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Optogenetics Guide


The field of optogenetics integrates optics and genetic engineering to measure and manipulate cells (frequently neurons) and their governing biomolecular processes. The tools and technologies developed for optogenetics research utilize light to detect, measure, and control molecular signals and cells in order to understand their functions.

expression of a channelrhodopsin in neurons

Example schematic of an optogenetics procedure. A channelrhodopsin, fused to mCherry, is expressed in neurons (red dots). When exposed to light of the correct wavelength, the pore opens, cations flow into the cell (yellow dots), and the neuron is activated.

Optogenetics tools can be broadly classified based on their functions into two groups:

  • Actuators are genetically-encoded tools for light-activated control of proteins; e.g., microbial opsins and optical switches
  • Sensors are genetically-encoded reporters of molecular signals; e.g., calcium indicators
In this overview, we will focus on the common actuators used in optogenetics. For information on sensors, check out our biosensors collection.

Microbial Opsins

Opsins are light-gated ion channels or pumps that absorb light at specific wavelengths. Upon activation by light, these channels and pumps respond by opening or closing, which conducts the flow of ions into or out of the cell. Scientists have identified a variety of naturally occurring microbial opsins that respond to different wavelengths of light, like blue or yellow light. These various opsins also initiate different electrochemical responses, such as nonspecific cation influx vs. proton efflux. Researchers have used genetic engineering to improve these natural opsins - by inducing point mutations to alter the absorption spectrum or adding trafficking signals to localize opsins to the cell membrane.

Microbial opsins, such as those described below, can be targeted and expressed in specific subsets of neurons, allowing precise spatiotemporal control of these neurons by turning on and off the light source. Optogenetics has been broadly applied to study the physiology of the brain and nervous system to better map and understand neuronal circuits. Optogenetic tools have also been used to trigger and study certain behavioral responses in model organisms like mice, zebrafish, and Drosophila. These tools have been instrumental in neurological disorder research, helping scientists to better understand Parkinson’s disease, depression, drug addiction, and more.

Below is a list of commonly used microbial opsins, including a brief overview of the wild-type opsin of each and some examples of relevant variants. This list is not exhaustive - please browse our curated list of opsins here to find the right optogenetics plasmids for your experiments.


Excitatory Channelrhodopsin

Channelrhodopsins are a foundational optogenetics tool - they typically allow the fast depolarization of neurons upon exposure to light through direct stimulation of ion channels. Naturally occurring channelrhodopsins were discovered in the green algae Chlamydomonas reinhardtii. Channelrhodopsin-1 (ChR1) is excited by blue light and permits nonspecific cation influx into the cell when stimulated. Channelrhodopsin-2 (ChR2), the first widely adopted optogenetic tool, is also a blue light activated cation channel. ChR2 is preferred over ChR1 because ChR2 has higher conductance at physiological pH and trafficks well to the membrane.

  • Excitatory (Depolarizing) ChR Variants. The optogenetic toolbox has been expanded by scientists through both the identification of novel ChRs from other algal species and the development of synthetic variants to enhance the functionality of ChR. Examples of ChRs from other species include: CsChR (from Chloromonas subdivisa), CoChR (from Chloromonas oogama), and SdChR (from Scherffelia dubia). Synthetic variants have been created via genetic point mutations, codon optimization, and chimeric fusion of domains from two different ChRs. These ChR variants still function as light-gated, cation channels resulting in excitation (depolarization) of the neuron. Feature enhancements include:
    • Increased photocurrent amplitude
      • Examples: ChR2(H134R), C1V1(t/t), ChIEF
    • Increased channel (on/off) kinetics:
      • Examples: ChETA, C1V1(t/t), ChrimsonR
    • Red-shifted peak action spectra
      • Examples: VChR1, C1V1(t/t), Chrimson, ChrimsonR, Chronos

  • Inhibitory Channelrhodopsin
  • Inhibitory (Hyperpolarizing) ChR Variants. Alternatively, ChR variants that inhibit neurons have been created and identified in other species - by acting as light-gated chloride channels, these variants result in the hyperpolarization of neurons. Examples of anion channel variants from other species include: GtACR1 and GtACR2 (from the cryptophyte Guillardia theta). Other feature enhancements include:
    • Increased photocurrent amplitude
      • Examples: iChloC, SwiChRca, Phobos, Aurora

Browse Channelrhodospin plasmids.


