Officially the nano world starts below the 100nm size, so at 200nm I normally refer to sub-micron particles. With forward scatter being one of the most variable signals between instruments I would also urge everyone to use side scatter triggering for smaller particles on most instruments. I actually routinely measure 190nm latex particles on the Elite for surface loading. For our workshop in Budapest Roy Bongaerts and myself were pleased that when trying out a couple of instruments on the exhibition floor to find that all the instruments we could test detected the 190nm particles on side scatter triggering. Depending on instrument cleanliness they had various amounts of interference / space below the 190 nm beads. For instruments that use diodes for side scatter like the EPICS-XL you might have to shift the side scatter to the first PMT to reduce electronic noise. Colloidal gold and silver can have more scatter signal than latex and their relative positions are wavelength dependent. Depending on the dye loading relative positions can also vary with for latex (see the light blue 632nm side scatter data from my Elite). With the Apogee A40 and my Elite I have analysed 60nm gold as the smallest particle. Most important is sheath and flow cell cleanliness. If you still have the Elite running were I added the disposable in line sheath filters and the alignment is fine your Elite should be able to do the job without an ultracentrifuge. Biggest problems seem to be air bubbles you have to get out of the system and unwanted micro particles / immune-complexes, protein precipitates etc. that come from your sample side. For improved sensitivity you might want to use a single colour detection mode removing the band passes but trying to avoid the Raman scattering. When analysing small particles it is essential to display all your signals against log side scatter as your fluorescence signals are also particle size dependent. Gerhard Nebe-von-Caron Research Scientist and Biomedical Engineer SPDspark Swiss Precision Diagnostics Priory Business Park Bedford, MK44 3UP, UK Mob+44(0)7792-116609 Tel+44(0)1234-835474 Fax+44(0)1234-835006 mailto:g.nebe-von-caron@spdspark.com > > On Jun 26, 2008, at 10:17 AM, Timothy Overton wrote: > >> Dear All, >> >> I just has a request from someone working in my department which I >> think is theoretically possible, but I wondered if anyone has tried >> anything like it. They have nanoparticles coated in biotin, to which >> are attached Streptavidin conjugated to Alexa 568. The size of the >> particles is around 200 nm across. They asked whether flow cytometry >> could be used to determine the proportion of particles with >> Strep-Alexa attached, and then if they could be sorted. Has anyone >> done anything like this? >> >> Thanks, >> >> Tim >> >> >> ***************************************** >> Dr Tim Overton >> Lecturer in Biochemical Engineering >> School of Chemical Engineering >> The University of Birmingham >> Birmingham B15 2TT >> t: +44 (0) 121 414 5306 >> e: t.w.overton@bham.ac.uk <mailto:t.w.overton@bham.ac.uk> A flow cytometer with a sensitive forward scatter channel, such as the Apogee or the Cytopeia/BD inFlux, should be able to detect and trigger on 200 nm particles, and many instruments should be able to use a side scatter signal as a trigger. Whether this will work with the nanoparticles in question depends on their size distribution (the narrower the better) and how much other stuff in the size range is in the sample and sheath. As to fluorescence signals, people have sorted microvesicles in the size range mentioned, but they were probably looking for at least a few thousand fluorescent molecules/particle. I agree with Mario that a few hundred molecules might be detectable in a more-or-less conventional sorter under near-ideal conditions. You could probably get down to single cell fluorescence in a slow flow instrument, but the millisecond range dwell time would limit your analysis (and sorting) to a few dozen particles/second. -Howard This attachment - 'image002.gif' - 8.76 KBytes - can be viewed at http://www.cyto.purdue.edu/MD-parts/4fe914dcd58cb66f547ad329e21b0f8bfbb59e6d.gifReceived on Tue Jul 1 16:38:00 2008
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