Last week I was planning my next optogenetics experiment, and I thought I’d try to find the optimal fibre placement. Normally I just aim them to point close to the site of interest, but I’ve had some less-than-optimal experiments in the past so definitely time for some optogenetics irradiance optimisation.
First of all, we need to start with the intensity of light needed to activate the opsin, which in this experiment will be ChR2-H134R. Lin et al. investigated some of the early opsins back in 20091, and found that you get approximately half of the full activation of ChR2-H134R (EC50) at about 1 mW/mm2. Now, I know that I get, on average, 7.5 mW of blue light out the end of a 200 µm optic cannula using our opto setup. So, dividing through, that gives an irradiance (ie. power per surface area) of 7.5/(π*0.12), which comes out around 239 mW/mm2.
Obviously, this is vastly more than enough to activate the ChR2, but how does it spread through the brain? To answer this question, I headed over to Karl Deisseroth’s optogenetics website, where he has an “irradiance calculator”, that will estimate the dissipation of light through brain matter (Figure 1)2.
The light tails off dramatically in the brain; so much so that it is hard to see how deep you can retain ChR2 activation. I plotted the data on a logarithmic scale (which you can also do on the irradiance calculator), and included the EC50 of 1 mW/mm2 as well as an upper “phototoxicity” limit (Figure 2). There isn’t really a clearcut limit for causing neuronal damage, but an early paper by Cardin et al. found that 100 mw/mm2 was capable of causing phototoxicity, so I’m taking that as my upper limit. This produces a nice “Goldilocks zone” between 1 mW/mm2 and 100 mW/mm2, where we expect good neuronal activation with limited damage.
This produces a “Goldilocks zone” between about 0.2 mm and 1.3 mm from the tip of the optic cannula. Given experimental variance, I would put the ideal range to aim for at about 0.4 mm to 1.1 mm (Figure 2).
So, taking this all together, I can plot the fibre and light scatter onto the mouse brain atlas (Figure 3). My neurone population of interest lies in the mediobasal hypothalamic area surrounding the VMH, but particularly on the side near the fornix. Plotting the expected irradiance, we see that the entirety of the neuronal population lies within the “Goldilocks zone”. Great.
However, I have drawn an estimate of the spread of light from a .22 NA fibre, and you can see that it doesn’t successfully hit all the neurone population laterally. But, this is based on the spread through air, and doesn’t take into account the scatter of light by brain tissue, which will necessarily cause some amount of lateral spread. So, how to quantify this?
This takes us to the final stage of optogenetics irradiance optimisation, which uses a freely available light scatter tool called optogenSIM3. I won’t go into details, but essentially you input similar parameters as for the irradiance calculator, but also including the position of the fibre in the brain. The program then runs a simulation to predict light scatter based on the absorption and scatter coefficients of different brain areas, and outputs something like this (Figure 4).
The images aren’t great for visualising details, but note the extent of the green 1 mW/mm2 threshold. The light scatters far wider than I had expected, especially given that this is a low divergence .22 NA fibre. Either way, this shows that I will definitely hit the vast majority of my targeted neurone population with my planned fibre placement.
One final note from Figure 4: see how there is backscatter, so the light goes dorsal to the end of the fibre. Which means that even if your fibre ends up level, or even slightly below, your region of interest, you might well still activate the neurones. The issue then becomes, are you causing damage due to the high irradiance at that point? I have seen, in the brains of previous opto mice, plenty of c-fos at the end of the fibre, even in control mice that don’t express ChR2.
Overall, I’m happy that this optogenetics irradiance optimisation has helped with my planned fibre placement, and hope for a good experiment.
1. Lin et al., Biophysical J 96, 1803-1814 (2009) Characterization of engineered channelrhodopsin variants with improved properties and kinetics.
2. https://web.stanford.edu/group/dlab/cgi-bin/graph/chart.php
3. Liu et al., Biomed Opt Express 6(12), 4859-4870 (2015) OptogenSIM: a 3D Monte Carlo simulation platform for light delivery design in optogenetics.