Halorhodopsin schematic

Halorhodopsins are light-gated inward chloride pumps isolated from halobacteria. Wild-type halorhodopsin, known as NpHR (from Natronomonas pharaoni), causes hyperpolarization (inhibition) of the cell when triggered with yellow light, thus inhibiting function of the neuron.

  • NpHR Variants. Variants have been engineered with enhancements such as:
    • Human codon optimization
      • Example: Halo
    • Increased photocurrent amplitude
      • Examples: eNpHR, eNpHR2.0, eNpHR3.0
    • Red-shifted peak action spectra
      • Example: Jaws

Browse Halorhodopsin plasmids.


Archaerhodopsin schematic

Archaerhodopsin-3 (Arch) from Halorubrum sodomense is also commonly used to inhibit neurons in optogenetic experiments. Arch is a light-activated outward proton pump that hyperpolarizes (inhibits) the cell when triggered by green-yellow light.

  • Arch Variants. Arch variants have been developed with the following enhancements:
    • Increased light sensitivity
      • Example: ArchT
    • Increased photocurrent amplitude
      • Examples: eArch3.0, eArchT3.0

Browse Archaerhodopsin plasmids.

Wild-Type Mac

Leptoshpaeria schematic

Leptosphaeria rhodopsin (Mac) is a blue-green light-activated proton pump derived from the fungus Leptosphaeria maculans. Mac and its variants allow for inhibition of neurons using blue-green light.

  • Mac Variants. Mac variants have been engineered to include enhancements such as:
    • Improved photocurrent amplitude
      • Example: eMac3.0

Browse Leptosphaeria plasmids.

Glossary of Microbial Opsin Variants

Opsin Type Variant Description Peak Response Spectra (nm)
Channelrhodopsins: cation channels from Chlamydomonas reinhardtii ChR2 Widely used light-gated cation channel from Chlamydomonas reinhardtii (CrChR2) 470
ChR2/H134 Widely used variant with larger photocurrent compared to CrChR2 450
ChETA E123T mutation; creates faster kinetics but reduces photocurrent amplitude 490
ChR/T159C T159C mutation; displays increased photocurrents 470
SFO/SSFO Step function Opsins and Stabilized Step Function Opsins. Mutations at C128 and D156 of CrChR delay the closing of the channel 470(activ.), 590(inact.)
ReaChR Red-activatable variant of CrChR2 590
Channelrhodopsins: cation channels from other species VChR1 Channelrhodopsin from Volvox carteri 570
Chronos High-speed, light-sensitive channel from Stigeoclonium helveticum 500
Chrimson, ChrimsonR Red-light drivable channel from Chlamydomonas noctigama. ChrimsonR carries K176R mutation. 590
PsChR2 High-efficiency blue-shifted channelrhodopsin from Platymonas subcordiformis 510
CoChR Channelrhodopsin from Chloromonas oogama
CsChR Channelrhodopsin from Chloromonas subdivisa
CheRiff Channelrhodopsin from Scherffelia dubia
Channelrhodopsins: cation channels- chimeras C1C2 ChR1-ChR2 chimera 470
ChIEF, ChEF, ChD Engineered by chimeragenesis of ChR1 and ChR2 450-470
C1V1 ChR1-VChR1 chimera with E122T and E162T mutations 540
Channelrhodopsins: chloride channels iChloC Chloride-conducting channel, CrChR2 with mutations E90R, D156N, T159C, CE83Q & E101S 465
SwiChRca Chloride-conducting channel, Step-waveform inhibitory ChR with mutation C128A (derived from C1C2 chimera) 475
GtACR Chloride-conducting channel from Guillardia theta 515(ACR1), 470(ACR2)
PsChR1 Red-shifted chloride-conducting channel from Proteomonas sulcata 540
Phobos Blue-shifted iC++ variant 467
Aurora Red-shifted anion-selective ReaChR variant 517
Halorhodopsins Jaws Red-shifted, light-driven inward chloride pump from Haloarcula (Halobacterium) salinarum (strain Shark), used to silence neuronal activity 632
Halo/NpHR Light-driven inward chloride pump from Natronomonas pharaonis, used to silence neuronal activity 589
eNpHR 3.0 NpHR with addition of trafficking signal from Kir2.1 and ER export signal provide improved membrane targeting 589
Archaerhodopsins Arch Light-activated outward proton pump from Halorubrum sodomense 566
eArch 3.0 Arch with addition of trafficking signal from Kir2.1 566
ArchT Light-activated outward proton pump from Halorubrum strain TP009. Improved light sensitivity over Arch. 566
eArchT 3.0 ArchT with addition of trafficking signal from Kir2.1 566
Leptosphaeria rhodopsins Mac Light-activated outward proton pump from Leptosphaeria maculans 540
eMac 3.0 Mac with addition of trafficking signal from Kir2.1 540

