Hello Flow'ers, A couple of points on the BD benchtops. 1. Unlike the FACsort & FACScalibur, the FACScan's filters are fixed and not easily replaced. 2. Sample voltage is probably one of the more critical readouts because it tells you if your sheath tank is properly pressurized. A particular voltage setting is meaningless because they vary from one instrument to the next. What is important is that a particular instrument have basically the same voltage readings from one day to the next. The voltage will vary during the regulation of sample pressure, so it will move around a little, but not much. It is a good thing to record this voltage in Hi and Lo flow rate, also in Standby. When you move the stage under a sample on the FACS benchtops to the side position (soft standby), your sample voltage should go to 10.23v very quickly. This tells you that you have enough pressure in the sheath tank. If you don't get 10.23v very quickly, then you have a pressure problem somewhere. 3. Actual sample pressures on the FACS benchtops are 4.6 psi in LO and 5.0 psi in HI. (assuming your instrument is properly setup by service person) The sheath tank sits at 4.5 psi, so Lo flow rate is .1 psi over tank pressure and HI flow rate is .5 psi over tank pressure. 4. Specification for flow rates are as follows; 12ul (+/-3) per minute in Lo and 60ul (+/-7) per minute in Hi. 5. Users have no control over the amount of pressure placed on the sample in the FACS benchtops. As soon as a sample is placed on the SIP (sample introduction probe), a burst of about 9 psi is utilized to initiate flow. Sample pressure is then regulated to HI or LO depending on your setting. It is this bursting phenomenon that requires one to wait for 20 seconds after placing the sample on the SIP before clicking aquire, thereby allowing the flow rate to stabilize and become uniform. Happy flowing, Tony Leger Automation Lab Technology 360-983-8690 oiyt@tds.net -----Original Message----- From: Marcus Reckermann [SMTP:recker@ftz-west.uni-kiel.de] Sent: Friday, March 16, 2001 1:51 AM To: Cytometry Mailing List Subject: PMT Voltage Responses Taking up Bunny?s suggestion, I will share with you the answers I received to my "PMT voltage" question (in chronological order). The question was whether the PMT units (mistakenly also threshold) are actually VOLTS or arbitrary units. Thanks to everybody who responded. - Threshold is not a voltage setting, it is a channel number and depends on the sensitivity, alignment, and gain setting of your particular instrument. In our FACScan, we had a common measurement with the FSC gain at 1.75 and threshold 120; after the instrument was serviced and optics cleaned and realigned, the same result was obtained with FSC gain of 1.0 instead of 1.75 on the same instrument. I prefer a description such as: "threshold at 120 in FSC, to include small lymphoid cells and to ignore most red cells". A PMT setting of "FL1 640V, Gain 1" is useful, but I also need to know what optical filters are in that channel. For instance, in a FACScan the FL1 filter is usually 530/30, but this may have been changed for this particular experiment. - Just be aware that voltage and gain settings as well as thresholds are not directly transferable between instruments, neither are threshold values. There are variations in PMT characteristics and even more important spectral characteristics of the optical arrangement. In the Elite the threshold is expressed as a channel number between 0 and 1023, indicating that the threshold is dependent on the voltage and gain settings of the PMT - I thought Threshold was in "Channel Numbers"--at least for the BD instruments. - If your data acquisition software writes list mode files that comply with the FCS specification then you will find a series of keywords and their values that take the form $PnV and $PnG where n is the parameter number and V and G stand for PMT voltage and gain, respectively. With log amplification, the gain will typically be set to 1 and the voltage applied to that PMT will determine its sensitivity -- the higher the voltage the greater the sensitivity. The second type of description that you mentioned, i.e., something like FL1 640V, gain 1, is an appropriate form. The threshold value is something different and refers to the signal level in some parameter that must be satisfied in order to trigger the electronics to capture the signals from that event. The threshold value is commonly set on forward scatter which is also most commonly set to acquire in linear mode thereby allowing various gain settings other than 1. Therefore, to describe the instrument settings, one should specify the voltages applied to the PMT's ($PnV values), the gains ($PnG values), log or linear amplification for each parameter and the parameter used for triggering or thresholding. Coulter and BD also permit one to set a "listgate" or "live gate", respectively, that will limit the data actually saved to the list mode file. These gates are commonly set on the projection of forward vs. side scatter or on fluorescence from anti-CD45 and side scatter. If such a collection gate is used, it too should be specified, at least the combination of parameters that