Validating in vivo optogenetics LED systems

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.

Stimulation wavelength and irradiance threshold for common opsins.

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:

Effective stimulation depth for Plexon Plexbright LED's for a range of common opsins.

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:

Effective stimulation depth for Prizmatix UHP LED's for a range of common opsins.

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:

Effective stimulation depth for Doric optogenetics LED's for a range of common opsins.


Mightex make a wide array of optogenetics equipment. Their in vivo LED’s are reported from a 400 µm 0.22 NA fibre:

Effective stimulation depth for Mightex optogenetics LED's for a range of common opsins.

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.

Effective stimulation depth for optogenetics LED's for a range of common opsins.

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

A Lamplight in dark places

Since its discovery over 15 years ago, optogenetics has exploded in popularity in research. Along with this increase in interest and use has been a coincident profusion of optogenetic tools. This includes excitatory and inhibitory opsins across a wide range of timescales and light sensitivities.

Optogenetics publications have been increasing for the past 15 years.

However, one type of opsin that has consistently failed to present itself is a long-term super-sensitive optogenetic silencer. All the *good* inhibitory opsins have very fast kinetics and low sensitivity in the 3-5 mW/mm2 range.

A recent paper by Rodgers et al. changes all that1. They have discovered a novel opsin from the lamprey, which they have named “Lamplight”. It’s a Gi-coupled receptor (unlike most opsins which are light-responsive channels), which means that it is slower to signal but orders of magnitude more sensitive. In fact, its EC50 of 2.4 µW/mm2 is 1000-fold more sensitive than the classic inhibitory opsins like Arch and eNphR3.0.

However, as always, the sensitivity of an opsin is inversely correlated to its kinetics. Therefore, and as expected, Rodgers et al. show that Lamplight has a long and slow activation time (little to no diminishing of effect after 90 seconds). In addition to its extremely high sensitivity, Lamplight also has some other interesting qualities (Figure 1):

  • Scalable response – increasing light levels will produce a higher (stable) response from the opsin.
  • Switchable – the opsin is activated by 405 nm light and inhibited by 525 nm light. This has the added benefit that it won’t be accidentally activated by ambient light, which has much more green than UV

It should also be noted that Lamplight will limit neuronal damage, both by phototoxicity and electrophysiological. Normal opsins can stress (and potentially damage) neurones following chronic activation. This is not an issue with Gi signalling, you really can’t overactivate it.

Based on these unique characteristics, I can imagine Lamplight being a useful opsin for specific uses:

  • Extremely sensitive and longterm inhibition would be useful for use with lower power output wireless optogenetics, or for a single-stim inhibition that could work similarly to injecting CNO with inhibitory DREADDs.
  • Scalable inhibition for probing relative importance of a neurone population to mediate different behaviours/physiology. For example, we had an experiment where increasing the ChR2 stimulation frequency would shift the response from increasing glucose levels to aggressive/escape behaviour.
  • Using 2-colour opto stimulation to turn neurone populations on/off over medium-long term time scales.

Overall, I think this is an interesting opsin with potentially important applications for in vivo research. It is not yet available on Addgene, so anyone who is interested in this opsin should contact the lead author Rob Lucas.

1. Rodgers et al. EMBO Rep 22, e51866 (2021) Using a bistable animal opsin for switchable and scalable optogenetic inhibition of neurons

Effective Stimulation Depth

The effective stimulation depth is one of the critical factors in determining the success of an optogenetics study. But it is routinely ignored, particularly by the vendors of optogenetic LED systems. Be wary of any vendor of optogenetic LED’s that loudly proclaims the mW power they can achieve. Particularly if that fibre has a high NA or diameter.

A dose of light

Quoting the power at the fibre tip is all well and good, but it doesn’t take into account the spread and scatter of light in the brain. So, unless you intend to have your optic fibre literally touching your neurone population of interest, you must consider how the irradiance (light power over area, mW/mm2) drops relative to distance from the fibre tip.