Plan Your Optogenetics Experiment

When designing your optogenetics experiment, you’ll need to pick both an opsin and a delivery system. Here are some key factors to consider:

Optogenetic excitation or optogenetic inhibition. First things first: do you want to turn ON or turn OFF neurons in your experiment? Depending on your answer, you’d pick an excitatory or inhibitory opsin, respectively.

Color of activation light. There are a variety of different activation wavelengths, ranging from blue to yellow to red. Red light exhibits better tissue penetrance, which may allow you to place the optic fiber outside of the brain, rendering the experimental procedure less invasive. Different activation wavelengths also make it possible to combine multiple opsins in the same experiment. For example, you could use different colors of light to activate/silence the same neuronal population, or activate different neural populations at distinct times.

Temporal considerations. Temporal precision is key in optogenetic experiments. Your experimental design will determine whether you’ll need short or long periods of neuronal activation/inactivation periods. These can range from msec (hChR2) to “long lasting,” e.g. seconds to minutes with stable step-function opsins (SSFOs).

Opsin delivery systems. Two factors determine which neuronal population is manipulated in a given experiment: the expression of the opsin and the area that is being illuminated. There are several different ways to control opsin expression. Generally, the most robust and stable expression is achieved in a transgenic mouse line, such as for example the VGAT-ChR2 mouse, where ChR2 is expressed under the control of the vesicular gamma aminobutyric acid (GABA) transporter (VGAT) and ChR2 is thus expressed in all GABAergic neurons. In this case, the subpopulation of GABAergic neurons being activated by the light is controlled via the placement of the optic fiber.

Using viral vectors for opsin delivery results in more localized expression of the opsin. Depending on the virus and promoter system used, there is an incubation time (days to weeks) until peak expression of the opsin is reached. This approach is especially powerful when combined with site specific recombinase technology like Cre-lox, which allows you to express an opsin ONLY in a genetically defined subpopulation of cells within the injection site of the viral vector, rather than in all cells. For example, using a viral vector with a floxed opsin in a VGAT-cre animal will result in expression of the opsin only in inhibitory neurons near the injection site.

Optical Switches

Optical Switch example

Researchers have harnessed plant and bacterial photoreceptors to create protein systems controlled by light. These “photoswitchable” proteins offer exquisite spatial and temporal control of protein activity. Phytochrome, cryptochome, and LOV (light oxygen voltage)-based systems have been used in many experimental contexts, including protein activation, membrane localization, and transcriptional activation.

In the widely used Cry2-CIB1 system, light induces a conformational change in cryptochrome Cry2 to permit CIB1 binding. As shown in the figure above, this light induced binding can be utilized to control the localization of a protein of interest. A genetically-encoded nuclear Cry2 fusion can thus direct a CIB1-protein fusion to the nucleus upon light activiation. Alternatively, when one half of a given protein, such as the Cre recombinase, is fused to Cry2 and the other half to CIB1, light-stimulated heterodimerization can reconstitute the protein. This principle has also permitted the design of synthetic two-part transcription factors - Cry2 and CIB1 are fused to a transcriptional activation domain and DNA binding domain, allowing light to activate transcription.

Phytochromes and light oxygen voltage (LOV) domains function similarly to cryptochromes with light-induced changes in protein conformation/dimerization. In addition to the applications described above, both LOV and Dronpa can control activity of a fused protein through allosteric interactions. Researchers continue to engineer new optobiology tools and improve upon previous tools through directed mutagenesis - use Addgene's Optical Switch Plasmid Table to search for plasmids for your next experiment. For a full description of different domains used in these plasmids, see the Glossary below.

