Oops, auto corrector mishap ... 2P excitation … sorry 

P.


> On Apr 28, 2016, at 9:37 AM, De Tombe, Pieter <[log in to unmask]> wrote:
> 
> Hi,
> 
> We used 2P exception to measure radiometric Indo-1 Ca2+ transients in cardiac myocytes; we needed red excitation to prevent breakdown of Blebbistatin, a compound that uncouples
> contractile proteins from Ca2+ activation but is photo-inactivated by blue light.
> see: Farman GP, Tachampa K, Mateja R, Cazorla O, Lacampagne A, de Tombe PP. Blebbistatin: use as inhibitor of muscle contraction. Pflugers Arch. 2008 Mar;455(6):995–1005. 
> 
> -----------------------------------------------------------
> Pieter P. de Tombe, Ph.D.
> James R. DePauw Professor of Physiology
> Chair, Department of Cell and Molecular Physiology
> Loyola University Chicago
> 2160 South First Ave.
> Stritch School of Medicine
> Maywood, IL  60153-5500
> (708) 216-1018
> (708) 216-6308 (FAX)
> [log in to unmask]
> 
>> On Apr 26, 2016, at 5:01 PM, Christian Wilms <[log in to unmask]> wrote:
>> 
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>> 
>> Ratiometric measurements using two-photon excitation is not easy. It helps to keep that in mind.
>> 
>> Fura-2 *might* work if you use a laser that allow fast enough  wavelength switching or use two lasers. But due to the broadened excitation bands for the separate peaks (when compared to one-photon excitation), it is by no means clear that it will be successful.
>> 
>> Emission-ratiometric indicators would be the way to go. But the calcium-bound form of Indo-1 has an extremely poor two-photon cross-section, making it unsuited for ratiometric imaging when using 2P-excitation.
>> 
>> FRET-based genetically encoded calcium indicators such as YC-2.60 can be used for ratiometric detection using 2P-excitation, but one needs to keep in mind that their calcium binding properties tend to be very non-linear, making calibration difficult.
>> 
>> An approach many people have been using, although mostly without the step to actually attempting to quantify the [Ca2+]  is to use a mix of dyes: one calcium indicator (e.g. Fluo-4) and one [Ca2+]-insensitive dye (e.g. Alexa 594). This combination yields ratiometric traces with a very good signal-to-noise ratio, but I would be very sceptical of papers trying to actually claim it reflects an absolute calcium concentration.
>> 
>> If actually quantifying calcium is what you want to achieve, there are approaches using single wavelength indicators such as Fluo-4 or OGB-1 that might be worth looking into. If you are working in a system that allows you to saturate the indicator in the cell at the end of an experiment, have a look at Miquel Maravall's 2000 Biophysical Journal paper (DOI: 10.1016/S0006-3495(00)76809-3). And finally, a shameless plug: fluorescence lifetime imaging enables ratiometric analysis of a single wavelength indicator. See Wilms et al. Cell Calcium, 2006 (DOI: 10.1016/j.ceca.2006.03.006)
>> 
>> Good luck with your experiments!
>> 
>> Best, Christian
>