Brightness of dyes

Richard Haugland richard.haugland at probes.com
Wed Mar 27 08:40:57 EST 2002


The spectral comparison I presented of Alexa Fluor 488 versus fluorescein was
in a steady state fluorometer with relatively low power and nonsaturating
conditions. Also, the excited state lifetimes of the two dyes (about 4 nsec
each) and the extinction coefficients of the two dyes (about 80,000-100,000
each) are very comparable as are the spectra so that their excitation by the
argon-ion laser is about the same.  The MAJOR difference beyond an
intrinsically higher quantum yield is the greater quenching of fluorescein
conjugates on antibodies when they are overlabeled. Also, of course, the
absorbance and fluorescence of fluorescein at 488 nm is strongly quenched below
pH 7 (pka about 6.3), but that of the Alexa Fluor 488 dye is not. The effect of
bleaching rate (and flow rate) and excited state lifetime in flow cytometry on
intensity ("brightness"), however, is of interest to at least Howard and
myself.

Another possible comparison that we have done in flow but not a cuvette is Cy2
versus Alexa Fluor 488. Our data indicate that the Alexa Fluor 488 dye is
brighter than Cy2 in a flow cytometry experiment but Cy2 has quite a bit
shorter excited state lifetime than does the Alexa Fluor 488 dye so it may
perform somewhat even better  in flow than it does in a cuvette. I believe that
its absorption peak, however, is a bit short for the 488 nm laser line and thus
the intensity difference may be, in part, due to a poorer extinction
coefficient at 488 nm. I don't know how many people are using Cy2 in flow.

Figure 1.13 Brightness comparison of Molecular Probes' Alexa Fluor 488 goat
anti–mouse IgG antibody with Cy2 goat anti–mouse IgG antibody from Jackson
ImmunoResearch. Human blood was blocked with normal goat serum and incubated
with an anti-CD3 mouse monoclonal antibody; cells were washed, resuspended and
incubated with either Alexa Fluor 488 or Cy2 goat anti–mouse IgG antibody at
equal concentration. Red blood cells were lysed and the samples were analyzed
with a flow cytometer equipped with a 488 nm argon-ion laser and a 525 ± 10 nm
bandpass emission filter.


[Image]



Howard Shapiro wrote:

> Mario Roederer wrote-
>
> >Indeed, for flow cytometry, "brightness" is a very different issue
> >than for microscopy.  Several people noted that Alexa488 is superior
> >to Fluorescein in microscopy--primarily because it bleaches less
> >quickly (therefore, it's not that it's necessarily "brighter" to
> >begin with--it just stays brighter over the integrated time of the
> >experiment). However, bleaching is much less of an issue for flow
> >cytometry, where the dye stays in the laser for a few microseconds.
>
> And gets exposed to a thousand or more times as many photons. If you look
> at the same cells twice, you'll see that bleaching occurs, even in
> relatively low-power instruments. But I would agree that you can't compare
> what you get by eye and what you get in the cytometer.
>
> >Others have reported variable results in comparing Alexa488 to
> >fluorescein by flow cytometric experiments.  This might be explained
> >by the different conditions of the flow cytometers, the two most
> >important being stream velocity (and thus how long the dye is in the
> >laser), and the laser power.
> >
> >One of the important brightness-related issues in flow cytometry,
> >where a dye is in the excitation beam for a very short time, is the
> >fluorescence lifetime of the dye (i.e., how long after it is excited
> >by the laser does it emit a photon).  The shorter the lifetime, the
> >faster the dye is available for re-excitation, and, thereby, the
> >"brighter" it will be during the time it is in the laser.  Note that
> >while quantum yield depends on fluorescence lifetime, it also depends
> >on other factors.  I think (but am not positive), that Dick's graph
> >of brightness really should be normalized by the fluorescence
> >lifetime to get a somewhat more accurate picture of brightness.
>
> Lifetime differences only start to get significant when you are working
> near photon saturation, when the time dye molecules spend in the excited
> state becomes "dead time" with respect to reexcitation. Dick Haugland and I
> have had a recent private correspondence on this issue and there is/will be
> a discussion of it in the 4th Edition of Practical Flow Cytometry.
>
> >In addition, the absorption coefficient (epsilon) of a dye can be
> >important to brightness--but much more so on low laser power systems
> >(like benchtop instruments) than high laser power systems (like
> >sorters).  This is because on high power systems, there are more than
> >enough photons present to excite the dye; it will always be in the
> >excited state; on the low power systems, the exciting light is
> >subsaturating and hence the absorption coefficient of the dye becomes
> >important for "brightness".  This might explain why different groups
> >see different relative brightnesses of Alexa488 and fluorescein:  the
> >relative brightness may well be dependent on the laser power.
>
> I don't think so. No cross-section, no absorption; no absorption, no
> fluorescence. The absorption coefficient has to figure into how much power
> it takes to saturate the dye.
>
> >But there are many other considerations that lead to "relative"
> >brightness.  For example, one reason we switched from using TexasRed
> >to Alexa595 is that the former dye generates conjugates that are much
> >more sticky than Alexa595.  Thus, while there may not have been any
> >difference in their "brightness", practically the Alexa595 was far
> >superior because the background was lower.  This increased the
> >"relative brightness" (signal of the positive cells vs. the negative
> >cells) of Alexa595 vs. TexasRed.
>
> Good point.  For labels, you're generally concerned with the ratio of
> intensities of stained and unstained cells, not the absolute intensities.
>
> >These issues simply illustrates that "brightness" on the flow
> >cytometer is very complex, and really must be determined empirically
> >for any particular conditions.  One need not conjugate dozens of
> >antibodies; one need only conjugate a single antibody.  However, to
> >properly do the comparison, one must do a titration of dye:protein
> >for both conjugates, optimize both conjugates, and then compare
> >them--on stained cells, on the flow cytometer, and taking into
> >account background binding (i.e., staining cells that do not express
> >the antigen).
> >
> >My conclusion is that the "brightness" of any dye, conjugate, or
> >staining system must be evaluated empirically for the type of
> >experiment that you do, utilizing your particular hardware.  What may
> >be better for some people could be worse for your system because of
> >hardware differences.  Rather than trying to model all of these
> >differences to come to some prediction of brightness, just do the
> >experiment once and determine the answer for your system.
> >
> >mr
> >
> >PS.  Howard Shapiro ("The Howard") has long criticized me for
> >referring to the relative signal intensity of positive and negative
> >cells as brightness--he is quite correct that brightness is not
> >really the right word here.  That's why I've tried to put the word in
> >quotation marks as much as possible.  By "brightness" in the above
> >discussion, I try to convey what we think of as "useful brightness"
> >or "biological brightness"--a value that is empirically determined
> >and is useful from a practical standpoint.  What chemists and
> >physicists think of as brightness is only partly useful to flow
> >cytometry experiments.
>
> I'm not quibbling with the use of "brightness" here.
>
> -Howard
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