3 Key Factors Influencing In-Vitro Optogenetics Experiment Design

3 Key Factors Influencing In-Vitro Optogenetics Experiment Design

Introduction

Optogenetics is a powerful technique that enables researchers to manipulate cellular activity using light. While in-vivo optogenetics focuses on studying neural circuits in living animals, in-vitro optogenetics allows for the investigation of cellular responses and signaling pathways in controlled laboratory settings. When designing in-vitro optogenetics experiments, several key factors come into play.

Opsin Sensitivity

Opsins, the light-sensitive proteins used in optogenetics, are one of the key factors to consider for in-vitro optogenetics research. Opsins are a class of photoreceptor proteins that have a selective sensitivity to various wavelengths of light. Opsins function by changing their molecular structure as a response to receiving specific wavelengths of light. The change in structure results in activation of the G protein, also known as guanine nucleotide-binding proteins, causing a signaling cascade and a physiological response in the target sample. (Shichida and Matsuyama, 2009). 

Different opsins exhibit varying sensitivities to light across the spectrum. For example, channelrhodopsin-2 (ChR2), a commonly used algal protein, is maximally activated by blue and violet light. Similarly, opsins with sensitivities to other wavelengths, such as green, red, or even ultraviolet (UV) light, can be utilized with in-vivo and in-vitro optogenetics experiments to selectively modulate cellular responses and investigate specific signaling pathways. 

The choice of opsins and the corresponding wavelengths used in in-vitro optogenetics experiments depend on your individual objectives and the specific cellular responses under investigation. Understanding opsin’s sensitivity to light is key to successful in-vitro optogenetics experiments and unlocking the mechanisms underlying cellular activity and signaling pathways.

Tissue Penetration

Tissue penetration is an important factor when deciding which wavelength of light to use for your in-vitro optogenetics experiment. Different wavelengths of light exhibit varying levels of tissue penetration. This can unlock the potential to target specific cell types or regions of interest.  

The exact penetration depth can be influenced by factors such as scattering, absorption, and the specific cell types or structures being targeted. Generally, shorter wavelengths, such as ultraviolet and blue light, have lower tissue penetration, meaning they can only reach superficial layers of tissue. On the other hand, longer wavelengths, such as red and infrared light, have greater tissue penetration and can reach deeper layers of tissue.

Here are some examples of different wavelengths and common opsins utilized with each:

Wavelength Penetration
 Light Color Wavelength Common Opsin Description
Ultra-violet365 nmOptoSTIM1Ultraviolet light at 365 nm has limited tissue penetration. It is primarily used for the precise excitation of UV-sensitive opsins, providing control over cellular activity in superficial layers of tissue.
Violet405 nm, 420 nmOptoSTIM1Violet light at 405 nm and 420 nm penetrates moderately into tissue. It enables precise control over cellular activity in both superficial and deeper layers.
Blue450 nmChannelrhodopsin-2 (ChR2)Blue light at 450 nm and 470 nm exhibits moderate tissue penetration. It is commonly employed to activate channelrhodopsin-2 (ChR2), inducing depolarization and increased neural activity in both superficial and deeper regions.
Green525 nmGreen-Activated Protein (GAP)Green light at 525 nm penetrates moderately into tissue. It offers a versatile option for activating and inhibiting various opsins, enabling control over neural circuits in both superficial and deeper tissue layers.
Yellow590 nmHalorhodopsin (eNpHR3.0)Yellow light at 590 nm exhibits moderate tissue penetration. It is commonly used to inhibit neuronal activity using opsins like halorhodopsin, facilitating neural suppression in both superficial and deeper tissue regions.
Red630 nm, 660 nm, 740 nmReaChRRed light at 630 nm, 660 nm, and 740 nm provides deeper tissue penetration compared to shorter wavelengths. It enables the activation or inhibition of neurons in deep brain regions due to its effective tissue penetration.
Infra-red940 nmJawsInfrared light at 940 nm possesses the deepest tissue penetration among the mentioned wavelengths. It finds applications in stimulating neurons in thick brain slices or reaching deeper brain regions.

