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Jason:

I think direct excitation of the channelrhodopsin2 by the 340/380
wavelengths used to excite fura will be a much bigger problem then the
effect described in your post (excitation via pathlength-dependent
reabsorption or proximity-dependnet FRET). This is because the number of
excitation photons impinging on the sample is several orders of
magnitude larger than the number of emitted photons, which more than
makes up for the smaller activation cross section at <400 nm compared to
488 nm.   According to Proc Natl Acad Sci U S A. 100:13940(2003), the
activation cross-section of channelrhodopsin2 in the 300-400 nm range is
less the 1 order of magnitude less than that at 488 nm.

To avoid direct excitation, you would have to use a calcium indicator
that can be excited beyond the long wavelength limit of the
channelrhodopsin2 action spectrum.  Unfortunately, such indicators
(rhod-2, X-rhod-1) lack the ratiometric reponse property of fura-2.
Another possible tactic might be to use two-photon excitation of fura-2.
Whether that is a viable approach depends on how the two-photon action
spectrum of the channelrhodopsin2 maps onto the two-photon excitation
spectrum of fura-2.

Iain  

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of JASON P WEICK
Sent: Monday, May 08, 2006 8:14 AM
To: [log in to unmask]
Subject: ratiometric calcium imaging

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Dear all,

We're looking to do some ratiometric calcium imaging on neuronal 
cultures using a dual excitation flurophore (we don't have the 
capabilities to use dual emission). However, we also want to control 
the excitability of our cultures using a light-gated cation channel 
(Channel Rhodopsin 2), which excites around 488, close to the emission 
of Fura-2 (~500nm). Thus, we're afraid that emission of Fura-2 will 
cause excitation of our channel (aberrantly) leading to a positive 
feedback loop. Therefore, we would like to find a dye that uses dual 
emission for ratiometric measurements, but is neither excited by, nor 
emits around 488nm. Any thoughts?

Sincerely,

Jason P. Weick, PhD
University of Wisconsin-Madison
Waisman Center
1500 Highland ave.
Madison WI, 53705