The effective stimulation depth is one of the critical factors in determining the success of an optogenetics study. But it is routinely ignored, particularly by the vendors of optogenetic LED systems. Be wary of any vendor of optogenetic LED’s that loudly proclaims the mW power they can achieve. Particularly if that fibre has a high NA or diameter.
A dose of light
Quoting the power at the fibre tip is all well and good, but it doesn’t take into account the spread and scatter of light in the brain. So, unless you intend to have your optic fibre literally touching your neurone population of interest, you must consider how the irradiance (light power over area, mW/mm2) drops relative to distance from the fibre tip.
Now that we have plotted the irradiance loss as we move further from the fibre tip, what next? We need to know at what point the light ceases to be effective in activating our optogenetics. If you are used to pharmacology, you can think of it as a drug dilution, but one that occurs spatially through the tissue. So in that case, we need to find the EC50 of the opsin. Then, we can determine an irradiance “dose” to aim for, below which we lose efficacy.
Opsin characterisation
Fortunately, the early pioneers of optogenetics went through a lot of effort to validate and characterise everything. A 2012 paper from Karl Diesseroth’s lab characterised a range of opsins in exhaustive detail1.
The important info for this post is the determination of irradiance needed for activation. Mattis et al. determined photocurrent at a range of irradiances, firstly for a selection of stimulatory opsins (Figure 1A). From this they were able to calculate the irradiance needed for half activation, or the effective power density for 50% activation (EPD50; Figure 1B). This is analogous to the EC50 for pharmacology.
The EPD50 is helpful in that it provides a measure of the sensitivity of the opsins regardless of the expression level in the cell. Having said that, I don’t think we should disregard the magnitude of the photocurrent. Particularly when measured in a directly comparable system as we see here. My takeaway here is that these ChR2-based opsins have an EPD50 around 0.8 – 1.5 mw/mm2; the exception is the CatCh and C1V1 variants which all have very slow (>50 ms) off kinetics.
Inhibitory opsins
Mattis et al. also investigated a number of inhibitory opsins in the same way (Figure 2). These universally have a much higher EPD50 than the excitatory ChR2-based opsin. My takeaway from this figure is that the eArchT3 seems to be the best of these. It has a comparable EPD50 to eNpHR3.0, but a much higher photocurrent. Also, Arch opsins have peak excitation at 520 nm, which is technically easier to obtain than the ~590 nm peak of eNpHR3.0.
Right, so now we have a good idea of the threshold irradiance needed to activate our opsin of choice. Ideally, you would back this up by validating in vitro, using patch-clamp electrophysiology of your neurone system.
Predicting effective stimulation depth
So now I can replot the predicted irradiance loss from the tip of the fibre. Only this time, if I add the threshold irradiance of 1 mW/mm2 (as tested for ChR2h134r in vitro) it will highlight how deep I can expect to activate my opsin. This gives us a predicted effective stimulation depth.
Based on this graph, it appears that I will produce effective stimulation of my neurones for just over 1.2 mm. This is good, as it will allow me to aim the fibre 0.5 mm away from my population of interest, while still having plenty of leeway for experimental variability and still expect to activate the entire population.
In order to simplify this process, I have put this effective stimulation depth calculation into a handy (and free) online tool. Please do try it out, as it should inform you about your experimental design.
1. Mattis et al., Nat Methods 9(2), 159-172 (2012) Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins
Nice discussion, it is something that comes up regularly at our group meetings.