 10A apart, and have the "proper" spatial orientation. Light energy absorbed by the short wavelength absorbing fluorochrome is not emitted as fluorescence, rather it can be transferred directly to the longer wavelength absorbing fluorochrome. (With PE-Cy5 conjugates there is some fluorescence emission of PE--coupling is not perfect. Carl Stewart has shown a nice overview of this.) PE-Cy5 pair is very effectively excited by 488nm light with good emission around 670nm. It works very well with air-cooled argon lasers. Conjugates are available from (by alphabetical order) Amac, CalTag, GenTrak, Gibco, and Pharmingen (among others). This coupling is a good example of how photosynthesis works. Chlorophyll absorbs in the blue and red ends of the spectrum; light in the green to near red parts of the spectrum is used very inefficiently. Accessory pigments allow the absorption of light in this region, and thus a selective advantage for those organisms that contain them. Accessory pigments in cyanobacteria, red algae, and cryptomonads include the phycobiliproteins: phycocyanin (PC) and allophycocyanin (APC); many also include phycoerythrin (PE). Arranged in order of absorption, they are PE-->PC-->APC-->chlorophyll. (PE can transfer directly to APC as well.) These fluorochromes allow the organism that contains them to harvest light in the green to red parts of the spectrum and transfer it directly to chlorophyll. In seawater, the deepest growing algae are the red algae since they are able to use green light which is the predominant wavelength at depths (blue light is scattered by particulates, red light is absorbed by water).
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