See also [[optogenetic-axon-guidance]] and [[biosight]].

# Optogenetics basics

* **Boyden et al., 2005 (ChR2 in neurons)** — the paper that showed a *microbial cation channel* (ChR2) can be expressed in mammalian neurons to drive spikes with millisecond precision using blue light.
  *Intuition:* light opens a non-selective cation channel (Na+, Ca\2+, H+) → fast depolarization → spikes. [Nature](https://www.nature.com/articles/nn1525?utm_source=chatgpt.com), [PubMed](https://pubmed.ncbi.nlm.nih.gov/16116447/?utm_source=chatgpt.com)
* **Zemelman et al., 2002–2003 (precursors)** — “genetically ChARGed neurons” and photochemical gating of heterologous receptors laid conceptual groundwork: make chosen cells light-sensitive by *introducing light-responsive proteins/ligands*. [ScienceDirect](https://www.sciencedirect.com/science/article/pii/S0896627301005748?utm_source=chatgpt.com), [PNAS](https://www.pnas.org/doi/10.1073/pnas.242738899?utm_source=chatgpt.com)
* **Halorhodopsin/Arch (optical silencing)** — pumps that move Cl⁻ in or H⁺ out to hyperpolarize and silence neurons; later refined (eNpHR3.0, ArchT).
  *Intuition:* light-driven ion pumps set membrane potential away from spike threshold; beware pump side-effects (e.g., chloride load). [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC6010559/?utm_source=chatgpt.com)

# Opsin families

* **Fast/bright ChRs for excitation:** Chronos, CheRiff, C1V1/Chrimson (red-shifted), ChRmine (ultrapotent).
  *Intuition:* mutations/species mining tune photocurrent, spectrum, and kinetics to balance *less scattering (red)* vs *fast gating and big currents*. Representative papers: Chronos/CheRiff/Chrimson (2015), ChRmine (2020). [Nature](https://www.nature.com/articles/s41598-023-31384-3?utm_source=chatgpt.com), [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC8435545/?utm_source=chatgpt.com)
* **Anion-conducting channelrhodopsins (GtACR1/2)** — channel (not pump) inhibitors with large, fast hyperpolarizing currents; often better inhibition than pumps.
  *Intuition:* opening a high-conductance Cl⁻ channel shunts/exerts E\_Cl-limited hyperpolarization; soma-targeting helps avoid axonal depolarization. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC12220681/?utm_source=chatgpt.com)
* **Step-function opsins (SFO/SSFO; SOUL)** — bistable ChR2 mutants where brief pulses toggle long-lived open/closed states.
  *Intuition:* mutations (e.g., C128S/D156A) stabilize the open state → much higher light sensitivity but reduced temporal precision; great for state setting. [PubMed](https://pubmed.ncbi.nlm.nih.gov/19079251/?utm_source=chatgpt.com), [ScienceDirect](https://www.sciencedirect.com/science/article/pii/S0896627320302397?utm_source=chatgpt.com)
* **Soma-targeted variants** — add Kv2.1 motifs to concentrate opsins at soma/dendrites → stronger, cleaner control; works for excitatory and inhibitory opsins (stCoChR, stGtACR).
  *Intuition:* trafficking tags change membrane localization to where you *want* the current. [eLife](https://elifesciences.org/articles/63359?utm_source=chatgpt.com), [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC6175909/?utm_source=chatgpt.com)
* **Bidirectional single-vector control (BiPOLES)** — fused Chrimson (excite, red) + GtACR2 (inhibit, blue) with balanced expression.
  *Intuition:* color-orthogonal push/pull in the *same* cells, controlled by wavelength. [PubMed](https://pubmed.ncbi.nlm.nih.gov/34312384/?utm_source=chatgpt.com)