Glossary of Optical Switches

Category Variant Description Peak Response Spectra (nm)
BLUF domains bPAC Light-activated adenylyl cyclase from Beggiatoa sp. for light-induced cAMP modulation. Utilizes BLUF (blue light utilizing FAD) domains. 441
Phytochromes BphP1–PpsR2 Bacterial phytochrome BphP1 and its natural partner PpsR2 from Rhodopseudomonas palustris bacteria. Key feature is the sensitivity to 740- to 780-nm near-infrared light. 740–780
Phy–PIF Light-controlled Phytochrome Interacting Factor 6 (PIF6) to PhyB (Phy) interaction 660
Cryptochromes Cryptochrome 2 and CIB1 Blue-light–mediated induction of protein interactions based on Arabidopsis thaliana cryptochrome 2 and CIB1 450
LARIAT Inhibits protein function by reversibly sequestering targets into large, optically assembled protein architectures in living mammalian cells.
LITE Two-hybrid system integrating the customizable TALE DNA-binding domain with cryptochrome 2 protein and its interacting partner CIB1 to control transcription effectors
OPTOSTIM PHR domain2 of cryptochrome 2 (Cry2) from Arabidopsis thaliana is fused to truncated forms of cytosolic STIM1 to control calcium channels ~488
LOV domains LOV-based Light-oxygen-voltage-sensing (LOV) domains are protein sensors used by many plants, microalgae, fungi and bacteria to sense environmental conditions. These domains are fused to different effector domains of proteins to generate novel engineered light-controlled molecules. ~450–495
BACCS light-activated calcium channel switch
iLID Improved light-inducible dimers; Bacterial SsrA peptide is embedded in the LOV2 domain (iLID plasmid), while its natural binding partner SspB is included in a separate plasmid; Blue light activation induces a conformational change in the LOV2 domain which allows SsrA to bind with SspB
LAD Light-activated dimerization, using proteins GIGANTEA (GI) and the light oxygen voltage (LOV) domain of FKF1
LITEZ Light-inducible transcription using engineered zinc finger proteins, using proteins GIGANTEA (GI) and the light oxygen voltage (LOV) domain of FKF1
LINUS light-inducible nuclear localization signal based on the LOV2 domain of Avena sativa phototropin 1
LOVETRAP reversibly sequester and release proteins from cellular membranes using light. light-induced protein dissociation using protein A fragments (called Zdk) that bind to the LOV domain only in the dark. attaching only one member of the Zdk/LOV2 pair to the target protein, and the other to the membrane where the target protein is to be sequestered
Lumitoxin Peptide toxin for blocking K + (Kv) channels fused to LOV2 photoswitch. Illumination causes the photoswitch to unfold, lowering the toxin's local concentration near the cell surface, and enabling the ion channel to function.
TULIPs Tunable light-inducible dimerization tags (TULIPs), plasmids allow cloning of protein coding sequences with GFP-LOVpep, cpPDZ, ePDZb and ePDZb1 as tags.
pDawn/pDusk Blue-light photoreceptor for light-controlled gene expression. These plasmids rely on the engineered two-component regulatory system YF1/FixJ. YF1 is a synthetic, photosensitive kinase, which uses the ubiquitous flavin mononucleotide as its chromophore, and phosphorylates the transcriptional activator FixJ in the absence of blue light. 470
Fluorescent Protein domains Dronpa based Light-inducible Dronpa mutant domains that associate and cage a protein in the dark, while dissociate and activate the protein in the light ~400 (on)/~500 (off)
PhoCl Photocleavable protein engineered from a green-to-red photoconvertible fluorescent protein 400
UVR8 domains UVR8 plant photoreceptor protein that forms photolabile homodimers with slow reversal kinetics and a UV-B absorption profile 282


Optogenetic Switches from OptoBase, W. Weber Lab

Edward Boyden's Lab: Synthetic Neurobiology Group Optogenetics Resources

Karl Deisseroth's Lab: Optogenetics Resource Center

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