Irradiance loss in the mouse brain

Now that we have plotted the irradiance loss as we move further from the fibre tip, what next? We need to know at what point the light ceases to be effective in activating our optogenetics. If you are used to pharmacology, you can think of it as a drug dilution, but one that occurs spatially through the tissue. So in that case, we need to find the EC50 of the opsin. Then, we can determine an irradiance “dose” to aim for, below which we lose efficacy.

Opsin characterisation

Fortunately, the early pioneers of optogenetics went through a lot of effort to validate and characterise everything. A 2012 paper from Karl Diesseroth’s lab characterised a range of opsins in exhaustive detail1.

The important info for this post is the determination of irradiance needed for activation. Mattis et al. determined photocurrent at a range of irradiances, firstly for a selection of stimulatory opsins (Figure 1A). From this they were able to calculate the irradiance needed for half activation, or the effective power density for 50% activation (EPD50; Figure 1B). This is analogous to the EC50 for pharmacology.

The EPD50 is helpful in that it provides a measure of the sensitivity of the opsins regardless of the expression level in the cell. Having said that, I don’t think we should disregard the magnitude of the photocurrent. Particularly when measured in a directly comparable system as we see here. My takeaway here is that these ChR2-based opsins have an EPD50 around 0.8 – 1.5 mw/mm2; the exception is the CatCh and C1V1 variants which all have very slow (>50 ms) off kinetics.

Inhibitory opsins

Mattis et al. also investigated a number of inhibitory opsins in the same way (Figure 2). These universally have a much higher EPD50 than the excitatory ChR2-based opsin. My takeaway from this figure is that the eArchT3 seems to be the best of these. It has a comparable EPD50 to eNpHR3.0, but a much higher photocurrent. Also, Arch opsins have peak excitation at 520 nm, which is technically easier to obtain than the ~590 nm peak of eNpHR3.0.

Right, so now we have a good idea of the threshold irradiance needed to activate our opsin of choice. Ideally, you would back this up by validating in vitro, using patch-clamp electrophysiology of your neurone system.

Predicting effective stimulation depth

So now I can replot the predicted irradiance loss from the tip of the fibre. Only this time, if I add the threshold irradiance of 1 mW/mm2 (as tested for ChR2h134r in vitro) it will highlight how deep I can expect to activate my opsin. This gives us a predicted effective stimulation depth.

Based on this graph, it appears that I will produce effective stimulation of my neurones for just over 1.2 mm. This is good, as it will allow me to aim the fibre 0.5 mm away from my population of interest, while still having plenty of leeway for experimental variability and still expect to activate the entire population.

In order to simplify this process, I have put this effective stimulation depth calculation into a handy (and free) online tool. Please do try it out, as it should inform you about your experimental design.

1. Mattis et al., Nat Methods 9(2), 159-172 (2012) Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins

A Ray of Insight

A paper came out recently that looks at optogenetics in a way I would have never thought of1. It’s funny how much effort we put into lasers and optics and everything in order to deliver light into a mouse’s brain, because visible light just doesn’t pass through tissue well enough for us to try activating from outside the brain (Figure 1A). But it never occurred to me to use non-visible wavelengths of light for the purpose; in this case it’s x-ray optogenetics.

Obviously, this comes with its own challenges – if a wavelength of light passes straight through tissue, it can’t interact with the proteins, which includes any opsins. So, how do you do this? Well, today we’ll find out from Matsubara et al.1

I actually remember an undergraduate practical that gave the answer to this, although my hazy memories suggest it was gamma waves from a radioactive isotope, rather than X-rays. Either way, the principle remains, which is the use of “scintillant”, which as far as I know is a fancy word for a chemical that is fluorescent under high energy light waves.

Anyway, Matsubara et al. show us the effects of UV and X-rays on their scintillant, which is Ce:GAGG, which emits yellow light upon stimulation (Figure 1B). They then test this scintillant for activating opsins in cultured cells, and find robust activation of the red-shifted opsins (Figure 1C/D), particularly of our favourite super-sensitive red-shifted opsin chRmine2.

After some optimisation in brain slices, Matsubara et al. then take their system in vivo, and inject AAV-DIO-chRmine and their scintillant into the VTA of a DAT-cre mouse (Figure 2A). They get nice c-fos induction from X-ray stimulation of their model system (Figure 2B/C).