 

In-Vitro LED Array

Ready to enhance your in-vitro optogenetics research? Discover the power of Amuza’s LED Array, designed for precise control and manipulation of cellular activity. Visit our website to explore our range of products and take your experiments to the next level.

Phototoxicity

Phototoxicity is an important concern with in-vitro optogenetics experiments due to the potential damage it can cause to cells. In 2018, Marvek et al. demonstrated blue-light decreases overall neuronal cell viability and can modify neuronal morphology. However, possible solutions have been proposed within the past few years to address causes of the decreased cell viability as a result of blue light. 

One approach to increasing cell viability during in-vitro research is decreasing blue-light fluorescence intensity. In 2019, Duke et al. observed that increasing blue-light fluorescence intensity was detrimental to cell viability. This was measured by exposing cells to various duty cycles and measuring the impact on cell viability. Duke defies a duty cycle as “light-on-time / total-time x 100.” As the light-on-time in each duty cycle increases, cell viability after 8 hours of exposure decreases. This was measured at duty cycles of 1.67%, 3.33%, and 6.67%.

Different opsins exhibit varying sensitivities to light across the spectrum. For example, channelrhodopsin-2 (ChR2), a commonly used algal protein, is maximally activated by blue and violet light. Similarly, opsins with sensitivities to other wavelengths, such as green, red, or even ultraviolet (UV) light, can be utilized with in-vivo and in-vitro optogenetics experiments to selectively modulate cellular responses and investigate specific signaling pathways. 

The choice of opsins and the corresponding wavelengths used in in-vitro optogenetics experiments depend on your individual objectives and the specific cellular responses under investigation. Understanding opsin’s sensitivity to light is key to successful in-vitro optogenetics experiments and unlocking the mechanisms underlying cellular activity and signaling pathways.

Recommended Case Studies

In the following case studies, a few example applications of some specific wavelengths of light in in-vitro optogenetics research will be highlighted, showcasing the potential of this technique in advancing our understanding of cellular behavior and contributing to the development of targeted interventions in various fields.

Blue Light (470 nm)

In Stierschneider’s publication, blue light was utilized as a tool for in-vitro optogenetics to activate the Toll-like receptor 4 (TLR4) signaling pathway and the NF-κB-Gluc reporter system in human pancreatic cells.

By exposing the cells to blue light, the researchers were able to precisely control the activation of these pathways, allowing for a detailed investigation of their dynamics and regulatory mechanisms in a controlled laboratory setting.

Yellow Light (590 nm)

In 2021, Amitrano et al. used in-vitro optogenetics emitting a yellow light to show that there was an increase in overall mitochondrial function during both human and mouse CD8+ T cell activation. The Amuza LED array (590 nm) was used to illuminate HEK293T cells and CD8+ T cells with the ATP assay.

Blue and Yellow Light in One System (470 nm and 590 nm)

Catanese et al. utilizes a combination of transcriptomics, proteomics, optogenetics, and pharmacological approaches, revealing the accumulation of aberrant aggresomes, reduced synaptic gene expression, loss of synaptic contacts, and dynamic MAL activation of the transcription factor CREB in ALS-related motoneurons. 

The observed pathological features of ALS-related motoneurons included the accumulation of aberrant aggresomes, reduced expression of synaptic genes, loss of synaptic contacts, and dynamic MAL activation of the transcription factor CREB.

Conclusion

The 3 key factors to consider during in-vivo research are opsin sensitivity, tissue penetration, and phototoxicity. By selecting the appropriate opsins and wavelengths of light, researchers are able to investigate cellular responses and signaling pathways in their target tissue while keeping phototoxicity to a minimum.

Through the recommended case studies, we have witnessed the ability of specific light wavelengths in studying signaling pathways, mitochondrial function, and disease-related features. Researchers are encouraged to explore tools like the Amuza LED Array to further enhance their experiments.

Looking for information discussing In-Vivo Optogenetics?

