Receive a Chapter Per Week via Email for Our eBook Optogenetic Stimulation: The Ultimate Guide
Optogenetic stimulation is a genetic technique that enables scientists to activate or inhibit the activity of specific neuron populations using light.
This guide explores questions related to optogenetics, including what’s equipment is necessary for optogenetics? What systems are available for optogenetics?
Click the links below to jump to a specific chapter and learn more about optogenetics.
Chapter 1: What is Optogenetics?
Chapter 2: What Equipment Do You Need to Perform Optogenetic Stimulation?
Chapter 3: Selecting an Optogenetic Light Source: LED vs. Laser
Chapter 4: Widefield Vs. Cellular-Resolution Optogenetics: What’s the Difference?
Chapter 5: What Systems are Available for Cellular-Resolution Optogenetics?
For years, neuroscientists have been fixated on understanding how behavior and cognition arise from neural circuit activity.
To begin investigating these questions, scientists originally employed slow pharmacological and imprecise electrical stimulation techniques to control neuron activity. However, with different classes of neurons communicating through vast, complex networks and fast electrical signals, it is essential to identify a method with both high spatial and high temporal resolution to precisely control select neurons and to causally decode the function of neural circuit activity.
In 1979, Francis Crick proposed a novel solution: use light to control neuron (Boyden et al. 2005). Derived from this very idea and with the effort from many other scientists, the revolutionary technique 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 the 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 enabled 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.
Optogenetics has been possible through the discovery of opsins, such as ChR2. Opsins are light sensitive channels that cause depolarization or hyperpolarization of the cell through mechanisms such as the influx of ions or protein signaling cascades (Kim et al. 2017). Opsins are sensitive to specific wavelengths of light.
For example, blue light (~470 nm) activates ChR2, leading to an influx of Na+ ions in turn depolarizing the neuron (Boyden et al. 2005). Viral expression has enabled researchers to express optogenetic opsins in the brain of animals, such as rodents.
Multi-disciplinary collaborations between neuroscientists, biologists, and engineers have led to the expansion of the optogenetic toolbox. Opsins have been discovered for manipulating neurons on or off at varying speeds and with different wavelengths of light (see Table 1).
For example, Halorhdopsin, an inhibitory opsin, was found to turn off neurons, and red-activated opsins, such as JAWS, were developed to penetrate deeper in the brain (Kim et al. 2017). Furthermore, viral strategies for expressing optogenetic proteins in the brain have advanced, enabling scientists to manipulate the activity of specific cell classes to explore brain-wide projections (Kim et al. 2017).
|Optogenetic Construct||Excitation Wavelength||Function|
Optogenetics has benefited many scientific fields by allowing scientists to control the activity of different cell-types with millisecond precision.
In neuroscience, optogenetics has enabled scientists to causally link neural circuits, behaviour, and function (Kim et al. 2017). In addition, optogenetics can be performed in both in vitro experiments with electrophysiology, or in a behaving animal to investigate the link between cell-types and behaviour. Of great benefit to neuroscientists, the use of 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 meticulously 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?
The first step to successfully perform optogenetics is grasping the biological components. This consists of selecting the appropriate optogenetic probe, expressing the optogenetic probe in the brain region and cells of interest, and implanting an optical cannula, if you are performing freely-behaving experiments.
A crucial first step for optogenetic experiments is selecting the appropriate optogenetic probe to use.
Do you want to activate or inhibit the neuron population of interest? How fast do you want to stimulate the population of interest? Do you need to use a specific wavelength for optogenetic stimulation (e.g. combining with imaging)? (see this blog post for discussion of optogenetics and calcium imaging). These are some of the questions that will help you choose the appropriate optogenetic probe for your experiments (see here for a great guide from Addgene summarizing currently available optogenetic probes).
Once you’ve selected the optogenetic probe, it must be expressed in the brain. Neuroscientists employ two methods to express constructs in the brain: viral expression and transgenic mouse models. Viral expression involves injecting a virus encoding an opsin in the brain (Mei & Zhang 2012). This virus is linked to a gene of interest to target expression in a specific cell-type (Mei & Zhang 2012).
