How Bright is Bright?

I have previously written about the importance of brightness for in vivo optogenetics experiments. It’s just as important for in vitro optogenetics, which is what I’ll be looking at today. This came about because we’re planning a publication, and we need quantification of the light irradiance that we get on the brain slices.

When I started in vitro optogenetics, I tested the brightness of the LED system I had bought for the purpose, but not with a light meter – instead I tested directly on ChR2-expressing neurones, and found that 2 % brightness of the 470 nm LED was sufficient to elicit action potentials.

This was enough for me at the time, and I never bothered doing the metred quantification because the light meter didn’t fit under the objective (even after removing the tissue perfusion bath). However, for publishing I wanted a proper irradiance value, which meant me and our ephys technician spent an afternoon trying to dismantle the condenser under the stage. I say trying to, because microscope has been in pretty heavy use for at least a decade without any kind of service, and we found a lot of salt residue from past aCSF leakages.

Hopefully y’all cringed at the thought of that, because salt build-up inevitably means corrosion of expensive microscope parts. And, surprise surprise, we found the screws and bolts holding the condenser together and onto the microscope are all rusted in place. In the end, we managed to unscrew the top part of the condenser’s lens and wiggle the light meter in place under the objective. Phew! So now we went through a range of LED brightness and measured the brightness coming out the bottom (Figure 1).

LED brightness on my electrophysiology rig.

As ever, the brightness is not the important parameter here. What matters for activating opsins is the irradiance hitting the slice (irradiance being intensity of light per unit area). So now comes the difficult bit – how do I know the area that the light is hitting on the slice? It is possible to get a microscope ruler, put that under the objective and measure the diameter of your field of view. However, how do I know the camera or eyepiece are visualising the entirety of the illuminated area?

The answer is to go back to physics, and field of view of the objective in use. I found a useful guide from the makers of my LED’s1:

Diagram for calculating irradiance for in vitro optogenetics.

A quick investigation shows me that the Field Number for my objective is 22. Dividing that by the magnification of 40 gives a diameter of 0.55 mm. I know the area of a circle is πr2, which gives me a surface area of 0.238 mm2. So, adjusting the brightness values obtained earlier gives the irradiance output from the LED’s (Figure 2).

LED irradiance on my electrophysiology rig.

I have also plotted a simple linear regression line on the irradiance graph to give an easy formula to give a rough estimate of the irradiance at any given LED brightness. However, I will still make sure to use actual measured values in any publication, rather than the estimates obtained from the regression. Anyway, this does match up nicely with my earlier test data, as the EC50 for ChR2h134r is 1 mW/mm2, and 2 % brightness on my blue LED gives an irradiance of just over 2 mW/mm2.

For any future in vitro optogenetics studies on this rig, I will aim to use 10 % LED intensity, as this will give a solid irradiance of 10-15 mW/mm2, without going into higher saturating irradiance levels.