Brightness of dyes

Howard Shapiro hms at
Tue Mar 26 00:01:14 EST 2002

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.
>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.


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