Purpose

When you want to make absolute, quantitative measurements with a ratiometric biosensor, we often find it difficult to choose a sensor well-suited for our experimental designs. Or, if we have already choosen a sensor, we usually want to know whether it is accurate enough for our experiments.

For example, it's generally accepted that roGFP1 is a good sensor to measure redox potential in C. elegans cytosol, but that roGFP1-iE might be a better choice for the ER.

This tool serves to quantify that intuition and help you make better-informed choices for your experiments.

Scope

This tool is appropriate for any ratiometric sensor with two states. It will be useful if you convert a ratiometric measurement into a real biochemical value, such as the concentration of a ligand or a redox potential.

This tool puts all sensors into three categories:

Redox: Sensors can be oxidized or reduced, and their ratio is converted to a voltage

pH: Sensors can be protenated or deprotenated, and their ratio is converted to pH

Ligand: Sensors can be bound or unbound, and their ratio can be converted to pLigand (-log10 of ligand concentration)

Instructions

To predict whether a sensor is well-suited to your needs, you need to know a few things:

How precise are your microscopy techniques?

Your microscope and analysis methods will produce noise in your ratiometric measurements. To determine that noise, run through your microscopy pipeline with GFP or another mock sensor, making measurements that should be consistent with each image (for example, 410/410 with a roGFP, or 410/470 with GFP).

Then, use those measurements to estimate your relative error. For example, if you find that 95% of your 410/410 measurements are between 1.02 and 0.98, your relative error is +/- 2.0%.

How accurate do you need your measurements to be?

For example, you may be trying to distinguish between a pH of 7.1 and 7.3, in which case you will need an accuracy of at least 0.2.

What are the biochemical and biophysical parameters of your sensor?

You can generally find estimates of these parameters in the literature. We have also aggregated these estimates in the supplementary notes of the associated manuscript.