Posted by
De Tombe, Pieter on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Ratiometric-measurements-for-multiphoton-microscopy-tp7585091p7585112.html
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.
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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)
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> On Apr 26, 2016, at 5:01 PM, Christian Wilms <
[hidden email]> wrote:
>
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>
> 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