After next showing that their scintillant particles are not cytotoxic when injected into mouse brains, they test their opto system in a conditioned place preference experiment with stimulation of the VTA (Figure 3A). Due to the nature of X-rays, they set up the in vivo experiment in lead-lined 2-compartment preference cages (Figure 3B/C).

They then show that the mice show increased place preference with chRmine stimulation (Figure 3D), and decreased place preference with stGtACR1 (Figure 3E), as expected.

I’m also happy to report that the authors checked for long-term damage to the mice caused by exposure to X-rays. They found no change to locomotor activity or blood-brain barrier function.

However, after prolonged stimulation with the high-dose X-rays, mice did have reduced numbers of immature neurones in the detate gyrus. The low dose flashing of X-rays had no impact though, so I think this method would be fine to use so long as you were careful with your experimental planning to limit the X-ray exposure of the animals.

Having said that, x-ray optogenetics is not a technique that I ever envisage myself actually wanting to use. There is a high level of difficulty and complexity, which I don’t think outweigh the improvements to animal behaviour. I think other wireless opto methods have a much better balance of complexity to impact on the animal.

1. Matsubara et al. Nat Commun 22(1), 4478 (2021) Remote control of neural function by X-ray-induced scintillation.


Mosquito vs Lamprey

A colleague sent me a paper about a novel opsin the other day, because he knows about my interest in optogenetics, and particularly in new tools that we can use to improve our experiments1. And then a few days later I received an email alert of a second paper2 that fulfils the same purpose as the first, namely producing new inhibitory opsins.

So, in this post I will investigate and compare these papers and what their results might mean for doing opto experiments. To begin, both papers aim to solve the same problem that has plagued optogenetics since its inception: the inability to optogenetically inhibit neurone terminals.

If this sounds untrue, let me quickly explain that while we have a number of inhibitory opsins available, none of them can produce reliable inhibition at the terminal. For example, ArchT is a proton pump, which causes hyperpolarisation, but in the tiny volume of the terminal also has a dramatic impact on the pH, which causes spontaneous neurotransmitter release.3

I’ll start with the common aspects of these new opsins: both are light responsive Gi/o-coupled GPCR’s, which means that they inhibit synaptic fusion by blocking production of cAMP and by suppression of Ca2+ release. However, the lamprey parapinopsin (PPO) is bistable, activated by UV and turned off by amber light (Figure 1A/B), whereas the mosquito panopsin homolog (OPN3; Mahn’s variant is called eOPN3) is activated by green light (Figure 1D/E).

Next, each paper goes on to demonstrate potent inhibition of neurone terminals in vitro. Both papers show extensive in vitro analysis, but for today I’m interested in the action at terminals, where they both show decreased amplitude of evoked post-synaptic currents (Figure 2A for Copits; Figure 2C for Mahn). They also both show they can decrease spontaneous post-synaptic current frequency without changing amplitude (Figure 2B for Copits; Figure 2D for Mahn).

Lastly, they both show they can impact animal behaviour in vivo by stimulating neurone terminals with their new opsins. For example, Copits et al. were able to block cocaine-induced conditioning in a VTA -> NAc projections (Figure 3A), whereas Mahn et al. managed to influence which direction mice were turning in an open field (Figure 3B).

All in all, I was very impressed by these new inhibitory opsins. If they ever become available, for example through Addgene, I would definitely look into them. It is important to be able to inhibit neurone projections like this.

However, from a purely practical point of view, I think I would lean towards the mosquito eOPN3 from Mahn et al, due to the stimulation wavelength of 500-550 nm as opposed to the UV stimulation of lamprey PPO from Copits et al.

1. Mahn et al. Neuron 109, 1621-1635 (2021) Efficient optogenetic silencing of neurotransmitter release with a mosquito rhodopsin.

2. Copits et al. Neuron 109, 1791-1809 (2021) A photoswitchable GPCR-based opsin for presynaptic inhibition.

3. Mahn et al. Nat Neurosci 19(4), 554-556 (2016) Biophysical constraints of optogenetic inhibition at presynaptic terminals.