In our free Wireless Optogenetics eBook, “The Wireless Way: Optogenetics” We explore the basics of in-vivo optogenetics and provide examples. Included are optogenetic suppression of medial prefrontal cortex -> paraventricular nucleus projections and optogenetic activation of parvalbumin inhibitory interneurons in the dorsal-medial prefrontal cortex.

References

Amitrano, A.M. (2021). Optical Control of CD8+ T Cell Metabolism and Effector Functions. Frontiers in Immunology, 12, 666231. https://doi.org/10.3389/fimmu.2021.666231

Catanese, A., Rajkumar, S., Sommer, D., Freisem, D., Wirth, A., Aly, A., Massa-López, D., Olivieri, A., Torelli, F., Ioannidis, V., Lipecka, J., Guerrera, I. C., Zytnicki, D., Ludolph, A., Kabashi, E., Mulaw, M. A., Roselli, F., & Böckers, T. M. (2021). Synaptic disruption and CREB-regulated transcription are restored by K+ channel blockers in ALS. EMBO Molecular Medicine, 13, e13131. https://doi.org/10.15252/emmm.202013131

Duke, Corey G., et al. “Blue Light-Induced Gene Expression Alterations in Cultured Neurons Are the Result of Phototoxic Interactions with Neuronal Culture Media.” Eneuro, vol. 7, no. 1, 2019, https://doi.org/10.1523/eneuro.0386-19.2019. 

Shichida, Y., & Matsuyama, T. (2009). Evolution of opsins and phototransduction. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 364(1531), 2881–2895. https://doi.org/10.1098/rstb.2009.0051

Stierschneider A, Grünstäudl P, Colleselli K, Atzler J, Klein CT, Hundsberger H, Wiesner C. Light-Inducible Spatio-Temporal Control of TLR4 and NF-κB-Gluc Reporter in Human Pancreatic Cell Line. International Journal of Molecular Sciences. 2021; 22(17):9232. https://doi.org/10.3390/ijms22179232

Marek, V., Potey, A., Réaux-Le-Goazigo, A., Reboussin, E., Charbonnier, A., Villette, T., Baudouin, C., Rostène, W., Denoyer, A., & Mélik Parsadaniantz, S. (2019). Blue light exposure in vitro causes toxicity to trigeminal neurons and glia through increased superoxide and hydrogen peroxide generation. Free radical biology & medicine, 131, 27–39. https://doi.org/10.1016/j.freeradbiomed.2018.11.029

Illuminating Neuroscience: The Power of Wireless Optogenetics

Illuminating Neuroscience: The Power of Wireless Optogenetics

Introduction

In the realm of neuroscience research, a groundbreaking technique has emerged known as wireless optogenetics. Wireless optogenetics is a fusion of wireless technology and light stimulation that allows for precise control and manipulation of neural activity in real time. In this blog, we will explore the remarkable applications of wireless optogenetics and its potential to advance our understanding of the brain. We will also delve into the limitations of traditional tethered systems and the numerous benefits offered by wireless optogenetic technology.

The Power of Optogenetics

Optogenetics utilizes light-sensitive opsins to precisely control and modulate specific neurons. By introducing genetically engineered opsins, researchers can manipulate neural activity with remarkable precision, enabling the study of neural circuits, brain activity, and neurological disorders.

Advancing Neurological Research

Wireless optogenetics holds promise in studying neurological disorders like Parkinson’s disease and epilepsy. With wireless technology, precise optical stimulation and neural modulation can be achieved, offering potential therapeutic approaches and novel treatment strategies.

Ready to Illuminate Your Neuroscience Journey? Download Our FREE eBook Now!

Understanding Brain Activity

Wireless optogenetics enables researchers to investigate neural circuits with exceptional accuracy, shedding light on the functional roles and connections between brain regions. This detailed understanding enhances our knowledge of brain function, information processing, and behavior generation.

Wireless Technology and Neurophysiology

Integration of wireless technology in optogenetics revolutionizes neurophysiology. Researchers can study neural activity and behavior in freely moving animals, acquiring real-time data for comprehensive analysis. This integration opens new avenues for studying the brain in natural contexts.

