However, gleaning the pertinent information from Diesseroth’s irradiance calculator can itself be challenging. I almost find that it provides too much information. In fact, you can see from my blog post the only information I actually take from it is the depth at which the irradiance remains above the activation threshold for the opsin I’m using.
The standard irradiance threshold I use is 1 mW/mm^2 for two reasons:
- It is the published EC50 for my opsin of choice ChR2H134R1
- I have tested the response using electrophysiology and found good consistent activation of ChR2-expressing neurones using 1mW/mm^2
Using the calculator
For the purposes of my depth calculator, there are 4 values for you to input:
For light power, my recommendation is to use 10 mW, or as high as your system goes if it can’t reach 10 mW. For the fibre core and NA, I recommend using 200 µm and 0.22 NA, respectively (I use these from Thorlabs; check out my blog post for further info). Finally, the threshold irradiance you expect from the opsin you have used. For the fibre details and irradiance, I have put in recommended preset values, but you can of course change them to suit your specific experimental setup.
Once you have input your values, simply press “Calculate”, and the predicted stimulation depth of opsin activation will be calculated. A typical value to aim for is about 1.0-1.5 mm, as this gives plenty of leeway for experimental variability.
I hope people find my depth calculator to be useful and easy to use. I have also developed a tool to do the opposite calculation, ie. to predict the required light power needed based on your experimental needs.
1. Lin et al., Biophysical J 96, 1803-1814 (2009) Characterization of engineered channelrhodopsin variants with improved properties and kinetics.