The Power of Red Shift

My recent attempts at making a red optogenetics stimulation system (in the 620 nm range) were partially successful, in that I was able to make something that would produce about 3 mW from the end of an implanted 200 µm fibre. Unfortunately, this is less power than I wanted (ideally more like 10 mW), but my research into which opsin to use led me to a recent paper from Karl Diesseroth’s group1, where they develop a new red-shifted super sensitive opsin.

In this work, Marshel et al. begin by data mining a marine microbes genetic database for potential channelrhodopsins (Figure 1A/B). This kind of largescale screening is not my forté, but it was very interesting the way they used previous channelrhodopsin crystal structure to inform their screen as to variants that had novel sequences specifically for the residues that form the ion pore, thereby increasing their chances of finding a variant with very different properties.

They then transfected these channelrhodopsin variants into cultured neurones and used patch clamping to determine the channel properties, which allowed them to narrow down to a the most promising super sensitive opsin, of a previously unknown class. They called it chRmine, supposedly to be pronounced carmine (like the colour), but my brain can’t call it anything other than chromine.

Anyway, naming aside, it seems to be an excellent opsin for use in optogenetics experiments. It shows huge excitatory photocurrents in the orange and red spectra, when compared to 2 other red-responsive opsins, Chrimson and bReaChES (Figure 1C). They then go through a series of opto-electrophysiology recordings to demonstrate the suitability for optogenetics experiments, beginning with the reversal potential, which demonstrates Na+/K+ permeability, which is great for driving neuronal spiking (Figure 1D).

ChRmine induces neuronal firing with very short pulses (Figure 1E) and very low irradiance (Figure 1F), both on an order of magnitude better than the compared opsins. This sensitivity is so important for optogenetics, as it makes it much easier to deliver the amount of light needed and you can limit the phototoxicity damage.

The other important quality of chRmine is that it has very quick rise and fall times –  essentially it opens and closes very quickly upon light activation, which allows very fast (40 Hz) spiking (see supplementary info from Marshel et al.).

The figure I’ve shown here is only half of the first of 6 figures (not including supplemental information) from this absolute behemoth of a paper. The complexity of the experiments is far beyond my experience and understanding in neuroscience, but they use their new red super sensitive opsin to control individual cortical neurones with millisecond precision for a series of learning and behaviours in mice. Please do check it out if that’s your bag, but it’s far too “neurosciencey” for me.

Anyway, back to my attempts at making a red-based opto system. I’ve managed to achieve 3 mW of 620 nm light from a 200µm fibre with an easy-to-use LED system, which is on the low end for classic red optogenetics, but should do great for the super-sensitive chRmine.

And to get an idea of how well it would work in a mouse’s brain, I went to Karl Diesseroth’s optogenetics website to use his irradiance prediction tool2. For those who’ve never used this, it’s quite handy – you input various parameters for your study, including the wavelength of light and details of your optic fibre cannula, and it shows you the light spread you can expect through the brain (Figure 2).

So, going back at the Marshel paper, they show 100% activation of chRmine at 0.08 mW/mm2. Looking at the predicted irradiance values, that comes out at a depth of 2.3 mm from the fibre tip, which is immense – in an experiment that would illuminate deeper than the entire hypothalamus. Clearly then, we could drop the intensity of the stimulation light, but by how much? Using Diesseroth’s irradiance calculator, I input a series of decreasing light intensities and noted how deep you can go and maintain 0.08 mW/mm2 (Figure 3).

Given experimental practicalities, I would be happy to have 1.2 mm of activation depth, which translates to about 0.3 mW of power, which is a 10-fold reduction even from my relatively low power LED system. This would obviously need validating in vivo, but it’s very promising, and should negate a lot of the classic difficulties with obtaining high light power for optogenetics. I’ve had a peek, and it seems that Addgene stock chRmine AAV’s, so I am excited to try this out.

