[FDS] Re: FDS sensitivity
Tom Laue
tml at cisunix.unh.edu
Mon Jun 26 13:14:15 CDT 2006
Hi-
Back from travel last week.
The rms of the intensity measurement is the third column of numbers in
the *.FI# output files.
The default absorbance file output will be 4 columns of numbers, r Int1
Rms1 Int2 Rms2, where the intensities are for the sample (#1) and its
reference (#2) and the rms are the corresponding standard deviations.
The reference can be any sample in the rotor (or none... it defaults to
the 2nd channel in a 2-channel cell).
Tom
Glen Ramsay wrote:
> Greetings FDSers:
>
> Some time ago I was faced with globally fitting circular dichroism, fluorescence, light scattering and absorbance data at various wavelengths. Signal to noise values were magnitudes apart for the different detectors. Fortunately I had control of the instrument software and collected the standard deviation as well as the signal averages for each data point. The deviations were used as the weighting factors and the results came out nicely. An added advantage is that some (not all) stray data points had higher deviations, so bad data was sometimes automatically accounted for.
>
> The FDS software default is to measure 5 revolutions. Tom will have to comment on whether these can be used to calculate a deviation for each data point. When the absorbance detector becomes available via AOS the same might be done with this detector. I think that actual quality measurements will be better than guessing what the weighting factors should be.
>
> Glen
>
>
> -----Original message-----
> From: Tom Laue tml at cisunix.unh.edu
> Date: Wed, 21 Jun 2006 16:34:37 -0400
> To: AUC Fluorescence Detection System List fds at biochem.uthscsa.edu
> Subject: Re: [FDS] Re: FDS sensitivity
>
>
>> Hi-
>> Yes, the FDS has an enormous dynamic range and you can work with
>> concentrations that are measurable by both systems. The ability to do
>> both FDS and absorbance simultaneously will be a big plus.
>> You do have to worry about the linearity of the fluorescence signal with
>> concentration due to: 1) inner filter effects, 2) dye-dye interactions
>> (at higher concentrations) and 3) nonlinearity of the gain setting (e.g.
>> going from 25% to 50% gain more than doubles the signal- this is setting
>> the PMT voltage, the PGA setting 1x, 2x, 4x and 8x is much, much closer
>> to the proper gain multiples).
>> For a given gain setting, and at concentrations where the inner filter
>> effect is small, the FDS signal is linear over a one to two log
>> concentration range. The concentration at which the inner filter effect
>> becomes significant depends on: 1) the objective lens focal point (how
>> deep it goes into the sample), 2) the alignment of the dichroic mirror
>> vis a vis the pin hole and 3) the extinction coefficient of the dye. For
>> our system, we found that the inner filter effect became significant in
>> the high micromolar range. At very high dye concentrations (tens of
>> micromolar), the fluorescence intensity will actually increase with
>> dilution due to the inner filter effect.
>> There was discussion of building a tabletop accessory for the FDS so
>> that samples could be analyzed (e.g. F versus c determined, gains set)
>> without having the system in the vacuum chamber. This would yield a
>> relative measure (similar to what you can get with the calibration
>> centerpiece, only without the trouble of loading the rotor, etc.) of
>> F<->C which would be sufficient for most work.
>> Measuring the QY accurately is bit more work than it may seem. To get
>> the incident intensity accurately, you have to determine the absolute
>> light source intensity. This is done using using a calibrated phosphor
>> (film), calibrated photocell or a calorimeter. The absolute sensitivity
>> of the detector(s) is then measured (e.g. uA/lumen) at various gains and
>> wavelengths. Then you measure the absorbance of a sample (again using a
>> standard sample... rhodamine is the one I recall being used) and the
>> fluorescence intensity. The detector sensitivity is usually fairly
>> stable with time. The optics and light source stability can drift with
>> time with wear and tear on the optics.
>> Tom
>>
>> Borries Demeler wrote:
>>
>>>> Hi all,
>>>>
>>>> Most fluorescent dyes will not show a significant absorbance signal at
>>>> the concentrations used for fluorescence experiments (nM-pM). So, I
>>>> don't think that you will learn much from doing multiple wavelength
>>>>
>>>>
>>> Correct me if I am wrong, but as far as I understand it, the dynamic
>>> range of the FDS is orders of magnitude higher than that of the UV
>>> detector. Hence you should be able to easily get into a concentration
>>> range that can also be measured with the UV detector and still get
>>> linear signal in the FDS. I don't think it matters that the lower end of
>>> the concentration spectrum can only be measured using the FDS system,
>>> as long as you can find a concentration region where both overlap. That's
>>> enough to establish the signal correlation.
>>>
>>>
>>>
>>>> scans. If you want to calibrate the fluorescence emission intensity I
>>>> think that you have to first measure the emission spectrum in the
>>>> presence and absence of protein in a fluorimeter. For the types of
>>>>
>>>>
>>> I actually would like to calibrate the FDS detector, and have a clear
>>> correlation for the various gain settings and what kind of response
>>> I get - is that perfectly linear? If I double either of the two
>>> gain settings, do I double the intensity counts?
>>>
>>>
>>>
>>>> probes being discussed, that are useful because they are extremely
>>>> environmentally sensitive, each will likely have a unique macromolecule
>>>> dependant signature. One protein may change the probes emission
>>>> intensity while another may shift the emission max. I think this is
>>>> going to have to be worked out on the bench and then load the same
>>>> samples into the AUC to calibrate the emission intensities in the FDS.
>>>>
>>>>
>>> What does a benchtop fluorimeter tell you that you cannot get from
>>> the AUC FDS? I don't have much feel for this, but I agree with Rachel
>>> and Tom that one would probably have to recalibrate each particular
>>> protein/dye combination to be absolutely certain. But how do you get
>>> absolute concentration? I think you can get closest to it with the known
>>> E280, which requires UV measurement, right? The basic problem with the
>>> fluorescence is that you never know how much quenching exists, and what
>>> your labeling efficiency is. Making a correlation with E280 should solve
>>> this, so you can possibly get a molar fluorescence emission constant
>>> for each protein/dye combination.
>>>
>>> How reliable are the Molecular Probes assays for labeling efficiency?
>>>
>>> -Borries
>>> _______________________________________________
>>> FDS mailing list
>>> FDS at biochem.uthscsa.edu
>>> http://biochem.uthscsa.edu/mailman/listinfo/fds
>>>
>>>
>>>
>> --
>> Department of Biochemistry and Molecular Biology
>> University of New Hampshire
>> Durham, NH 03824-3544
>> Phone: 603-862-2459
>> FAX: 603-862-0031
>> E-mail: Tom.Laue at unh.edu
>> www.bitc.unh.edu
>> www.camis.unh.edu
>> _______________________________________________
>> FDS mailing list
>> FDS at biochem.uthscsa.edu
>> http://biochem.uthscsa.edu/mailman/listinfo/fds
>>
>>
>
> Glen Ramsay
> Chief Scientist
> Aviv Biomedical, Inc.
> Lakewood, NJ 08701
> USA
> (732) 370-1300
> (732) 370-1303 FAX
> glen at avivbiomedical.com
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>
>
--
Department of Biochemistry and Molecular Biology
University of New Hampshire
Durham, NH 03824-3544
Phone: 603-862-2459
FAX: 603-862-0031
E-mail: Tom.Laue at unh.edu
www.bitc.unh.edu
www.camis.unh.edu
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