Neuroscientists employ viral expression to regulate opsin expression or restrict it to a particular brain region. This is useful because expression can vary depending on the brain region, cell-type, or virus. In addition, neuroscientists can use viral expression to express opsin in brain projections to map neural circuits across brain regions (Mei & Zhang 2012).
In comparison to viral injections, transgenic mice models are designed to express the opsin throughout the entire brain (Mei & Zhang 2012). Depending on the transgenic model, opsin expression can vary from region to region, such that one region may express the opsin more than the other. Transgenic mouse models may be used to produce more stably reproduced mouse lines for optogenetic expression in specific-cell types. Or, neuroscientists examining large cortical areas use these models as they require much more widespread expression, rather than a single region of interest.
Optogenetic probes can be expressed through viral expression (on the left) and this limits expression to a specific cell-type and localizes in a specific brain region. In comparison, transgenic expression (on the right) is widespread throughout the brain.
For freely-behaving optogenetic experiments, light needs to be transmitted into the brain while the animal is moving. This is accomplished by surgically implanting an optical cannula into the brain region expressing the optogenetic probe. An important step in selecting an optical cannula is selecting the appropriate length to reach the target brain region.
Once implanted, a light source is connected via a fiber-optic cable (see below for more details) and the area below the cannula will be illuminated. The spread of light and penetration depth will be dependent on tissue and light source wavelength (this is a helpful calculator from the Deisseroth lab: https://web.stanford.edu/group/dlab/cgi-bin/graph/chart.php).
An optical cannula is implanted into the region expressing the optogenetic probe.
Two experimental setups are used for optogenetic experiments: microscopy and freely-behaving. These two different approaches for optogenetic stimulation have distinct components and setups.
Optogenetics can be performed during microscopy experiments and synchronized with other microscopy methods.
For neuroscience experiments, researchers commonly integrate optogenetics into their electrophysiology rigs—usually on a standard epi-flourescence microscope—to probe neural activity during their recordings (Andrasi et al. 2017) or they integrate optogenetics with their two-photon imaging experiments to probe neural activity during imaging (Forli et al. 2018).
For cell biology, researchers will integrate optogenetics into the confocal imaging experiments to probe different genetic perturbations, for example (van Haren et al. 2018).
For these microscopy setups, there are two main components required to integrate optogenetics: 1) a light source and 2) a filter set. The light source is used to illuminate the sample and activate the optogenetic probe, and it is important to select the appropriate light source for your optogenetic probe (this will be discussed in the next post).
For most microscopy setups, a collimated light source will be connected to the back epi-fluorescence port to illuminate the sample. Depending on the light source, it will be connected via direct coupling to the backport or coupled via lightguide/fiber to the epi-fluorescence port. Light will travel through the epi-fluorescence port to the filter turret, where it will be directed onto your sample through the objective. The objective will dictate the field of view and power of the light source for optogenetic stimulation (i.e. lower magnification leads to a larger field of view but also lower light intensity).
As mentioned above, the light will be directed to the filter turret prior to being reflected onto the sample. Thus, it is crucial to select the appropriate filter set to direct the correct wavelength to the sample, and potentially block it from reaching the camera depending on your experiment.
Another method to couple a light source for optogenetics experiments is to externally illuminate the sample independent of the microscope. A fiber-coupled light source or spotlight light source may be used for this particular application. This method may be used because the epi-fluorescence port is already occupied or you want to target part of your sample (methods for cellular-resolution optogenetics will be discussed in a future post).
Of particular importance, researchers want to accurately synchronize their optogenetic stimulation with either electrophysiology or imaging equipment. The ability to synchronize and control the light source via analog or TTL signals is crucial for proper synchronization. For example, a research may want to illuminate their sample for a specific amount of time during an electrophysiology recording.
Example microscope optogenetics setup.
Optogenetics provides a causal method to test the link between neural circuits and behaviour. Thus, researchers have the 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: 1) an optical cannula , 2) a fiber-optic cable, and 3) 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 (how to select a light source will be discussed in the next post.). 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 behaviour, researchers may use a commutator, which prevents the fiber from getting twisted and from restricting the animal’s behaviour.
Similar to microscopy optogenetics experiments, researchers may want to synchronize optogenetic stimulation with certain behavioral events or equipment. For example, if an animal moves into a certain region, they may want to illuminate 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 will 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 output to elicit a 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?