1. Marshel et al., Science 365, eaaw5202 (2019) Cortical layer-specific critical dynamics triggering perception.


Lockdown Expansion

Covid has hit every one of us, and all in different ways. During the first national lockdown (in the UK we’re now on number 3), my daily life changed a lot. The University had shut down, so I was no longer going in to do research, and the nursery had shut down so the little man was kept home. And the wife was doing online learning for her studies, so I had a lot of time on my hands, most of which was spent trying to tire out and otherwise distract a toddler.

One of the things I did to stay motivated in science was to sign up to interesting webinars. This week I’ll be talking about one that I found particularly interesting hosted by the people at Inscopix (who make head-mounted miniscopes; Ed Boyden talking about some of the tools he’s developed.

Ed Boyden, along with Karl Deisseroth, literally invented the field of optogenetics1, so this was bound to be an interesting listen. In fact, Inscopix archived the presentation, so I would urge anyone reading this to go check it out2. He went into a number of his recent advances for interrogating neuronal circuits, including optimising genetically-encoded voltage sensors and soma-targeted GCaMP.

The development that I found most interesting was expansion microscopy, a method for “nanoscale microscopy over extended scales”3. Not the most obvious description for a technique that lets you improve your microscope resolution by expanding the sample from the inside. Essentially, you infuse your sample with a swellable acrylamide polymer, anchor to cellular proteins as a scaffold, polymerise then add water to expand equally in all directions at once.

This enables you to drastically improve your imaging resolution without having to use ludicrously advanced (and expensive and difficult) microscopes (Figure 1 shows successive zooms of the same sample pre- and post-expansion). They managed to improve the imaging resolution to the point of distinguishing pre- and post-synapses in a brain section; this is otherwise extremely challenging to do using light microscopy, due to the diffraction limit of light causing blurriness at such limited distances.

However, this was not enough for Ed Boyden, so he decided to take expansion microscopy a step further with iterative expansion. He described this as “like PCR, but for expanding samples”. Essentially you do iterative rounds of polymer infusions and swelling to produce massive increases in sample volume and separation between particles. You can see the progression of improved resolution of “Brainbow” neuronal circuitry imaging in Figure 2.

Once again, this was not enough for Ed Boyden, who has since developed a technique for sequencing RNA in situ on a slice, leading to the precise mapping of RNA sequences across a section5. However, this leads me to my point for presenting this methodology (beyond my interest in cool new techniques), which is to emphasise the need to only use methods or technology for relevant applications. By which, I mean that it is all too easy to get lured in by some fancy new tech, and then spend a lot of time and money getting them working in your own research (and I promise you, it will take you a lot longer than you think, it always looks a lot easier and cleaner in the pioneering papers than it will be to do it yourself).

In fact, my first thought upon seeing these methods was to think of how I might use it in my own research. However, this level of resolution is just not necessary for me at all; there have only been a few occasions over the past decade that I actually needed any microscopy more advanced than “normal” epifluorescence, and that could be achieved with pretty basic confocal imaging.

I’ve come to realise that it is always better to stick with what you know (obviously doing iterative improvements as and when needed), and only progress to more advanced techniques when there is a definitive need for it, and have a plan for the work you plan to do with it and how it will improve your research impact. And assume it will take twice as long to get it running well compared to what you think.

Finally, always make sure you find a friend who can show you how to do a new technique before diving in for yourself; if you don’t know anyone personally, there are plenty of ways to find someone who can help, such as at conferences or workshops, or even just email people who’ve published in your field (academics tend to be happy to help, even someone who is technically a rival).

If you seek help, you will not only save yourself potentially huge amounts of cash on suboptimal equipment, but also huge amounts of wasted time and resources learning and optimising the technique. This is a lesson I wish I had learned years ago; I would have saved a lot of time and effort, so I hope you will too.

1. Boyden et al., Nature Neuroscience 8, 1263-1268 (2005) Millisecond timescale, genetically targeted optical control of neural activity.


3. Chen et al., Science 346(6221), 543 (2015) Expansion Microscopy.

4. Chang et al., Nature Methods 14, 593-599 (2017) Iterative expansion microscopy.

5. Alon et al., BioRxiv (2020) Expansion Sequencing: Spatially Precise In Situ Transcriptomics in Intact Biological Systems.