Posted by
Mark Cannell-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Ratiometric-measurements-for-multiphoton-microscopy-tp7585091p7585099.html
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Hi All
When we used info-1 with 690 nm 2P excitation, I thought that the ratio image was quite reasonable (see Jones, K.T., Soeller, C., Cannell, M.B., 1998. The passage of Ca2+ and fluorescent markers between the sperm and egg after fusion in the mouse. Development 125, 4627–4635). Also, using a second Ca insensitive dye to construct a “ratio" measurement is unwise -the point of the second wavelength is to control for bleaching and dye distribution etc.
my 2c
Cheers
On 26/04/2016, at 10:27 am, Christian Wilms <
[hidden email]> wrote:
>
> 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!
Mark B. Cannell Ph.D. FRSNZ FISHR
Professor of Cardiac Cell Biology
School of Physiology & Pharmacology
Faculty of Biomedical Sciences
University of Bristol
Bristol
BS8 1TD UK
[hidden email]