# Deep light delivery

* **Upconversion nanoparticles (UCNPs)** — NIR in, visible out locally to drive opsins; demonstrated transcranial behavior modulation in mice.
  *Intuition:* exploit NIR tissue windows; UCNPs convert NIR to blue/green/red *in situ*, cutting scattering/absorption losses from surface. [Science](https://www.science.org/doi/10.1126/science.aaq1144?utm_source=chatgpt.com), [CBP](https://cbp.brain.riken.jp/files/Chen-2018-science-679.full.pdf?utm_source=chatgpt.com)
* **Ultrapotent/red-shifted opsins + surface illumination** — systemic AAV + ChRmine enabled *implant-free* deep optogenetics in mice (mm-scale).
  *Intuition:* combine very light-sensitive opsin + red-shifted excitation + skull illumination → sufficient photons reach targets. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC7878426/?utm_source=chatgpt.com)
* **Hardware** — wireless µLED implants; flexible/waveguide photonics.
  *Intuition:* reduce tethering and heat, deliver patterned light close to targets. Good overviews: wireless optoelectronics/photonic probes. [ScienceDirect](https://www.sciencedirect.com/science/article/pii/S0896627316310042?utm_source=chatgpt.com), [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC5640477/?utm_source=chatgpt.com)

# Opsin-free optogenetics (without microbial opsins)

Some of these are not optogenetics. Sorry.

* **Infrared neural stimulation (INS)/mid-IR modulation** — pulsed IR heats membranes → membrane capacitance changes → spikes; no gene transfer.
  *Intuition:* dT/dt at the bilayer changes capacitance and currents (optocapacitance). Power/thermal safety are the limiting factors. [Nature](https://www.nature.com/articles/s41378-020-0153-3?utm_source=chatgpt.com), [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC8115038/?utm_source=chatgpt.com)
* **Photothermal/optocapacitive nanoparticles** — gold or other absorbers tethered to membranes convert light to heat locally to excite neurons.
  *Intuition:* nanoscale heaters generate fast capacitive currents in nearby membrane patches. [PubMed](https://pubmed.ncbi.nlm.nih.gov/25772189/?utm_source=chatgpt.com), [ScienceDirect](https://www.sciencedirect.com/science/article/pii/S000634951731247X?utm_source=chatgpt.com), [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC9042976/?utm_source=chatgpt.com)
* **Photopharmacology (azobenzene photoswitches, PORTLs/PTLs, LiGluR)** — covalently tether photoswitchable ligands to endogenous receptors (e.g., iGluRs, GPCRs) to make *native* proteins light-responsive.
  *Intuition:* light flips azobenzene (trans↔cis) to present/withdraw the ligand from the binding site; fully genetic options exist via UAA incorporation. Great for subcellular, receptor-specific control. [ScienceDirect](https://www.sciencedirect.com/science/article/pii/S0896627307003443?utm_source=chatgpt.com), [American Chemical Society Publications](https://pubs.acs.org/doi/10.1021/acscentsci.5b00260?utm_source=chatgpt.com)

# Optogenetic components beyond ion flux

* **Opto-signaling modules** — CRY2–CIB (blue-light dimerization/oligomerization), LOV-based iLID/LOVTRAP, PhyB–PIF (red/far-red). Used for optoSOS (Ras/ERK), opto-Rho/Rac, opto-Wnt, opto-Trk, etc.
  *Intuition:* light toggles protein–protein interactions and localization; signaling follows. Exemplars: optoSOS; CRY2/CIB; iLID; LOVTRAP. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC3925772/?utm_source=chatgpt.com), [PubMed](https://pubmed.ncbi.nlm.nih.gov/21037589/?utm_source=chatgpt.com), [Nature](https://www.nature.com/articles/nmeth.3926?utm_source=chatgpt.com)
* **Opto-GPCRs (OptoXRs; 2024 bistable inhibitory Opto-GPCR)** — chimeric rhodopsins driving GPCR cascades with light; recent bistable inhibitory variants expand multiplexing.
  *Intuition:* graft intracellular loops from the GPCR of interest onto a light-activated rhodopsin scaffold. [ScienceDirect](https://www.sciencedirect.com/science/article/abs/pii/S0959440X19300557?utm_source=chatgpt.com), [Nature](https://www.nature.com/articles/s41592-024-02285-8?utm_source=chatgpt.com)
* **Light-tunable CRISPR** — photoactivatable Cas9 (split-Cas9 reassembled by light), LACE/dCas9-based transcriptional control, light-caged or light-expressed gRNAs.
  *Intuition:* restrict editing/activation to illuminated spatiotemporal windows. [PubMed](https://pubmed.ncbi.nlm.nih.gov/26076431/?utm_source=chatgpt.com), [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC4412021/?utm_source=chatgpt.com)

