Have a look at SNARF indicator product information there equation (1) describes the connection between the proton concentration (not activity), the pK value and the fluorescence signal ratios

There's the problem. Let's assume I pick the acidic calibration point A to be pH = 6. At this point the denominator in the bracket after Ka becomes Ra-Ra and the fraction becomes infinite. It is, however, possible I missed something important about the two limiting endpoints A and B but the protocol makes no mention about how to pick them.

Hey Jan, so although I've never used the SNARFs I've had a lot of experience using ratiometric genetically encoded pH sensors which work pretty much the same way. If you really want to understand the equations from first principles I highly recommend the Grynkiewicz et al 1985 paper which I've attached to this comment. It's for Ca2+ indicators but explains how you calculate ion concentration from any ratiometric indicator. see Discussion and equations on page 3446 and 3447. The equations in the SNARF product information are derived / exactly the same as the Grynkiewicz equations - just without the helpful explanations.

But back to equation (1) in the product info. It might be helpful when considering R approaching Ra in the denominator if you replaced R with the fluorescence ratio Fa(λ1)/Fa(λ2). When substituting Fa(λ1)/Fa(λ2) in for R you'll see that the Fa(λ2) cancels with the term on the right - ie so it doesn't tend to infinity - hope this helps.

Hi Joseph, that's very helpful indeed! Thanks. If I understand it correctly, Ra and Rb are the fluorescence ratios for completely protonated, resp. completely deprotonated forms of the indicator, correct? Those, however, could be quite difficult to measure in live cells...Would you say it might be better simply to construct an experimental calibration curve and read the pH values from it rather than to use the formula?

Hi Jan, you're completely correct about what Ra and Rb are. And yes you're right I think the best tactic is to construct an experimental calibration curve, ie collect fluorescence ratios at different pHs during a calibration experiment, fit the data using the equations we've been talking about, then during future experiments read pH off the calibration curve for particular measured ratios. For example calibration curves, curve fitting etc check our paper: http://www.ncbi.nlm.nih.gov/pubmed/22666186 - see fig 2

The measurement of pH in single cells was done using micro-electrodes. Daniel Ammann (Ion-selective microelectrodes:

principles, design and application)

I agree with Mr. Raimondo's advice about constructing a full calibration curve. Endpoints are not really all that useful when it comes to pH (or calcium or any other standard ratiometric calculation) for the reason that it is best to do your calibration curves in permeabilized cells with carefully controlled media constituents that you can control pH in to do the measurements. Various factors can influence the shape of the curve and therefore the accuracy of your pH calculations.

Hi, in our lab we routinely calibrate our pH imaging data, either measured with SNARF or BCECF, by utilisation of nigericin. This is an antibiotic molecul with K+/H+ exchanger properties. For calibration, we use an extracellular solution containing the estimated intracellular K+-concentration. Since most animal cell membranes show a permeability for K+ at resting conditions, the intracellular pH will adjust to the extracellular.

I don't use SNARF but do use ratiometric imaging with intracellular calibration. Using a perfusion system, directly after your live cell imaging experiment is finished, you perfuse a set of high potassium/ nigericin containing pH calibrated solutions. This then allows the application of a pH value on a pixel by pixel basis by finding Rmax Rmin and pK.

Dear posters, very interesting thread.

Anybody willing to check also the question of Terézia Kisková :

How to measure pH in tumors and the tumor micro-environment of animals during the experiment? to be found

@ https://www.researchgate.net/post/How_do_you_measure_pH_in_tumor_micro-environment?

Any comments for her problem?

Thank you, best regards, Wolfgang

For calcium we use Ionomycin in 10 mM Ca2+ containing buffer. May be using a similar buffer might help to create a reference scale of pH activity. Say using ionomycin in pH 6 buffer and then pH 8 buffer and use the difference in signal as a pH 6-8 scale. Just a suggestion. You have to try to see if it works at all.

try their procedure

Biochem. J. (1996) 313, 957-962 

Intracellular pH governs the subcellular distribution of hexokinase

in a glioma cell line /  Laurent MICCOLI*, Stephane OUDARD*, Franck SUREAU, Florence POIRSON*, Bernard DUTRILLAUX* and Marie-France POUPON

Jan Trnka and Joseph Valentino Raimondo How can I get the SNARF fluorescence ratio ? Is it by dividing the Median value of Pe/ Median value of PerCP ? And this Median can be obtained form the flow cytometry listmode files (As attached file). Am I correct ?

Thank you in advance.