Future Directions

While wireless optogenetics has revolutionized neuroscience, ongoing efforts focus on miniaturizing wireless devices, optimizing power consumption, and developing advanced data analysis techniques. Integration with other cutting-edge technologies holds promise for unraveling the complexities of the brain.

In conclusion, wireless optogenetics with its precise control of neural activity through light stimulation transforms neuroscience research. It provides unprecedented insights into neural circuits, brain activity, and neurological disorders. By combining wireless technology and optogenetics, we are poised to unlock groundbreaking discoveries that will reshape our understanding of the brain.

2021 Optimizations to Wireless Fiber Photometry and Wireless Optogenetics Products, Based on Customer Feedback

2021 Optimizations to Wireless Fiber Photometry and Wireless Optogenetics Products, Based on Customer Feedback

Amuza has spent the last year listening to researchers who use optogenetics and imaging techniques with mice and rats and learning from their experiences. As customers use our products their suggestions for how to optimize our products have been invaluable, which sent our engineers back to work. The results are six new upgrades, options, and improvements for our wireless products, making them easier to use, allowing experiments to run longer, and providing a better customer experience.

Updates to Wireless Neuroscience Products

1. Removable, Rechargeable Batteries to Keep Experiments Running

One of the first questions researchers ask us about our wireless neuroscience products is how long the batteries will last, since this determines how long the experiment can run before the headstage needs to be swapped for recharging. To date, Amuza wireless neuroscience systems have been powered by integrated rechargeable batteries. We have offered versions with larger batteries, but this does increase the weight.

Our wireless systems are now offered with removable rechargeable batteries in several sizes. The batteries connect using a simple plug: To continue your experiment, just swap the battery for a recharged one. Choose a standard battery for mice (total weight for TeleFipho, 3.3 g), or a larger battery (total weight 5 g) with 60% longer battery life for rats.

TeleFipho headstage (left) and removable batteries (center, right).

2. Wired, but better.

Not entirely an update to our wireless products, but still highly relevant for long experiments and experiments where switching the battery would disturb the animal. Amuza now offers a wired version of our fiber photometry system. Unlike fiber photometry using optical patch cords, the headstage still contains the light source and fluorescence detection system. For the tether: instead of an optical signal, power and the amplified data signal are sent through a slim electrical cable. Because of this, compared to other wired systems, the wired version of TeleFipho does not degrade the signal by passing it through multiple optical connections or a rotary joint on the way to the detector. The cable is also very flexible, and bending it will not introduce artifacts into the signal. Just let us know what type of plug we should use on the cable, and we can make certain it will be compatible with the commutator you choose, preventing the cable from twisting or tangling.

Wired TeleFipho

3. Superior protection from interference for cleaner data on fiber photometry

To optimize the data transmission of our wireless fiber photometry system, we improved the shielding and the antenna on the TeleFipho headstage. This cuts down on both electrical noise in the data, and also makes the radio communication more robust in environments where radio interference has been an issue. In addition to the shielding and antenna improvements, a new directional antenna for TeleFipho provides a great solution for radio interference in more complex environments. If you experienced signal dropouts with TeleFipho’s standard antenna, this antenna should completely eliminate them.

4. New fiber optic cannula sizes for fiber photometry and optogenetics

TeleFipho has now been tested with 200 μm (core) diameter fibers as well as 400 μm. Narrower fibers offer the benefit of causing less physical trauma, as well as being able to target smaller structures. But they collect much less light from the target region, so bright, well expressed fluorescent biosensors are required to get high-quality data. Many of our customers in Japan have now used 200-micron fibers with TeleFipho to detect calcium using newer versions of GCaMP and reported excellent results. We now offer fiber optic cannula with either 200 or 400-micron fibers, cut to the length you specify. The cannula can also be used with our optogenetics system. Please see our blog post on fluorescent biosensors for updated information on sensors used for fiber photometry.

5. Scaling up fiber photometry for more animals

At launch, TeleFipho had four separate radio channels, allowing up to four animals to be monitored in the same room. We added 4 more radio channels to TeleFipho, so now up to 8 systems can be used together simultaneously.

