Hello Everyone,
To elaborate on a few of Tony's points:
1. Even though the filters in the Facsort and FacsCaliber are changeable,
the mirrors are not. Assuming you remove the filters in front of the PMT's,
the wavelength range is limited to, 488-560nM for FL1, 560-640nM for FL2,
640
to ~900nM for FL3 and FL4. The only way to get around these limitations is
to make both the filters and the mirrors interchangeable. I am curious to
know how many people would be interested in a custom optic set-up and at
what wavelengths.
2. I have found that actual sample pressure at the flow cell to be approx..
4.1 PSI
for Low, and approx.. 4.6 PSI for High, and the square root of the product
of these
two for Medium. I have yet to find a regular use for Medium flow rate.
While it is my experience to see the sample voltage go to 10.23V in soft
standby, it doesn't always go above 10 V in hard standby (Standby on the
mode selector switch). This is while measuring 4.5 PSI at the sheath tank.
This measuring of 4.5 PSI at the sheath tank is the most accurate way to
measure sheath tank air pressure. The 10.23V method measures sheath fluid
pressure at the pressure transducer. If there is a clog in the sheath line
between the sheath tank and the pressure transducer, the voltage reading may
not accurately reflect the actual sheath tank pressure. Knowing both values
is helpful in troubleshooting fluidic/pneumatic problems. If anyone is
interested in
getting a custom pressure gauge for measuring air pressures please contact
me via e-mail.
5. There is a way to adjust the sample pressure on the BD benchtops. This is
for the user who is willing to remove the side cover and turn a small
screwdriver. The sample Pressure Regulator PCB is easily visible on the
bottom right side of the instrument after removing the cover. On this PCB
there are two (Facscan), or three (Facsort, FacsCaliber) small blue
potentiometers that can vary the sample pressure when turned with a jewelers
screw driver. Please e-mail me if you would like me to put a procedure up on
our website for doing this adjustment.
It has been my experience that some flow users like to tinker with their
instruments while other just want a service engineer to fix their problem.
For those who like to tinker, please let me know if there are tools that you
need to do this. I will be happy to make them available.
Regards,
Raymond Lannigan
Tritech Inc.
2014 Renard Ct. Suite I
Annapolis, MD 21401
1-410-266-1522
1-800-886-7004
www.tritechinc.com
-----Original Message-----
From: oiyt <oiyt@tds.net>
To: cyto-inbox
Date: Monday, March 19, 2001 8:25 PM
Subject: RE: PMT Voltage Responses
>
>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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