# How Long is Bright Enough?

Last blog post I had a revelation about the best numerical aperture to use for in vivo implanted optical fibres. Today, as part of my indepth study planning, I’ll be investigating the best opto flash time. My default has always been 10 ms, because a) it seems to be what most others use and b) it’s always worked well for me.

However, I like to be sure, and it never hurts to optimise your methodology. But where to start? I’ve mentioned in the past about the EC50 of ChR2 being 1 mW/mm2. However, this is actually misleading, as it doesn’t take into account the duration of illumination.

## Power, energy and time

The important point here is that mW is a unit of Power (Watts), which is energy (Joules) over time (seconds). And the thing that actually determines activation of the opsin is the energy that it is exposed to. What this means is that, in principle, you could have wildly differing power output activating ChR2 to the same extent, so long as you adjusted the length of time of illumination accordingly.

If we think of a typical in vivo light flash of 10 mW for 10 ms from a 200 µm fibre, we can calculate the energy emitted in this flash with the equation Power (W) = Energy (J)/Time (s):

Energy (J)             = 0.01 W x 0.01 s

= 0.0001 J

So we can say that 0.0001 J (or 100 µJ) of 470 nm light is enough energy from a 200 µm diameter fibre to robustly activate ChR2 in the brain in an experimental setting.

## Low opto power

Now let’s say we could only produce 100 µW from our fibre (100-fold less than in the previous example). We could theoretically activate ChR2 by adjusting the illumination time accordingly:

Time (s)                = 0.0001 J / 0.0001 W

= 1 s

What this means is that if we had a pitifully weak light source, we could still activate ChR2. Although, I’m not sure how useful 1 Hz neuronal stimulation would be biologically. However, there is a way to make this dim level of illumination biologically relevant, as Anpilov et al. did in their recent wireless opto study1. They did this by using a stabilised step-function opsin (SSFO), which acts more like a toggle switch – a single activation turns it on for 30 mins or so.

## Fast opto flashing

We can also look in the other direction, power wise. Let’s say you were interested in making neurones fire at 100 Hz. To maintain a 10 % duty cycle (to allow the neurones to recover electrically and to limit tissue warming), we might want a 1 ms light flash, and we could calculate the required optical power like this:

Power (W)           = 0.0001 J / 0.001 s

= 0.1 W

So, to drive a fast-frequency neurone like this with an equivalently robust activation of ChR2, we would need to be able to produce 100 mW out the end of a 200 µm fibre, which would be possible with a laser system. A quick note: 100 mW is actually a lot of light power to pump into a mouse’s brain. So, I would not advise aiming that high. I would worry about heating or damaging the tissue, so better to limit yourself to 15 mW or so, and validate your experiment accordingly.

## Measured opto flash times

Anyway, back to my planned experiment. The question was: do I need my full 10 ms flash time to produce the firing I want? A recent paper by Herman et al. investigated the silencing of ChR2-expressing neurones at higher light exposures2. It includes a nice overall picture of light pulse duration-dependent spike probabilities in a variety of neurones (Figure 1).

What they find, flashing various neurone types at 20 Hz, is that with light pulses of 5 or 10 ms they have increasing spike probabilities up to 95 – 100 % depending on the neurone type. Then at on-times of 25 ms or longer, the spiking fidelity drops in all neurone types except for fast-spiking neurones in the cortex. Based on this work, I would suggest 5-10 ms appear to be optimal across various neurone types. At any pulse length above or below that, the spiking falls away.

Right, while 5-10 ms looks like a good time duration, that study was performed at a single light intensity, so only provides a partial answer. However, I found an early paper that investigated the threshold light power needed to stimulate an action potential at various distances from the end of the fibre, across a range of pulse widths (Figure 2)3.

A couple of things are clear from Figure 2:

1. Longer pulse widths drop the power threshold needed to trigger an action potential.
2. The threshold power needed to trigger an action potential increases with distance from the fibre tip.

It’s difficult to tell from the tiny scale on this graph, but it looks like 5 ms might just be enough to trigger an action potential at 1 mm from fibre tip at the ~9 mW power we get from our system. However, this is dependent on other factors, such as the NA of the implanted fibre.

## The best opto flash time

My verdict form this investigation is that 5 ms would likely be fine to trigger a response. However, increasing the flash duration to 10 ms would increase your likelihood of triggering action potentials without any noticeable drawbacks. So after all that, we come back to 10 ms as the best opto flash time (in my opinion).

1. Anplilov et al. Neuron 107(4), 644-655 (2020) Wireless Optogenetic Stimulation of Oxytocin Neurons in a Semi-natural Setup Dynamically Elevates Both Pro-social and Agonistic Behaviors

2. Herman et al. eLife 3, e01481 (2014) Cell type-specific and time-dependent light exposure contribute to silencing in neurons expressing Channelrhodopsin-2

3. Foutz et al. J Neurophysiol 107, 3235-3245 (2012) Theoretical principles underlying optical stimulation of a channelrhodopsin-2positive pyramidal neuron