6. Headstage protection chamber prevents equipment damage

Group housing gives animals the opportunity to nibble on or dislodge headstages, plus some cages have wire lids and other surfaces which can catch head-mounted equipment. Our new protection chamber isolates the headstage from the environment but is easy to remove between experiments. We recommend using the protection chamber with rats, non-human primates (NHPs), and other larger animals.

For more information on customizing wireless neuroscience products for your experiments to obtain better data from your wireless system, visit our wireless neuroscience products page.

Which upgrade is the most helpful for your research? What other upgrades would be valuable to you? Leave a comment below.

Optogenetics In Vitro: Illuminating Cells in Microplates

Optogenetics In Vitro: Illuminating Cells in Microplates

Teleopto LED illumination for 96 well plates from Amuza

Amuza’s Teleopto LEDA array: an incubator compatible illumination system for optogenetics, photobiology, and photochemistry in microplates.

Several years ago we found that while more and more work in optogenetics was being done in vitro, there was little in the way of equipment specifically designed to fill this role. Many labs built their own multiwell stimulation systems. These custom designs often performed brilliantly, but they took time to design and build and often made it more difficult to share protocols with other labs. We decided to provide a light source that is reproducible from well to well and from experiment to experiment, one that is ready to use in any lab.

The LEDA arrays are the result and now cell biologists, developmental biologists, chemists, neuroscientists, and others are using this array for optogenetics and to invent new techniques.

Original LEDA-B

Our most popular LED array is the LEDA-B: it is perfectly sized for a single 96 well plate. It has 96 LEDs with emission centered at a 470 nm wavelength. A lip surrounding the array locks your plate into place so that the wells stay centered over the LEDs. It can also be used with other microplates sharing the 96 well plate footprint. The cable is 2 m long, so only the array needs to be inside the incubator.

The LAD LED driver can be controlled manually, and you can vary the irradiance from zero up to roughly 1.5 mW/mm2, depending on wavelength. You can also use low voltage TTL pulses to trigger illumination. This way you can use a programmable pulse generator to control the LED array. Lowering the voltage of the pulse lowers the voltage sent to the LED array proportionately, allowing programmatic control of not just the timing but also the intensity of illumination. Our STO pulse generator is easy to program and use, but if you already have your own pulse generator or stimulator you should be able to use it with Teleopto. We now also offer programmable multichannel  LED drivers. These drivers do not require a pulse generator, they can be programmed via software on your PC.

Our LED arrays are available in many colors to match the ever-increasing variety of light-controlled tools available in life science, including:

White 

UV 365 nm

Violet 405 and 420 nm

Blue 450 and 470 nm

Green 525 nm

Yellow / orange 590 nm

Red 630, 660, & 740 nm

Infra-Red 850 & 940 nm

If you don’t see the wavelength you need listed on the Amuza website, please let us know, we may be able to find the correct LED for your application.

Our two-color arrays have two different color LEDs under each well. For example, a blue and yellow array allows you to work with step function (bistable) options: a blue pulse of light opens the channel and a yellow pulse closes it again.

Our LEDA4 and LEDA6 arrays allow you to control four or six different sections of the plate independently. This way you can trial different intensities or pulse trains simultaneously, saving your lab time and space when developing new protocols.

We also make these arrays in different sizes so that you can illuminate multiple plates at once, or change the placement of the LEDs to match the format of different well plates. These arrays are also perfect for illuminating Petri dishes, tissue culture bottles, flasks, and other vessels.

Our LED arrays are being used for a wide range of applications, including Cry2 based biomolecular condensates, developmental biology, Stem cell differentiation, Cardiac optogenetics, receptor activation, and protein expression, oncology, ophthalmology, development of novel opsins and other optogenetic tools, photochemistry, and neuroscience.

Questions?

Wireless Optogenetics Basics

Wireless Optogenetics Basics

Teleopto Wireless Optogenetics System Introduction

Introduction to Teleopto Wireless Optogenetic stimulation of mice, rats, and other small animals during behavioral testing.

