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:
| Light Color | Wavelength | Common Opsin | Description |
| Ultra-violet | 365 nm | OptoSTIM1 | Ultraviolet 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. |
| Violet | 405 nm, 420 nm | OptoSTIM1 | Violet light at 405 nm and 420 nm penetrates moderately into tissue. It enables precise control over cellular activity in both superficial and deeper layers. |
| Blue | 450 nm | Channelrhodopsin-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. |
| Green | 525 nm | Green-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. |
| Yellow | 590 nm | Halorhodopsin (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. |
| Red | 630 nm, 660 nm, 740 nm | ReaChR | Red 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-red | 940 nm | Jaws | Infrared 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. |
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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
