[FDS] Re: FDS sensitivity
Borries Demeler
demeler at biochem.uthscsa.edu
Wed Jun 21 16:44:08 CDT 2006
>
> Hi FDSers,
> An "extinction coefficient" (mass conc scale) can be
> specified for each species in a multi-species system either as a
> weight average, based on the stoichiometry, or separately in case of
> hyper- or hypo-chromicity or in this case changes in quantum yield on
> association. A special run would have to be done with two optical
> systems such as interference and fluorescence to estimate the
> "extinction coefficient" for the species in question. Right now there
> is no provision for a concentration dependent extinction coefficient
> for any given species; but I am sure that could be added without too
> much difficulty (he said confidently without having spoken to the
> programmer).
I know what you mean :-) UltraScan also doesn't currently have this
provision, and I guess right now there are actually two issues: 1. first
we need to determine what this function looks like with respect to
instrument and settings (different gain settings, dyes, buffers, etc),
and how much variability there is across instruments, etc... and 2)
what the dynamic effect is due to hyper- or hypo-chromicity at different
concentrations. Once it is known, it should be fairly straightforward
to include it in the fitting functions.
What I have taken away from this discussion is that this function
may have to be determined individually for each new experiment. My guess
is we will come up with some calibration routine that will generate a
polynomial that can then be included into the program to dynamically
calculate a molar emission coefficient dependent on gain settings and
other factors.
Thanks, -Borries
>
> Walter
>
> >From: Borries Demeler <demeler at biochem.uthscsa.edu>
> >Subject: Re: [FDS] Re: FDS sensitivity
> >To: Tom.Laue at unh.edu
> >
> >Walter - Tom suggested to add you to our FDS list (discussion
> >of the fluorescence optical detector for the AUC). Some questions
> >have come up as to what Sedanal can do to deal with variable
> >quantum yield - perhaps you can shed some more light on this.
> >Jack has already done a good job explaining most of it.
> >
> >You can find all the subscription options at, incl. an archive
> >of what has already transpired at:
> >
> >http://www.biochem.uthscsa.edu/mailman/listinfo/fds
> >
> >Regards, -Borries
> > >
> > > Hi-
> > > Walter's software will fit for the "extinction coefficient." Of course,
> > > you cannot do that and fit for concentration simultaneously. I'll defer
> > > any further discussion to Jack. We may want to add Walter
> > > (STAFFORD at bbri.org) to this discussion board, too.
> > > There are two inner filter effects. The primary effect is a consequence
> > > of absorbance of the excitation beam between the point of incidence on
> > > the sample and the point where emission detection occurs. It is easier
> > > to visualize this if you think of a standard 1x1 cm cuvette. The
> > > emission optics are focused on a relatively small volume at the center
> > > of the cuvette. Some of the excitation beam intensity is absorbed as it
> > > traverses the distance from the entrance wall to the center. Hence, as
> > > the concentration of dye goes up, a greater amount of the beam intensity
> > > will be absorbed before exciting the dye at the center of the cuvette.
> > > The secondary effect is that the emitted light also can be absorbed,
> > > thus the intensity, normalized to concentration, will go down as the
> > > concentration goes up. The FDS uses front face excitation and emission.
> > > The distance traveled by the excitation and emission beams is ~ 1 mm (or
> > > less) below the window, minimizing both the primary and secondary inner
> > > filter effects (but not eliminating them).
> > > Yes- using the absorbance + FDS will be very useful. I am working on the
> > > rapid scan absorbance, and plan to have the next major release of AOS
> > > software take absorbance data from the XLA to help with this sort of
> > > calibration.
> > > By the way, it will be best to fit for the quantity of fluorescence
> > > intensity that actually sediments. Using the intensity directly from the
> > > FDS would include the background fluorescence from the windows and solvent.
> > >
> > >
> > > Borries Demeler wrote:
> > > > Tom,
> > > >
> > > > thanks very much for this very helpful explanation of all the factors
> > > > that affect linearity - this is important to know for any calibration
> > > > experiments we might want to do, and software calibration modules
> > > > that we want to design for UltraScan. So are you saying that Walter's
> > > > software can do all of these calibrations already & reliably? Or were
> > > > you refering to the ability to enter a molar emission constant?
> > > > The latter of course is available in SedAnal, UltraScan, Sedfit, etc,
> > > > the former is what my question was actually about - sorry for not being
> > > > clearer.
> > > >
> > > > I also would like to know what this inner filter effect is - I never
> > > > heard of this term. Are you talking about the 500 nm filter? What effect
> > > > has it?
> > > >
> > > > With respect the high micromolar concentration where this seems to
> > > > be important, I don't think we need to go that high, at least not for
> > > > somewhat larger molecules, because the absorbance optics are sensitive
> > > > enough to measure in the nanomolar range when you can measure at lower
> > > > wavelength (appropriate buffer required - which brings up another issue -
> > > > what effect has the buffer on intensity). So from your answer I gather
> > > > that I probably don't have to worry about this too much. Mostly the
> > > > interesting spectrum of proteins is below 235 nm where the extinction of
> > > > protein is high enough to measure most proteins in the nanomolar range,
> > > > so the wavelength measurement approach I proposed earlier should still
> > > > be a sufficiently accurate way to measure absolute concentration in
> > > > comparison of FDS to UV.
> > > >
> > > >
> > > >> 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.
> > > >>
> > > >
> > > > That's good to know - because other that this approach I am not sure
> > > > how one could get a good handle on this problem.
> > > >
> > > >
> > > >> 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).
> > > >>
> > > >
> > > > Would you expect each instrument to have a different calibration curve,
> > > > or would one calibration be appropriate for all detectors? (this
> > > > affects the design of the planned UltraScan modules)
> > > >
> > > >
> > > >> 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.
> > > >>
> > > >
> > > > Hmmm, you obviously can't do anything about (3), but are there
> >ways inside
> > > > the AOS where we can set (1) and (2)?
> > > >
> > > >
> > > >> 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.
> > > >>
> > > >
> > > > I don't think I need to know the absolute QY or even the labeling
> > > > efficiency. As long as I know how many counts I get from a particular
> > > > sample per mol at a given instrument setting I should be OK. I would
> > > > be satisfied to know that with the same accuracy with which I can
> > > > determine absolute concentration in a UV spectrophotometer. That seems
> > > > like work enough :-(
> > > >
> > > > Thanks again to everyone for this very helpful discussion!
> > > >
> > > > -Borries
> > > >
> > > >
> > >
> > > --
> > > 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
> > >
> --
> -----
> Walter Stafford
> mailto:stafford at bbri.org
> direct dial: 617-658-7808
> receptionist: 617-658-7700
> -----
> Boston Biomedical Research Institute... Today's Research for Tomorrow's Health.
> Please visit us at www.bbri.org
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