One of the most challenging aspects of starting in vivo optogenetics is the equipment. In particular, how do you know which optogenetics stimulation systems will work for your purpose? I’m a big fan of LED’s, because of how cheap and easy they are to use compared with lasers. However, the high degree of scattering can make it challenging to obtain sufficient brightness for in vivo optogenetics.
Today, I will be investigating the most common commercially available in vivo optogenetics LED systems. Specifically, predicting the effective stimulation depth of their LED’s against the most commonly used opsins.
See below the opsins I’m investigating, along with the peak wavelengths and typical activation thresholds. Included are the papers I referenced for the irradiance thresholds.
Now I have the reference values to aim for. Next step is to check the manufacturers’ websites for light power output from their in vivo optogenetics LED systems, find the most appropriate LED for each opsin, and run it through the Depth calculator.
A brief note on my analysis: I use the published fibre characteristics from each vendor and estimate effective stimulation depth in “mixed” brain matter. In each case, I have picked the nearest/brightest LED to the opsin. I have also colour coded the reported stimulation depths to give an easy indication of experimental effectiveness.
I purchased the Plexbright system back in 2016, and it has worked well for activation of blue-responsive opsins. They also sell a wide range of colours to target different opsins. I have picked out their reported light power output from a 200 µm 0.66 NA fibre:
Really only the blue 465 nm LED is bright enough to have a stimulation depth approaching 1 mm for classic opsins. stGtACR2 and ChRmine are so super sensitive you can easily stimulate them even with relatively dim LED’s. Hence why they are the favourites for people wanting to do bidirectional optogenetics or with wireless opto’s7.
The Prizmatix UHP LED is the other in vivo optogenetics LED system that I have used (purchased by a collaborator). Again, I’ve only used the blue LED, which worked well. I have picked out their reported light power output from a 200 µm 0.66 NA fibre:
Same as Plexon, the blue LED is the best. Although, in this case the green 520 nm LED provides decent activation of inhibitory eOPN3.
Doric are well known for their photometry system, maybe not so much for in vivo optogenetics. They only sell a high powered in vivo optogenetics LED in blue. This time, the reported power values are from a 0.63 NA fibre:
Mightex make a wide array of optogenetics equipment. Their in vivo LED’s are reported from a 400 µm 0.22 NA fibre:
A caveat for these Mightex figures: their published power output figures weren’t explicitly clear that the power is from the end of an optic fibre cannula. It’s possible they are reporting the output from the optic cable, which means the experimentally usable value will be lower.
So which system should you buy?
I think it’s clear from my analysis here that most of the optogenetics LED systems you can buy for in vivo optogenetics are, quite simply, not fit for purpose. And I have only selected the most relevant wavelengths for my analysis; most of the vendors sell a much wider range of colours.
Looking at the effective stimulation depth, I can understand if people would want to use stGtACR2 and ChRmine, and forget about any other opsins. However, those come with their own limitations: stGtACR2 is soma targeted, so you can’t investigate circuits, while ChRmine has super slow kinetics which makes it unusable for many optogenetic applications. My point here is to be careful with your selection of equipment and opsins to match your experimental requirements.
I will happily recommend Plexon’s Plexbright LED’s and Prizmatix UHP LED’s for in vivo optogenetic stimulation for blue wavelengths. If you get those and use ChR2(h134r) for activating and stGtACR2 for inhibiting neurones, you should be fine. For other colours or other opsins? It’s not so clear cut. Currently, the best option is probably to buy a laser. In fact, Doric sell an interesting thing called a Liser, which is kind of like a hybrid between and LED and a laser, and I would definitely investigate it for non-blue opto’s.
1. Mattis et al. Nat Methods 9(2), 159-172 (2012) Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins
2. Mahn et al. Nat Comms 9, 4125 (2018) High-efficiency optogenetic silencing with soma-targeted anion-conducting channelrhodopsins
3. Mahn et al. Neuron 109, 1621-1635 (2021) Efficient optogenetic silencing of neurotransmitter release with a mosquito rhodopsin
4. Marshel et al. Science 365, eaaw5202 (2019) Cortical layer–specific critical dynamics triggering perception
5. Klapoetke et al. Nat Methods 11(3), 338-346 (2014) Independent optical excitation of distinct neural populations
6. Chuong et al. Nat Neurosci 17(8), 1123-1129 (2014) Noninvasive optical inhibition with a red-shifted microbial rhodopsin
7. Li et al. Nat Comms 13, 839 (2022) Colocalized, bidirectional optogenetic modulations in freely behaving mice with a wireless dual-color optoelectronic probe