Teleopto is a turnkey solution for optogenetic stimulation of mice, rats, and other small animals during behavioral experiments. Most systems use a stationary laser or LED and a long fiber optic cable for stimulation, limiting the ability of your animals to move during behavioral experiments. But since Teleopto is wireless, your research animals have complete freedom of movement and can move through doorways, tunnels, mazes, and even large open field environments.

The Teleopto system consists of remote control, wireless receiver headstages, and LED fiber-optic implants. The whole system is ready to use right out of the box. The remote sends an infrared signal to the receivers to turn the LEDs on and off. The receivers weigh as little as one gram and can be recharged between experiments. The implants are made to order with many options for light colors, single or bilateral fibers, fiber length, and fiber diameter.  Two-color implants are available for use with bistable opsins or stimulation and inhibition at the same site.

You can adjust the light power with a screwdriver. For two-channel receivers, the power for each channel is adjusted independently. Using our LPM light meter to measure the output lets you ensure that each mouse or rat in a group receives the same irradiance at the target.

Between experiments, receivers are recharged. Battery life depends on how frequently and how intensely you stimulate your mice or rats. We offer larger receivers with larger batteries for use with rats and smaller receivers for use with mice. It’s easy to switch receivers during an experiment, so chronic experiments lasting many days are possible.

Teleopto can be controlled manually, but normally a programmable pulse generator is used to send pulse trains. Our pulse generator is easy to program and use, but if you already have one it will probably be compatible with Teleopto

The pulse generator can, in turn, be triggered by behavioral events. A nose poke, lever, video tracking system, or other equipment can easily be used to trigger the start or stop of a pulse train. Amuza can work with you to make certain your behavioral equipment is compatible with Teleopto.

Teleopto is great for scaleup: a single IR emitter can control multiple receivers on multiple mice or rats. With additional emitters, one remote can be used with multiple operant conditioning chambers or cages. Additional emitters can also make certain there are no dead spots in a maze or other complex environment. The emitters have a range of 1-2 meters depending on lighting, but we also have high power IR emitters for use in large open field environments.

Questions?

Forgetfulness at the touch of a button: Teleopto Wireless Optogenetics in Science Magazine and The New York Times.

Forgetfulness at the touch of a button: Teleopto Wireless Optogenetics in Science Magazine and The New York Times.

Melanin-concentrating hormone (MCH) neurons are unlike most neurons: they are most active during sleep. Scientists have studied their role in regulating sleep and feeding behavior for some time, but the Yamanaka lab at Nagoya University in Japan has found that they may also have a role in preventing the consolidation of memories during sleep.  

Prof. Yamanaka (a co-developer of Teleopto) found that MCH neurons can suppress neurons in the hippocampus responsible for memory consolidation. His lab confirmed this role by using 

Teleopto Wireless Optogenetics: blue light and channelrhodopsin 2 were used to activate MCH neurons; green light/archaerhodopsin were used to inhibit them. This was done bilaterally during both memory consolidation (REM sleep) and awake periods. Teleopto was used so that the animals were able to move freely and interact naturally with objects during Novel Object Recognition (NOR) tests.

When mice had MCH neurons activated during sleep, their ability to remember events decreased: they forgot which objects they had encountered before sleeping and treated them the same way they treated novel objects. Conversely, when their MCH neurons were inhibited they were able to remember which objects they had already interacted with. They ignored the familiar objects and explored the novel objects instead.

When Teleopto was used to illuminate MCH neurons during awake periods, there was no effect on hippocampal-dependent memory.

 When interviewed by The New York Times, Prof. Yamanaka explained

“These results suggest that hypothalamic M.C.H. neurons help the brain actively forget new information that is not important.” And because the neurons are most active during R.E.M. sleep, they may explain why humans usually do not remember their dreams when they wake up. “The neurons may be clearing up memory resources for the next day,” Dr. Yamanaka said.

The article, “REM sleep–active MCH neurons are involved in forgetting hippocampus-dependent memories.” is available at:

https://science.sciencemag.org/content/365/6459/1308