LEDs are a prime choice for optogenetic experiments for a multitude of reasons. To start, LEDs are low cost, eye safe and have a long lifetime, making them a great choice for any lab looking to perform optogenetics.
In addition, LEDs come in a wide-range of wavelengths (UV to NIR), and hence scientists can usually find the appropriate LED that best fit their choice of opsins. Depending on the wavelength, the available output power can vary significantly between LEDs. In the visible range, LEDs can produce a large amount of power, making them an ideal solution for most optogenetic experiments.
Compared to other typical microscopy light sources, such as halogen or xenon lamps, LEDs have a low operating temperature and do not require any heat up or cool down time. This allows LEDs to be easily turned on and off for easy use during experiments, and scientists can easily modulate the intensity or pulse the LEDs without worry about their stability or causing damage. In addition, LEDs have a much longer lifetime than traditional light sources, and they do not require frequent replacement of bulbs, making them a more sound investment in the long-term.
Importantly for optogenetic experiments, LEDs can be switched on and off with microsecond timing for pulsing protocols. LEDs are easily triggered via TTL to synchronize pulsing with external equipment.
Compared to a laser source, LEDs have relatively high divergence, causing the power of the LED to be spread across the field of view, although on this front LEDs are far better than the traditional light sources such as a mercury lamp or a halogen lamp. As a result, LEDs are not ideal for applications such as laser-scanning systems in which light must be tightly focused into a tiny spot in order to provide the necessary spatial resolution for point-by-point scanning across the field of view. Secondly, LEDs, compared to lasers, also have a wider spectral bandwidth. This may or may not have a negative impact on your experiments, as you can easily block off the tails of the LED using optical filters. However, using optical filters could lead to a lower overall output power from the LED, as the output power is based on the full spectrum of the LED light source.
LEDs are suitable for a wide-range of optogenetic experiments. A popular use of LEDs is for widefield optogenetics on a microscope. LEDs are easily integrated into upright and inverted microscopes, and they provide sufficient output power for stimulation of all optogenetic probes. In addition, LEDs are easily synchronized with electrophysiological equipment.
LEDs are also ideal for freely-behaving optogenetics experiments, as they can be coupled with an optical fiber while providing sufficient power for such experiments, long lifetime, and low cost.
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. In addition, lasers can be well collimated to fit on a microscope, or can be efficiently coupled to 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, 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. In addition, for microscopy, the beam must be expanded to cover across the field of view and must be corrected for laser speckle.
Unlike LEDs, lasers have a limited availability of wavelengths; they are also much more expensive than LEDs, and may require eye safety measures such as interlock etc.
Due to a 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 as a technique to manipulate defined-neuronal activity with light has two main advantages:
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 explicit genetically-defined neuron populations (e.g. inhibitory neurons). This provides a direct 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 stimulated leading to activation or inhibition of specific neural activity (depending on the probe expressed).
However, standard optogenetic light sources can only illuminate the entire field of view and, as a result, stimulate all neurons expressing the optogenetic probe. Such a system may be adequate for some experiments in which all optogenetic-expressing cells are stimulated at the same time, but more advanced experiments may require the specificity to selectively stimulate individual neurons.
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 per say, 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 have no ability to control where they illuminate to target individual neurons.
The development of spatially targeted light technologies have enabled researchers to control where the light illuminates the sample (technologies for cellular-resolution optogenetics will be discussed in a later post), 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 select 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 is endless.
The ability to illuminate select neurons within a population of optogenetic-expressing neurons is called cellular-resolution optogenetics (Shemesh et al. 2017). Using 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 have enabled researchers to control where the 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?
The simplest form of patterned illumination is done using a serial scanning method. A focused beam of light is directed to a specific region of the sample using a pair of galvanometer mirrors.
This “galvo-based scanning” technique typically uses a high powered laser beam (either tunable or fixed-wavelength) for stimulation of opsins. The illumination is restricted to a single spot at a time, and the intensity of the spot follows a Gaussian distribution. Patterns more complex than a single spot are traced out on the sample by illuminating different points sequentially, commonly by raster or spiral scanning, which are each useful for different experimental conditions.
