# Optogenetics Depth Calculator

#### Calculate Effective Stimulation Depth

Irradiance drops rapidly in the brain, so how do you know you will successfully activate your neurones in vivo? This calculator predicts the effective stimulation depth for your study. Just input parameters according to your planned study and press Calculate. Scroll down for details and an explanation of effective stimulation depth.

INPUTS
Type of brain matter:
• Grey matter eg. cortex
• Mixed eg. thalamus
• White matter eg. brainstem
• Light power (mW):
Fibre core diameter (um):
Numerical aperture (NA):
Irradiance threshold (mW/mm^2):

OUTPUT
Effective stimulation depth (mm):

## How to use the depth calculator

Input the following parameters according to your planned study (some typical values have been preset, feel free to change):

1. Type of brain matter – the default option is a “mixed” intermediate density brain matter such as the thalamus. At the extremes are grey matter such as the cortex and white matter such as the brainstem.
2. Light power (mW) – the light power (in milliWatts) that you get out the end of the optic fibre cannula ie. the light power that will actually be entering the mouse’s brain. I recommend aiming for 10-15 mW, or if your system can’t achieve that then as high as it will go.
3. Fibre core diameter (µm) – the diameter of your fibre, usually 200 µm for in vivo optogenetics. I recommend these from Thorlabs.
4. Numerical aperture (NA) – the numerical aperture of your implanted fibre. I recommend 0.22 NA fibres for lasers or LED’s.
5. Irradiance threshold (mW/mm^2) – the irradiance (ie. power per surface area) needed to activate your opsin of choice. 1 mW/mm2 is a good value to start with, particularly for any ChR2-based opsins.

Then press “Calculate”, and it will predict your effective stimulation depth. This is the distance from the tip of the fibre that you can realistically expect your opsin to be activated. You can then use this value to inform your fibre placement in the brain, relative to the stimulation site. If your predicted stimulation depth is less than 1 mm, I would suggest rethinking some of your experimental parameters.

## Irradiance threshold

Irradiance is light power (mW) over a given surface area (mm^2). As you move away from the fibre tip, the irradiance drops rapidly due to geometric spread and tissue scattering. This means you a less likely to induce an action potential in target neurone, the further you get from the fibre tip.

Irradiance threshold is the irradiance needed to produce robust activation of your opsin. 1 mW/mm^2 is a well established irradiance threshold for ChR2(h134r), and holds true for many ChR2 variants.

Here you can see predicted irradiance relative to distance from fibre tip with standard opto settings. The different colours represent theoretical light power output from the fibre. The dotted horizontal line represents the threshold irradiance for activation of ChR2(h134r).

The higher the light power, the further the light will travel before the irradiance drops below the threshold. The depth calculator just gives you that depth reading given the settings you have chosen. As a visual example, here is a typical irradiance loss over distance aligned with the action potentials produced in neurones along the way:

Inhibitory opsins (eg. Arch and eNpHR3.0) tend to require higher thresholds of 3-5 mW/mm^2. Step-function opsins, however, require tiny activation irradiance in the low uW/mm^2 range. If in doubt, check the literature and, if you can, test the irradiance needed by electrophysiology.

## Effect of numerical aperture

The numerical aperture determines the acceptance angle of an optic fibre. Basically, the higher the NA, the more LED light goes in, and the more it scatters out the end of the fibre. Here you can see irradiance with standard opto settings, with only the NA changing. Increasing the NA dramatically decreases the distance from the fibre tip before the irradiance drops below the threshold.

## Scattering in different brain regions

Here you can see irradiance penetrance in different brain regions, with the 1 mW/mm^2 threshold drawn as a horizontal dotted line. Depending on the brain regions you are illuminating, depth of activation can be dramatically different. My recommendation is to use the intermediate scattering selection, unless your region of interest is particularly “grey” or “white”.

## Scattering from different wavelengths

The actual impact of changing visible light wavelengths on scattering in brain tissue is negligible, so I have left such options out. As you can see here, changing the wavelength from 470 nm (blue) to 620 nm (red) increases your effective ChR2 stimulation distance by about 0.1 mm with standard conditions.

## Further info

I will reiterate what I have said in my blog posts: this optogenetics depth calculator provides a predicted estimate of the effective stimulation depth according to the parameters you have input. So, please don’t take it as an absolute truth, but it should be helpful as a guide.

For further info about development of the optogenetics depth calculator tool, please check out my blog posts. To summarise the science, it is based on calculations by Aravanis et al.1 and uses scattering coefficients from Aravanis et al. and Yaroslavsky et al.1,2:

• Grey matter: 11.2
• Thalamus (intermediate scattering): 20
• White matter: 40

If you use my calculator to inform your experiments, please acknowledge it as: https://nicneuro.net/optogenetics-depth-calculator

1. Aravanis et al. J Neural Eng 4, S143-S156 (2007) An optical neural interface: in vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology
2. Yaroslavsky et al. Phys Med Biol 47, 2059 (2002) Optical properties of selected native and coagulated human brain tissues in vitro in the visible and near infrared spectral range

© NicNeuroNet April 2022