# Gene delivery and gene editing for in vivo optogenetics

* **AAV as the workhorse** — tropism/serotype choice (e.g., AAV9/PHP variants), promoters (hSyn, CaMKIIα), and soma-targeting tags (Kv2.1) determine where/how much opsin you get. Review overviews are useful. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC10319350/?utm_source=chatgpt.com)
* **Knock-in strategies in post-mitotic neurons** — SLENDR/vSLENDR (HDR with AAV donors), HITI (NHEJ-based insertion) when HDR is rare; these let you place opsins/tags at defined loci or label endogenous proteins.
  *Intuition:* pair Cas9 cut with donor template (HDR) or directionally designed NHEJ donors (HITI) to install payloads in adult brain. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC5691606/?utm_source=chatgpt.com)
* **Why it matters** — precise knock-ins can stabilize expression, match endogenous regulation, or achieve cell-type specificity without large promoters; HITI works in non-dividing neurons. [PubMed](https://pubmed.ncbi.nlm.nih.gov/27851729/?utm_source=chatgpt.com)

# Emerging techniques

* **Transcranial control with NIR + materials or ultra-sensitive opsins** — UCNPs and ChRmine push depth while avoiding craniotomies in mice; translation will hinge on safety, dosing, and light budgets in large brains. [Science](https://www.science.org/doi/10.1126/science.aaq1144?utm_source=chatgpt.com), [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC7878426/?utm_source=chatgpt.com)
* **Wavefront-shaping & virtual waveguides** — scatter-engineered light delivery to guide photons deeper with less damage. [Nature](https://www.nature.com/articles/s41467-023-40864-z?utm_source=chatgpt.com)
* **Nanophotonics & wireless implants** — on-chip beam shaping and battery-free µLEDs promise dense, patterned deep-brain illumination with minimal tethers. [Nature](https://www.nature.com/articles/s44328-025-00024-3?utm_source=chatgpt.com), [ScienceDirect](https://www.sciencedirect.com/science/article/pii/S0896627316310042?utm_source=chatgpt.com)

---

# References

**Foundational & reviews**

* Boyden et al., 2005 (ChR2 in neurons). [Nature](https://www.nature.com/articles/nn1525?utm_source=chatgpt.com)
* Yizhar et al., 2011 (Neuron primer). [Cell](https://www.cell.com/neuron/pdf/S0896-6273%2811%2900504-6.pdf?utm_source=chatgpt.com)
* Deisseroth, 2017 (form & function of ChR). [Science](https://www.science.org/doi/10.1126/science.aan5544?utm_source=chatgpt.com)

**Excitation & inhibition toolsets**

* Chronos/CheRiff/Chrimson review set (2015). [Nature](https://www.nature.com/articles/s41598-023-31384-3?utm_source=chatgpt.com), [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC8435545/?utm_source=chatgpt.com)
* ChRmine (ultrapotent, red-shifted). [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC9042976/?utm_source=chatgpt.com)
* GtACR1/2 (anion channels). [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC12220681/?utm_source=chatgpt.com)
* SSFO/SOUL (bistable). [PubMed](https://pubmed.ncbi.nlm.nih.gov/19079251/?utm_source=chatgpt.com), [ScienceDirect](https://www.sciencedirect.com/science/article/pii/S0896627320302397?utm_source=chatgpt.com)
* Soma-targeting (stGtACR, stCoChR). [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC6175909/?utm_source=chatgpt.com), [eLife](https://elifesciences.org/articles/63359?utm_source=chatgpt.com)
* BiPOLES (bidirectional, dual-color). [PubMed](https://pubmed.ncbi.nlm.nih.gov/34312384/?utm_source=chatgpt.com)

**All-optical & deep access**

* Cellular-resolution 2P holography reviews. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC10234433/?utm_source=chatgpt.com), [Nature](https://www.nature.com/articles/s41392-022-01234-1?utm_source=chatgpt.com)
* UCNP-mediated NIR optogenetics (Science 2018). [Science](https://www.science.org/doi/10.1126/science.aaq1144?utm_source=chatgpt.com)
* Implant-free deep optogenetics with ChRmine. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC7878426/?utm_source=chatgpt.com)
* Wireless/flexible photonics for optogenetics. [ScienceDirect](https://www.sciencedirect.com/science/article/pii/S0896627316310042?utm_source=chatgpt.com), [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC5640477/?utm_source=chatgpt.com)