As patterns are generated by serial scanning, the temporal precision of the system is primarily restricted by the positioning actuators of the galvanometer mirrors. Conventional galvo-based scanning systems take ~100 μs to redirect the beam to a new ROI.
Consequently, pattern generation by scanning through the full field of view can take a significant amount of time. Such limitations of temporal precision in this system present an issue for stimulating and recording fast physiological events, such as the generation of action potentials, which fire on the order of 100s of Hz.
Improvements on the temporal precision of the galvo-based scanning systems have been made using resonant scanning mirrors (Wang et al. 2007; Packer et al. 2012), or acousto-optic deflectors (AODs), which can allow scanning access to any single point in the field of view within microseconds (Zhu et al. 2009). However, the minimum dwell time required for sufficient illumination to elicit a physiological response – an intrinsic property of the opsin molecules – limits the extent to which scan speed may be increased.
A galvo-scanning setup.
Galvo-based scanning is amenable to either one- or two-photon systems.
One-photon photostimulation can offer spatial resolution in the tens of microns in the lateral dimension, sufficient for excitation of small groups of neighbouring neurons. One-photon galvo-based scanning systems are primarily used in mapping studies, wherein light is directed to a small population of labeled neurons and the activity of their downstream targets is measured (Petreanu et al. 2009; Wang et al. 2007).
In two-photon systems, fine enough spatial precision can be achieved to stimulate single cells, or even single dendritic spines (Zhu et al. 2009; Packer et al. 2012). There may be limited applications of this illumination strategy, for two reasons. First, not all opsins are easily activated using two-photon excitation. Second, the fine spatial resolution offered by two-photon excitation using galvo-scanning can only be applied to a single neuron at a time. This setup is used in studies mapping anatomical features of cell types and projections (Rickgauer & Tank 2009; Prafash et al. 2012).
A key limitation in galvo-based scanning systems is that only a single, small spot can be illuminated at a given time. This inability to illuminate multiple regions of interest simultaneously fundamentally restricts the applications of the system, as it cannot address questions about how multiple neurons behave simultaneously. An approximate simultaneous measure of neural activity can be given when two nearby neurons are stimulated in quick succession, due to the kinetics of opsins yielding long opening times (Lin et al. 2009). However, the number of neurons that can be “simultaneously” activated in this manner is quite limited.
Galvo-based scanning is particularly advantageous in that there is minimal loss of light intensity along the optical pathway. Therefore, the focused beam offers good illumination intensity onto the sample allowing the same system to be used for both light delivery and imaging. Furthermore, the illumination is fairly uniform and is not susceptible to background interference from other light sources.
Patterned illumination using a galvo-based scanning system is generally user-friendly and easy to integrate with existing systems, and can be used for both in vitro and in vivo head-fixed experiments.
While the galvanometer mirrors themselves are not particularly expensive, the requirement for high powered lasers, which are a significant cost, as well as software for controlling the galvos mean that a fully constructed system can be expensive.
Since Galvo-based scanning systems offer high illumination intensity at a single point it is therefore best for studies requiring a high degree of illumination in a small area, as with neurotransmitter photolysis and in optogenetic mapping studies.
To address the issues presented by serial pattern illumination, parallel excitation, as in phase- and amplitude, spatial light modulation techniques, may be used. 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 over galvo-based scanning, the holographic system offers far greater temporal resolution than can be achieved by serial scanning methods. When using pre-calculated patterns holographic projections can switch between multiple discrete patterns at rates of 60-200 Hz in standard liquid crystal on Silicon (LCoS) SLM systems, and up to kHz rates in high end ferroelectric LC-SLM systems (Reutsky-Gefen et al. 2013; Papagiakoumou 2013).
Holographic systems will 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 a new holographic pattern may take minutes to produce, this method is not suitable for applications requiring real-time pattern generation, uploading, and activation.
A holographic projection setup.
Both one- and two-photon systems can be used with holographic patterned illumination.
With one-photon microscopy, hundreds to thousands of neurons can be stimulated simultaneously. This technique is commonly used to address the effects of activation of cell types with specific spatial patterns (Reutsky-Gefen et al. 2013; Nikolenko et al. 2010).
