Optogenetic stimulation is a genetic technique that enables scientists to activate or inhibit the activity of specific neuron populations using light. In this guide, we will explore the key questions related to optogenetics.

For years, neuroscientists have been fixated on understanding how behavior and cognition arise from neural circuit activity.

To begin investigating these questions, scientists have been employing slow pharmacological and imprecise electrical stimulation techniques to control neural activity. However, with different classes of neurons communicating through vast, complex networks and fast electrical signals, it is essential to develop a method that can control neurons with precision to decode the function of neural circuit activity.

In 1979, Francis Crick proposed a novel solution: use light to control neurons (Boyden et al. 2005). Derived from this very idea and with the effort of many scientists, the revolutionary technique of optogenetics was born.

Optogenetics, a genetic method to turn select neurons on or off with light, was invented in 2005 by Karl Deisseroth and Edward Boyden (Boyden et al. 2005). Notably, their development of optogenetics began with a discovery by Peter Hegemann who successfully expressed the blue light-depolarizing opsin, channelrhodopsin-2 (ChR2), in cell culture (Nagel et al. 2003).

Expanding upon these findings, Deisseroth and Boyden virally expressed ChR2 in neurons, and, miraculously, they demonstrated for the first time that neurons could be activated using blue light (Boyden et al. 2005).

With millisecond precision, optogenetics enables the fast control of neuronal spiking. As a technique using both optics and genetics, the term opto-genetics was coined from their extraordinary findings

Optogenetic-expressing cells (in blue) are activated by blue light illumination.

What are the Applications of Optogenetics?

Optogenetics has benefited many scientific fields by providing the ability to control the activity of different cell-types with millisecond precision.

In neuroscience, optogenetics has enabled scientists to link neural circuits, behaviour, and function (Kim et al. 2017). Optogenetics can be performed both in vitro with electrophysiology and in a behaving animal with calcium imaging. Of great benefit to neuroscientists, optogenetics has been adapted for use in rodents, primates, C.elegans, drosophila, and zebrafish to study the neural correlates of cognition and behavior.

As a precise method to control cellular activity, optogenetics has impacted scientific fields beyond neuroscience with recent contributions to cell biology and cardiac research (Repina et al. 2017; Ferenczi et al. 2019).

From a clinical perspective, the application of optogenetics has begun to be used for exploring vision restoration and deep-brain stimulation in motor diseases (Towne & Thompson 2016).

With a causal method to analyze the function of neuron activity in real-time, optogenetics has advanced our understanding of the brain and may one day have significant clinical implications.

An example demonstrating the use of optogenetics in slice electrophysiology.

Optogenetics unlocks the ability to activate or inhibit select populations of neurons. With this technique, researchers have been able to investigate the causal relationship between neural activity, function, and behaviour. The popularity of optogenetics is evident from the surge in publications featuring the technique since its invention in 2005 (Boyden et al. 2005).

Labs that are new to the technique are probably wondering, what equipment do you need to perform optogenetics?

Example microscope optogenetics setup.

Freely-Behaving Optogenetics

Optogenetics provides a causal method to test the link between neural circuits and behaviour. Thus, researchers need to perform this method in a freely-behaving animal, which requires a different setup compared to microscopy experiments.

There are three main components for freely-behaving experiments: an optical cannula, a fiber-optic cable, and a light source.

As previously mentioned, the optical cannula is implanted into the region of interest expressing the optogenetic probe to illuminate light into the brain. Generally, the optical fiber is coupled to the optical cannula and connected to a fiber-coupled light source. This enables light to travel from the light source to the brain of the freely-behaving animal. A flexible optical fiber allows the animal to behave freely and illuminate the brain for optogenetic stimulation.

Depending on the complexity of the animal’s behaviour, researchers may use a commutator, which prevents the fiber from getting twisted and restricting the animal’s behaviour.

Researchers may want to synchronize optogenetic stimulation with certain behavioral events or equipment, similar to microscopy optogenetics experiments. For example, if an animal moves into a certain region, researchers may want to perform optogenetics for a specific amount of time. This is possible through synchronization with TTL or analog signals.

Example setup for freely-behaving optogenetics.

When selecting an optogenetic light source, you need to consider two key factors: the required light wavelength and intensity.

First, the opsin you select has a specific activation spectrum, such that an opsin will only be activated by certain wavelengths of light. An opsin usually has a peak wavelength at which it will require the least power to elicit a response. Wavelengths farther away from the peak wavelength will require a more powerful light output to elicit an optogenetic response. Therefore, it’s best to choose a light source closer to the peak wavelength to achieve optimal activation. For example, most researchers will use wavelengths close to 470nm for optogenetic activation of ChR2.

Second, the required light intensity for your experiment is dependent on the field of view and the opsin you select. The field of view, which is dependent on the optical fiber (for in vivo experiments) or the microscope objective, will determine how large of an area you need to cover with your light source. Opsins have an intensity threshold for eliciting a response, resulting from illumination. These two variables are directly coupled, such that optimal illumination power is required to elicit a response, and increasing the field of view requires more power as you have to cover a larger area with a given minimum intensity.

Now that you understand the requirements for an optogenetic light source, let’s discuss the two most commonly used light sources for this application: LED light sources and laser sources. 

You may be wondering, what are the differences between these light sources? Which light source is best for my optogenetics experiment?

Laser Source

Lasers are commonly used for optogenetic experiments that require higher intensity and/or tighter focus of light. Lasers provide very high intensity in a single spot, and hence, they are often used for laser-scanning applications. 

Compared to LEDs, lasers have a very narrow spectral width, enabling users to gain high intensity at a specific wavelength. Lasers can be well collimated to fit on a microscope, or they can be efficiently coupled into an optical fiber for optogenetic experiments that require high optical power.

