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> 1. Quantum efficiency is a measure of energy transfer.
"Energy transfer" in fluorescence spectroscopy refers to the
non-radiative transfer that occurs between coupled dyes. Quantum
efficiency is simply the
#photons out divided by the #photons in. I just asked four people
who have been in fluorescence for decades (I've been doing it for
15); none of them have heard of "energy transfer" being used in the
context of an absoprtion/re-emission event.
I did mis-state the important features of PE and APC, in that not
only doe they have high quantum efficiency but also a high absorption
coefficient as well
(compared to FITC). The product of these two numbers is related to
the brightness.
> 2. The FACScalibur can run CY5 without using PE as a
> tandem.
Sure, but I wasn't saying the FACScalibur doesn't have advantages
over the XL.
I was just addressing your point that the XL can only do one
highly-sensitive fluor (PE) vs the FACScalibur (PE + APC). i.e.,
the XL can do two (PE +
Cy5PE).
> 3.PE/CY5 conjugates have two major problems, one, every
> conjugate has to be individually compensated, unlike
> PerCP, and two, CY5 is nonspecifically taken up by cells
> of myeloid origin. (APC requires no compensation when
> used with a second laser)
The myeloid problem is a disadvantage, but only plays a role if you
are studying low-density antigens on monocytes/macrophages. As for
the compensation, this is minimized if you get your conjugates from
one place (i.e., only from PharMingen) and that place makes all of
the Cy5PE's the same... In PharMingen's case (with whose reagents I
am most familiar), they make an effort to release batches made from
the same Cy5PE conjugation. But you are correct that this problem
needs to be watched carefully.
> 4,.The sorting capability on the FACScalibur works
> extremely well for molecular applications in our
> laboratory. We sort cells for PCR, and cytospins for
> FISH. I would never use the FACScalibur for rare cell
> applications. These are reserved for the FACStar Plus.
> The FACScalibur works very well for sorting most
> populations > 10% of total to 99% purity.
Yes, I hadn't thought of molecular applications; it is good for that
as well.
Note that BD, when they designed this instrument, specifically had
rare cell applications in mind. A majority of the sorting that goes
on here at Stanford, however, could not use the FACScalibur, because
most of the applications want larger numbers of cells recovered than
the FACScalibur can accomplish in reasonable times.
> 5. In my experience, nothing is more sensitive for low
> density antigens than PE, as it requires minimal
> compensation when used with FITC, PerCP and APC.
> Sensitivity is limited only when using PE with PE tandem
> conjugates such as PE/Texas Red and PE/Cy5. This is the
> very reason a second laser to excite APC is so
> desirable.
Cy5PE is more sensitive than PE; primarily because autofluorescence
is so low in that part of the spectrum.
> 6. PE>APC>PerCP>FITC>Autoflurescence!
Autofluorescence does not belong in this categorization. A very dim
antigen on any fluor could be less bright than autofluorescence.
Otherwise, the order is correct (however, for many of our
applications the APC is brighter than the PE; this may be because we
use considerably more power to excite the APC than does the
FACScalibur).
In assessing brightness of a reagent, the important value is the
separation of the stained (i.e., fluor signal plus autofluorescence)
from negative (i.e., autofluorescence only). Thus, the ratio of the
stained to the unstained cells is the relevant value. This is how we
obtain the characterization
"PE>APC>PerCP>FITC"; and it is why Cy5PE is generally brighter than
PE: not because it generates more photons (it does not), but because
the AF spectrum in the 680 region is so low.
Mario,
If I remember quantum mechanics correctly, a photon is a packet/wave
of energy.
Any sample that contains myeloid elements (Leukemias, Stem Cell
Assays..........any sample with granulocytic and/or monocytic
contamination of a lymphocyte gate) will be problematic when using
PE/Cy5 or Cy5.
Compensation is a subtractive process. The unavoidable high
compensation of PE/Cy5 ( or any tandem) reduces it's utility.
As for PE>APC>PerCP>FITC>Autofluorescence!....I was responding
facetiously to your pedanticism.
I've been doing it (Flow) for 16 years, (Fluorescence for 21). We'll
have to swap stories sometime.
Happy Holidays!
Mark
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