was used. - As far as I know, the PMT settings on BD cytometers are reported in volts, so you could say "the FL1 PMT was set to 500V" or "the FL1 PMT voltage was 500". Threshold is NOT reported in volts. You may be able to use a formula to convert the threshold setting into a voltage, but I think the setting is more universal than voltage for reporting a threshold. BD usually uses 8 volts full-scale, so I would assume that setting the threshold to 1024 would be 8V (i.e., voltage = 8 * 1024 / setting). Since this is not common knowledge, reporting threshold as a voltage would be meaningless for most. (Actually, I think on linear scales the 8V is set to channel 1000 and not 1024 because before the FACS Vantage the BD sorters used only 3 digits [i.e., 000 to 999] to set manual sort gates.) Also, since the settings on one cytometer don't really mean anything on another cytometer it doesn't make sense to try to convert settings into some sort of "real" unit. Simply recording the settings as you see them on the cytometer is usually the best. "The threshold was set to 20." (Notice the lack of units.) One thing that annoys me is the "Sample Pressure" reading on BD benchtop cytometers (FACScan, etc.). It is displayed as "Volts", because the cytometer uses an electronic device that changes voltage corresponding to pressure changes. (The same device can be used in digital tire pressure gauges.) BD decided not to calibrate these devices, so instead of reporting an actual pressure they report the voltage of the electronic device. It makes training difficult when you have to say things like "try to keep the sample pressure around 6.15 volts". - Yes, the PMT voltage is volts applied to the PMT. As you probably know, the sensitivity of the PMT is proportional to the voltage applied. BD instruments have a maximum PMT voltage of 1000, I believe some other brands go up to 1500 or 2000 volts; most common PMT tubes have a maximum rating of about 1500 volts. I don't know the accuracy of the voltage reading, but I don't think it's important; I use published PMT volts as a starting guide only, because PMTs vary widely in sensitivity. - These are actual volt units. If you set 700volts for a PMT on a Vantage and check the cable going to the PMT, it will show -700 +/- 5V if properly calibrated. - it's volts. - Documenting the conditions under which an experiment is run is a always a good idea, but it is necessary to understand its use. Primarily, especially in conjunction with a well understood standards such a bead, the settings can help someone who knows the flow instruments at a particular site determine whether problems with experimental data are due to instrument problems or sample problems. Secondarily, these settings will allow an instrument user to recreate the conditions under which previous data were run. Because of alignment changes between runs, the instrument usually will not give the same results, but will be a good starting place for setup. And thirdly, knowledgeable users at other sites may also use these settings as starting points for their own work, but the variability from instrument to instrument is too high to do this successfully without some other portable standard, such as a bead. The actual instrument values set are "real" electronically, but may not have much meaning to a researcher. There is too much detail to include here - I would recommend reading one of the many good books on flow cytometry that describe the instrumentation. Briefly, the threshold levels may be a percent of full scale, a channel number, or turns on a knob. PMT voltages determine the amplification of the photomultiplier tube that is converting photons to electrons, which is proportional to the voltage raised to some power, and varies from PMT to PMT. The gain describes what kind of electronic amplification is following the PMT, either linear with a certain gain factor, or logarithmic. Thus, your example of "FL1 640V gain1" say that measurment channel is in linear gain 1 with its PMT set to 640V. - The PMT volt number, is the measurement of the actual voltage applied to the PMT. Depending upon the configuration of the PMT, this may be a positive or a negative voltage. If the voltage is applied to the photocathode (e.g. BD Benchtops) then it will be negative (with respect to the anode), repelling the photons converted to electrons, to the anode (output). If the voltage is applied to the anode, it will be positive (with respect to the photocathode) attracting the electrons to the output. The operating range of most flow cytometery PMT's is in the 300 to 1000V range. ___________________________________________________________ Marcus Reckermann Forschungs- und Technologiezentrum Westkuste der Universitat Kiel (FTZ) (Research and Technology Centre Westcoast of Kiel University) Hafentoern D-25761 Buesum Tel. +49 (0)4834-604-204 or -261 Fax. +49 (0)4834-604-299 E-Mail: recker@ftz-west.uni-kiel.de FTZ Web Site: www.uni-kiel.de/ftzwest/ FTZ Flow Cytometry: www.uni-kiel.de/ftzwest/flow/flowhome.htm ________________________________________________ Keep your computer busy with www.setiathome.ssl.berkeley.edu
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