>We are going to purchase a flow cytometer with sorting capabilities >for use in aquatic microbial ecology. Our main task is to measure >bacteria from natural samples (DAPI, SYTO13, CY3,...). > >Unfortunately these organisms are very small (down to 0.2 microns) >and they do not live in the water all by themselves - there will >be lots of algae, flagellates, detritus etc. > >So we need to have good sorting capabilities to check what we are >actually measuring in the microscope, but the big problem (I feel) >is the instrument's sensitivity. > >I would like to ask experienced users out there: > >1- Which features, do you think, are of particular importance to our >application. (PMT for forward scatter is certainly one). Other >important features? > >2- What do you think of jet in air vs. cuvette sensing. Flat surfaces >should enhance scatter measurement, but seemingly only Coulter is >ready to provide a sorting(!) cuvette. Other important features? > Light source noise is at least as important as jet in air vs. cuvette where forward scatter sensitivity is concerned. Some older instruments with jet in air sensing could detect smaller particles than could systems with cuvettes, because the older instruments use water-cooled argon lasers, which have low noise (0.2% RMS or less) because they use linear power supplies, while the newer ones, with more efficient light collection, use air-cooled argon lasers, which have switching power supp, lies and are therefore noisier (1% RMS). According to measurements I have made on the bench, the improvement in forward scatter sensitivity of a cuvette over a jet in air is only about twofold; however, cuvettes provide much better sensitivity (at least tens of times) than jets in air for orthogonal scatter measurements. You will actually improve sensitivity if you use a low noise diode or frequency doubled YAG laser; these typically have less than 0.1% RMS noise. If Coulter is willing to provide a PMT for forward scatter measurement on the Elite, it would probably be more sensitive than the Vantage or the MoFlo as far as scatter measurements go. The Elite also has an apparent advantage for small particle sorting in that it lets you set simultaneous trigger thresholds on more than one parameter; I have been using double thresholds (scatter and fluorescence) in the Cytomutts I build for bacterial analysis for many years, and find that this is advantageous in discriminating small signals from bacteria. As far as I know, neither the MoFlo nor the Vantage offers more than a single parameter trigger threshold. Whichever system you choose, bear in mind that having a PMT instead of a diode as a forward scatter detector won't help at all if your scatter noise is high. In the interest of avoiding a flame war on this issue, I should stress that: 1) I haven't used any of the commercial sorters myself. I build my own apparatus, but am familiar with what's out there. 2) I don't get paid by Coulter; in fact, I have consulted for at least two of their competitors and currently consult for one of them. 3) The practical approach to your problem is to ask the candidate manufacturers to demonstrate the performance of one of their instruments on your samples and pick the one which works best (always good advice to people contemplating purchase of a cytometer) With jet sorting, we know there are aerosol generation problems; there can also be vaporware problems. >3- We need three excitation wavelengths (UV, 488, 514 or higher) for >various experiments, not all three at the same time but all possible >combinations of two of them. This will involve a lot of laser >adjustment. How complicated is it on different instruments e.g. to >shift a laser beam from second to first sensing position? > Apparently harder than it should be. -Howard
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