Re: Near UV laser in FACS Aria II

Joe_Trotter wrote-

>	Right - the 375 nm laser is essentially as you describe; but as 
> far as Indo-1 not working goes, don't forget that ratiometric Ca++ 
> detection with a single loaded dye may be nicely done also with Fura Red 
> using two excitation lines (same emission near 670 nm). In fact, as I 
> recall the Fura dye was the preferred Ca++ dye before the Indo-1 method, 
> and it took some time for Indo-1 to be accepted. The old alternative 
> 'combo' method using fluo-3 with Fura Red was devised simply because there 
> were no violet lasers in those days on benchtops - you don't need the 
> fluo-3 if you have the standard violet laser (ie, violet excited Fura Red 
> replaces 488 excited Fluo-3 for the hi Ca++).

A few points: the reason the 375 nm UV laser won't work for indo-1 has
nothing to do with emission; the problem is that the excitation spectrum
of indo-1 with bound calcium has its peak at 325 nm, so, although you
can get some excitation at 350 or even 360, there is essentially none at
375. Indo-1 has been used with arc lamp systems, because the mercury
emission that peaks at 366 nm does extend down to shorter wavelengths.

The dye people used for calcium before indo-1 (which was first described
for flow cytometric use by Guenther Valet et al in 1985) was quin-2,
which could not be used for ratiometric measurements. The fura dye
developed by Tsien et al around the same time as indo-1 was fura-2; this
could be used ratiometrically in microscopy, with excitation at 335 and
362 nm and emission at 510 nm, but the excitation wavelengths were
problematic for flow (in theory, one could have used a He-Cd laser and
an argon laser or arc lamp, but I'm not sure anybody ever did this).

Fura Red didn't come into play until 1994; it can be used for
ratiometric measurements, paired with fluo-3 or calcium green, with
either of these dyes and Fura Red excited at 488 and the ratio of green
to red fluorescence measured. Fura Red red fluorescence decreases on
binding calcium; the fluorescence of the green dyes increases. Although
the use of these dyes in combination does tie up some useful
fluorescence channels, their ratio actually changes more rapidly in
response to calcium transients than does the indo-1 emission ratio.

Finally, one should always bear in mind that flow cytometric measurement
of calcium transients is only useful when the response of the cells of
interest is temporally homogeneous, i.e., when cell n at time n exhibits
more or less the same behavior as cell 1 would have exhibited at time n.
It has been shown using static cytometry, which permits the same cells
to be examined repeatedly over time, that the responses of many cell
types, including lymphocytes, may be temporally heterogeneous, with
cells differing from one another in the time course or pattern of
response (or both) to a given stimulus. Some people are now using
widefield fluorescence imagers to look at calcium transients in cell
populations.

-Howard