**Opsin-free**

* INS/mid-IR modulation (opsin-independent). [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC8115038/?utm_source=chatgpt.com)
* Optocapacitance/nanoparticle photothermal stimulation. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC9042976/?utm_source=chatgpt.com), [PubMed](https://pubmed.ncbi.nlm.nih.gov/25772189/?utm_source=chatgpt.com)
* Photopharmacology (LiGluR; PORTLs/PTLs; in vivo reviews). [ScienceDirect](https://www.sciencedirect.com/science/article/pii/S0896627307003443?utm_source=chatgpt.com), [American Chemical Society Publications](https://pubs.acs.org/doi/10.1021/acscentsci.5b00260?utm_source=chatgpt.com)
* Critical perspective on magnetogenetics. [PubMed](https://pubmed.ncbi.nlm.nih.gov/25532138/?utm_source=chatgpt.com)

**Genetically encoded pathway control**

* CRY2–CIB (light dimerization), iLID, LOVTRAP. [PubMed](https://pubmed.ncbi.nlm.nih.gov/21037589/?utm_source=chatgpt.com), [Nature](https://www.nature.com/articles/nmeth.3926?utm_source=chatgpt.com)
* OptoSOS (Ras/ERK). [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC3925772/?utm_source=chatgpt.com)
* OptoXRs & new opto-GPCRs. [ScienceDirect](https://www.sciencedirect.com/science/article/abs/pii/S0959440X19300557?utm_source=chatgpt.com), [Nature](https://www.nature.com/articles/s41592-024-02285-8?utm_source=chatgpt.com)
* Light-tunable CRISPR (paCas9; LACE; caged gRNAs). [PubMed](https://pubmed.ncbi.nlm.nih.gov/26076431/?utm_source=chatgpt.com), [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC4412021/?utm_source=chatgpt.com)

**Editing & delivery enabling opto**

* SLENDR/vSLENDR (AAV-HDR in adult brain). [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC5691606/?utm_source=chatgpt.com)
* HITI (NHEJ knock-in in non-dividing cells). [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC5331785/?utm_source=chatgpt.com)
* Practical AAV delivery considerations (overview). [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC10319350/?utm_source=chatgpt.com)

Other:

* **Two-photon holography & cellular-resolution photostimulation** — pattern light in 3D while imaging calcium/voltage.
  *Intuition:* scan‐less holography or multiplexed 2P focuses light onto many ROIs simultaneously, enabling precise ensemble control + imaging. Start here: Packer et al., Emiliani/Papagiakoumou reviews. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC10234433/?utm_source=chatgpt.com), [Nature](https://www.nature.com/articles/s41392-022-01234-1?utm_source=chatgpt.com)

---

# Caveats

* **Light budgets & heat:** red/NIR scatters less but most opsins need visible photons; UCNP/ChRmine help but dose & safety scale badly with brain size. [Science](https://www.science.org/doi/10.1126/science.aaq1144?utm_source=chatgpt.com), [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC7878426/?utm_source=chatgpt.com)
* **Inhibition gotchas:** Cl⁻ tools can change intracellular chloride; soma targeting mitigates axonal depolarization. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC6010559/?utm_source=chatgpt.com)
* **Opsin-free approaches:** impressive demos, but many require high irradiances or nanoparticles; mechanisms (thermal vs. capacitive vs. mechanosensory) must be quantified carefully. [Nature](https://www.nature.com/articles/s41378-020-0153-3?utm_source=chatgpt.com), [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC9042976/?utm_source=chatgpt.com)