Increased spatial precision can be achieved with two-photon holographic stimulation, achieving spatial resolution on the order of nanometers (Papagiokoumou et al. 2010), allowing activation of multiple single, discrete neurons simultaneously (Packer et al. 2012). However, this increase in spatial resolution comes at the cost of the temporal coherence of the laser beam, as the two photons are less likely to be in phase. Consequently, contrast between illumination spots and the background will be diminished.
Studies investigating the effects of manipulating the neural code of single neurons are particularly well addressed using two-photon holographic patterned illumination (Packer et al. 2012). The holographic system can be used both in vitro and in head-fixed animals for in vivo experiments.
While this system theoretically achieves good illumination intensity, in practice there are some concerns with efficiency. 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.
Holographic systems are also susceptible to background noise which can cause speckling and interference – resulting in undesired illumination points on the sample. This can be particularly problematic if the undesired illumination will stimulate neurons whose activity will have downstream effects interfering with the target.
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 component 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 in order to create a working system for experimental use.
Another drawback of the holographic projection is the fact that it can only work with one specific optical wavelength at a time, as each holographic pattern must be designed for a predefined wavelength. This means that holographic projection may not work well for applications that require multiple wavelengths.
A third patterned illumination system commonly used in optogenetic experiments employs a digital micromirror device (DMD), such as Mightex’s Polygon DMD Illuminator. A DMD is an array of many microscopic mirrors which can each be independently controlled.
When illuminated, each micromirror corresponds to a pixel in the illumination pattern that is defined by the user and it can be individually and independently controlled to reflect light onto the sample; mirrors are rotated approximately 12 degrees to either an on- or off-state (Knapcsyk & Krishnan 2005). When rotated to the “on” position, the micromirror directs light into the optical pathway of the microscope to illuminate the sample. When rotated to the “off” position, light is directed away from the optical path, resulting in a dark pixel at the corresponding point on the sample.
As with holographic pattern projection, DMD systems are able to stimulate multiple discrete ROIs simultaneously, allowing for parallel excitation of multiple cells while maintaining high contrast against the background. Because the micromirror array acts to reflect single pixels of light onto the sample, the illumination spot size on the sample is limited only by the objective lens and the number of micromirrors on the DMD chip.
With large arrays of many micromirrors, and high magnification (≥40X) objective lens, a single pixel can illuminate a region as small as 0.4μm x 0.4μm on the sample, small enough to stimulate select parts of the cell.
A DMD illuminator setup.
An advantage of pixelation with high contrast is that the illumination will be well contained within the pattern area. The sharp illumination edge helps ensure that light is only directed to the area of interest and that there is no spill over into unwanted regions. This can help provide more control to the excitation experiment.
A key advantage of the system is the temporal precision – at update rates of 1-10 kHz, DMD systems can be fast enough to stimulate in a physiologically realistic timescale (Wilt et al. 2009). Real-time pattern generation can also be achieved, and this is especially useful in closed loop experiments where a moving target is being tracked and a precise illumination pattern must be generated and delivered to the target.
Unlike the galvo-scanner or the holographic projector, with a DMD illuminator virtually any light source (e.g. lasers, LEDs, Xenon, metal halide, etc.) can be used to illuminate the sample, making it the most versatile system that makes it easier for users to choose pretty much any wavelength for their application. With the advance of super-high powered LEDs and other high power illumination sources, for example, a DMD illuminator can provide patterned illumination at any wavelength with ample illumination intensity to activate various opsins.
A key advantage of DMD-based systems is the high degree of uniformity of illumination across the sample, meaning not only is uniformity maintained over a given illumination area, but also that intensity is consistent over discrete illumination areas, regardless of position in the field of view. Moreover, there is very little background noise (contrast ratio of 1000:1) to cause interference in the illumination and no unwanted illumination patterns are generated, as is the case with galvo-scanning systems.
Typically DMD systems are most useful in studying the effect of activation of cell types in specific patterns (Blumhagen et al. 2011; Munch et al. 2009). DMDs have been widely used in in vitro, as well as in in vivo optogenetic experiments using organisms such as C. elegans (Leifer et al. 2011), zebrafish (Zhu et al. 2012), and mice, wherein optical access to the live, behaving animal is possible.
Of the three systems discussed in this paper, the DMD system offers the greatest ease of use at the lowest cost. It is easily integrated with existing commercial microscopes and is highly adaptable.