Steve,

we have developed from 1994 - 1996 on a LSM410 in our lab the method of
doing confocal Ca2+ imaging with fura-2 by using an AOTF for switching
the wavelength between 351 and 364. This was published in a paper in
1997:

R. Nitschke, S. Wilhelm, R. Borlinghaus, R. J. M. Bindels, J. Leipziger,
and R. Greger. A modified confocal laser scanning microscope allows fast
ultraviolet ratio imaging of intracellular Ca2+ activity using fura-2.
Pflügers Arch. 433:653-663, 1997.

Before we had done something quite similar for BCECF on a NORAN Odyssey
in 1995:

R. Nitschke and K. R. Spring. Electro-optical wavelength selection
enables confocal ratio imaging at low light levels. J.Micro.Soc.Am.
1:1-11, 1995.

This successfull implementations of an AOTF into a confocal for ratio
imaging was then brought into the market by ZEISS with the LSM 510 for
the VIS and UV and
LEICA (VIS and UV) and BIORAD (I think only VIS) followed as you might
know.

We have published more articles were confocal fura-2 imaging was
performed:

R. Nitschke, A. Henger, S. Ricken, J. Gloy, V. Müller, R. Greger, and H.
Pavenstädt. Angiotensin increases intracellular Ca2+ activity in
podocytes of the intact rat glomerulus. Kidney Int. Jan. 2000

S. Ricken, J. Leipziger, R. Greger, and R. Nitschke. Simultaneous
measurements of cytosolic and mitochondrial Ca2+ transients in HT29
cells. J.Biol.Chem. 273(52):34961-34969, 1998.



Fura-2 ratio imaging with the LSM 510 works in our lab quite nice in 2
and 3D even
with opposite responses for 351 and 364, as 364 is far enough away from
the isosbestic point away and the confocal uses sharp absolutely defined
laser lines
with an extrem small bandwidth. The maximal intensity changes are 30 -40
% for 351.
In our hands the 351 changes are about twice the changes of the 364.
In an imaging system this is different
if you choose 364 with a filter wheel or a monochromator then in reality
you usually have light with a bandwidth of 5 or 10 nm so you might not
see a change there. Christian is right that the sensitivity might be
lower due to not using optimal excitation, but it also depends a lot on
your S/N ratio how small the changes can be, which you want to pick up.

We also work with Indo-1 from time to time, but there bleaching is from
our experience more a problem than with fura-2 and the maximal changes
are comparable to what we see with fura-2.

We have seen that in some cells there can be problems with UV in
general, which means they are so sensitive that the signal transduction
is damaged easily and quickly by UV so that no Ca2+ changes can be seen.
However, these cells can have similar problems with VIS light when using
BCECF or fluo3 or 4. So a general rule is: go as low as possible with
your excitation
light in the confocal when doing live cell imaging. We use the 63x
c-Apochroamt water immersion lens, the laser is running at 40 % power,
the AOTF is usually at 1 to 1.5 % at both wavelength, for a pinhole of 1
to 2.0 airy units you
need usually a gain starting at 800 to 900 to max. The maximal number of
images you can do before you have to increase the gain is approximately
200-300. This depends mostly on the volume of your cells and the zoom
factor you are using.

I hope this helps and is also of some use for others from the group.

I am glad to get some input from other people who tried the fura-2
imaging with the LSM510UV or the LEICA-SP System.

Roland


___________________________

Nitschke, Roland Dr.

Institute of Physiology

Albert-Ludwigs-University Freiburg

Hermann-Herder-Str.7

D-79104 Freiburg

Germany
____________________________

E-Mail: [log in to unmask]
phone: 49-761-2035195
fax: 49-761-2035191