Although lasers provide high intensity for optogenetics experiments, there is a list of potential drawbacks. In itself, the high output power may be a pitfall for specific optogenetic experiments. In particular, for standard microscopy experiments, lasers may provide too much output power and cause tissue damage. For microscopy, the laser beam must be expanded to cover the field of view and must be corrected for laser speckle.

Unlike LEDs, lasers have limited availability of wavelengths; they are also much more expensive than LEDs, and they may require eye safety measures, such as interlock.

Due to higher optical coupling efficiency, lasers are particularly useful for performing optogenetics across a large area, such as a macroscope. Lasers can also be used for in vivo optogenetic applications where high intensity may be needed; however, most LEDs are suitable for in vivo optogenetics applications.

Where lasers can outperform LEDs is for laser-scanning and two-photon microscopy optogenetic experiments, as they can provide high-intensity in a small spot at longer wavelengths.

Optogenetics is a technique to manipulate genetically-defined neural activity with light, and it has two main advantages:

1. Millisecond Temporal Precision 
2. Genetically-Defined Spatial Precision

Temporal precision enables researchers to turn neurons on or off with millisecond timing. This fast timing can closely mimic the firing rates of neurons in the brain. 

Spatial precision enables researchers to manipulate the activity of genetically-defined neuron populations (e.g., inhibitory neurons). This provides a causal link between the manipulation of the genetically-defined population and the variable being measured.

In optogenetic experiments, only neurons expressing the optogenetic probe are controlled, leading to activation or inhibition of those neurons.

However, standard optogenetic light sources can only illuminate the entire field of view and, as a result, they stimulate all neurons expressing the optogenetic probe. Such a system may be adequate for some experiments in which all optogenetic-expressing neurons can be stimulated at the same time, but more advanced experiments may require the specificity to selectively stimulate individual neurons within the field of view.

The ability to illuminate select neurons within a population of optogenetic-expressing neurons is called cellular-resolution optogenetics (Shemesh et al. 2017). This method requires more sophisticated spatial specificity to selectively stimulate individual neurons within an optogenetic-expressing population.

Comparison between widefield and cellular-resolution optogenetic stimulation.

The ability to illuminate certain individual neurons is not a limitation of optogenetics, but a limitation of the standard optogenetic light sources used in most optogenetic systems. Standard light sources can only illuminate all cells within the field of view, and they cannot control where they illuminate to target individual neurons.

The development of spatially targeted light technologies has enabled researchers to control where light illuminates the sample, such as an individual cell, making cellular-resolution optogenetics possible (Ronzitti et al. 2017). For example, a digital micromirror device (DMD), such as Mightex’s Polygon, allows researchers to illuminate multiple individual neurons or regions simultaneously to perform cellular-resolution optogenetics.

Cellular-resolution optogenetics has multiple applications in the field of neuroscience. For example, scientists can study neural circuits and decode neural patterns at the level of individual neurons (Anastasiades et al. 2020; Tran et al. 2019). These types of studies have been carried out both in vitro and in vivo using different technologies (Shemesh et al. 2017; Anastasiades et al. 2020; Tran et al. 2019; Chen et al. 2019). The experimental applications for cellular-resolution optogenetics are endless.

The ability to illuminate select neurons within a population of optogenetic-expressing neurons is called cellular-resolution optogenetics (Shemesh et al. 2017). To use this technique, researchers require more sophisticated spatial specificity to selectively stimulate individual neurons within an optogenetic-expressing population.

The development of spatially targeted light technologies has enabled the control of where light illuminates the sample, such as an individual cell, making cellular-resolution optogenetics possible (Ronzitti et al. 2017).

What systems are available for cellular-resolution optogenetics?

Holographic Projection

Parallel excitation, as in phase and amplitude, spatial light modulation techniques may be used for cellular-resolution optogenetics. Parallel excitation methods are more adaptable to a variety of experimental needs, as they allow simultaneous illumination of multiple diffraction-limited spots on the sample.

Phase modulation commonly takes advantage of computer-generated holograms (CGHs) to stimulate multiple points at once. The desired illumination pattern is designed on a computer, after which the projection for patterned illumination is generated by a numerical algorithm that calculates the appropriate phase hologram. This hologram is then projected onto a spatial light modulator (SLM). A reference beam is reflected onto the SLM and through a set of imaging optics, to deliver a reconstructed illumination pattern onto the sample.

Holographic systems have the advantage of being able to stimulate any number and shape of three-dimensional patterns simultaneously onto the sample. Another advantage compared to galvo-based scanning is the holographic system offers far greater temporal resolution than serial scanning methods.

Holographic systems require up to several minutes to calculate and generate new patterns. This means for applications requiring quick or real-time pattern generation the holographic method would not be the most suitable. As the computation of new holographic patterns may take minutes to produce, this method is not suitable for applications requiring real-time pattern generation, uploading, and activation.

A holographic projection setup.

While this system theoretically achieves good illumination intensity, in practice, there are some efficiency concerns. Illumination spots are non-uniform, characterized by a bright focus with continuously diminishing intensity towards edges (Lutz et al. 2009). This results in poor contrast between illumination points and background and, hence, poorly defined edges of illumination.

The holographic system is the most costly and most difficult to integrate with existing systems. Holographic systems require a fair amount of expertise to use properly and can be very expensive, due in large part to the high cost of the parts (lasers and SLM, as well as computer hardware and software). There are also concerns with the lack of reliable precision and repeatability of the patterned illumination for the system, in particular with unintended speckling patterns. Holographic systems, therefore, require a significant level of expertise to create a working system for experimental use.