# Recent overviews or reviews for optogenetics for freely moving / freely behaving animals

* **Wireless optogenetics hardware** (compact survey): *Wireless Devices for Optical Brain Stimulation: A Review of Current Developments for Optogenetic Applications in Freely Moving Mice* (Cellular & Molecular Bioengineering, 2024/25 e-collection). Focuses on wireless powering, comms, device classes, trade-offs for unconstrained behavior. [PubMed](https://pubmed.ncbi.nlm.nih.gov/39949492/?utm_source=chatgpt.com)
* **Patterned light in behaving mice**: *Recent advances in light-patterned optogenetic photostimulation in freely moving mice* (Neurophotonics, 2024). Covers head-mounted optics, near single-cell patterning on the move, road map for next steps. [PubMed](https://pubmed.ncbi.nlm.nih.gov/38404422/?utm_source=chatgpt.com)
* **Tetherless optical neuromodulation** (orange-red → NIR/MIR): Review of red-shifted opsins, upconversion nanoparticle-mediated approaches, and photothermal routes enabling less-tethered experiments in vivo. Great for understanding behavior-compatible wavelengths and trade-offs. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC11306867/)
* **Integrated bioelectronics + optogenetics for brain–body circuits** (perspective with behaving-animal emphasis): *Integrated Bioelectronic and Optogenetic Methods to Study Brain–Body Circuits* (ACS Nano, 2024). Summarizes ultrapotent/red-shifted opsins and wireless μLED systems used in freely behaving animals. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC11544702/)
* Optical delivery tech that pairs well with free behavior: Review of optical fiber technologies for optogenetics, including flexible/biodegradable interfaces that reduce tethers and bulk. [ScienceDirect](https://www.sciencedirect.com/science/article/abs/pii/S0030399224007904?utm_source=chatgpt.com)

# Optogenetic stimulation across in vitro and in vivo (broad reviews)

* **Comprehensive primer across cells -> organisms**: *Optogenetics for light control of biological systems* (Nat Rev Methods Primers, 2022). Covers actuators (ion channels, GPCRs, cyclases), targeting, illumination strategies (1p/2p, holography), applications from cultures/slices to behaving animals. [PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC10627578/)
* **Biophysics & opsin fundamentals**: *Optogenetic control of neural activity: the biophysics of microbial rhodopsins in neuroscience* (Quarterly Reviews of Biophysics, 2024). Excellent for mechanism-level understanding that informs both in vitro and in vivo stimulation design. [Cambridge University Press & Assessment](https://www.cambridge.org/core/journals/quarterly-reviews-of-biophysics/article/optogenetic-control-of-neural-activity-the-biophysics-of-microbial-rhodopsins-in-neuroscience/6F9E422992D9418D9418B5F5B5DC5178?utm_source=chatgpt.com)
* Neurophotonics landscape (control + readout): *Neurophotonics: a comprehensive review, current challenges, and future trends* (Frontiers in Neuroscience, 2024). Broad survey including optogenetic stimulation modalities and how they integrate with optical recording—useful for planning full experiments. [Frontiers](https://www.frontiersin.org/journals/neuroscience/articles/10.3389/fnins.2024.1382341/full?utm_source=chatgpt.com)
* **Practical experimental design for behavior**: *Theoretical Considerations for Optimizing the Use of Optogenetics with Complex Behavior* (Current Protocols, 2023). Not a device review—rather, a clear guide to designing stimulation paradigms that translate from slices/cultures to behaving tasks. [Current Protocols](https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpz1.836?utm_source=chatgpt.com)
* Application-focused review: *Toward Optogenetic Hearing Restoration* (Annual Review of Neuroscience, 2024). Domain-specific, but an excellent model for translating opsins + light delivery from bench to in vivo function. [Annual Reviews](https://www.annualreviews.org/content/journals/10.1146/annurev-neuro-070623-103247?crawler=true&utm_source=chatgpt.com)

---

Subtopics include wireless μLED implants, red-light opsins for deep targets, or two-photon patterned stimulation.


[Optogenetic control of apical constriction induces synthetic morphogenesis in mammalian tissues](https://www.nature.com/articles/s41467-022-33115-0)

Optogenetics can also be used for some form of neuronal circuit mapping or connectome mapping, see: [Rapid learning of neural circuitry from holographic ensemble stimulation enabled by model-based compressed sensing](https://www.biorxiv.org/content/10.1101/2022.09.14.507926v3.abstract) or this [compressive sensing ref](https://www.cell.com/patterns/fulltext/S2666-3899(23)00224-6).


Besides using optogenetics to stimulate cells or neurons, you can also use GFP and other techniques to get data out of cells, such as genetically encoded voltage indicators (which are an alternative to various fluorescent dyes). See [Voltron2: Sensitivity optimization of a rhodopsin-based genetically encoded fluorescent voltage indicator](https://www.sciencedirect.com/science/article/pii/S0896627323002052).


See also [[morphogenesis]].