Significant technological and scientific breakthroughs are often the trigger for complimentary developments, making the initial pioneering work even more impactful. Optogenetics seem to be no exception to the tendency, with many more opsin variants and chimeric proteins being developed as well as significant improvements in experimental protocols, evolving the technique to much wider and more sophisticated applications.
Part of this trend is the development of more appropriate light delivery system or light sources (for the sake of simplicity and convention we’ll call it light sources) for different experimental requirements involving optogenetics. Five top considerations for selecting the ideal light source for in vitro preparations are discussed here. The current commercially available options are typically divided between laser (including 2-P activation) or LED-based systems, with different design options as to how the light is ultimately delivered to the sample and how light is controlled spatially and temporally. We’re hoping to discuss pros and cons of the different solutions on this blog space in coming weeks.
1) CHOICE OF OPSIN (OR OTHER LIGHT ACTIVATED PROTEIN) VARIANTS
Of course, the ideal light source needs to be suitable for the light sensitive protein you’ve selected for your project, which is dictated by the research question you’re targeting. Several mutational techniques have delivered a variety of opsins and related proteins, ranging from depolarising, repolarising or hyperpolarizing channels to proteins with a specific effect on cellular metabolic processes, like G-protein coupled pathways. The goal is for these entities to have an instantaneous and predictable effect after exposure to activation light. Expressing the protein at high enough densities in the desired cell type is essential for this to happen.
Expression density is determined by the insertion technique, which has been standardised through a variety of techniques including breeding transgenic mice, in utero electroporation and the use of viral approaches. Each of these methods poses its own challenge, with the most commonly used strategy being viral transduction. The ultimate challenge with all of these techniques is to achieve sufficient expression levels in the targeted cells with low levels of damage and cross contamination.
The spike in the research community’s interest in especially the Channelrhodopsin and Halorhodopsin families and more sophisticated genetic modulation tools are leading to an expanding variety in the optogenetics toolbox. Different characteristics for these proteins, which will ultimately influence your choice of opsin and the appropriate light source, include channel conductance, ion selectivity, channel kinetics, including desensitization and recovery from excited and desensitized state. Channels also differ in light sensitivity, requiring more or less intensity on sample for activation. Choice of light source will also depend on the spectral response, like the width of the excitation peak – irrelevant light which doesn’t contribute to activation should be avoided to prevent damage to your sample.
The trend is also for labs to combine different optogenetic proteins with regular fluorescence reporters or with light activated caged compounds. This requirement increases the usefulness of having a light source with the capacity of switching (quickly, when required) between different wavelengths, or which has the capacity of being upgraded in future when this requirement may become necessary.
Your choice of light sensitive protein will dictate the wavelength peak intensity you require and influence the minimum intensity level you will need on the sample. New developments will deliver channel kinetics requiring new activation protocols and new demands on how light sources are controlled.
2) FIELD OF ILLUMINATION
Activation of light sensitive proteins for in vitro experiments are typically done through the optical path of the microscope. Your experimental requirements will determine whether it’s best to activate a wide field of the sample, exposing a sample field typically wider than your microscope detector’s field of view (https://www.mci-neuroscience.com/product-category/optogenetics-light-sources/), or whether more local spot illumination is appropriate. Spot illumination allows spatial targeting of activation and offers the additional benefit of not exposing cells outside the target area to unnecessary light.
This more local activation can be achieved by delivering a spot of light to the sample, e.g. a laser spot or LED light projected through a pinhole inserted at a conjugate plane in your microscope’s light path. The sample can be moved around on the sample stage in order to manipulate the sample’s position relative to the spot, thereby controlling the area of activation. This is sometimes restrictive or not feasible within the experimental protocol, the solution to which is to project the laser spot onto the sample reflected from a set of XY galvo mirrors or to use a digital light projection (DLP)-based micromirror array or uLED array in the light path. An alternative approach is to position an optical fiber close to the area of interest (sometimes enclosed in a glass electrode), which typically results in a wider and less defined area of illumination, but offers the possibility of projecting light onto a sample area remote from the microscope’s objective lens.
With spot illumination, having flexibility in spot diameter is useful since you can play around more with wider spots in order to achieve adequate activation where smaller spots don’t achieve the activation threshold.
3) DEPTH OF ILLUMINATION
Light sensitive protein activation in brain slices demands the additional consideration of depth and the volume of tissue that need to be exposed to activation light. Light intensity emitted into brain tissue tends to decline exponentially with increasing depth. Different brain areas also have different scattering properties, and different colour bands of light is scattered in different degrees, where longer wavelengths have deeper penetration capacity than short wavelengths. This characteristic created the focus on developing opsins with red-shifted activation peaks, and the popularity in using 2-P microscopy for optogenetics activation.
The depth of the target area therefore contributes in determining the required intensity of light hitting the sample, which needs to be balanced with the consideration of limiting phototoxicity and photobleaching as much as possible.
4) REQUIRED INTENSITY
The magnitude of opsin response and the resulting current (with the Channelrhodopsin and Halorhodopsin families) are directly linked to the intensity of the light reaching the channel. Light power per unit area (mW/mm2) delivered on the sample is more important than overall output power (in mW) when comparing light delivery systems. Overall power combined with adequate collimating optics are the determining factors here, where scattered light and light lost in the microscope’s optical path should be discounted.
Without careful consideration of all the different variables, it’s impossible to suggest an absolute number for required intensity. A further complication is that only a few light source developers publish directly comparable and relevant figures, which means that borrowing demo equipment and testing it in your lab remains the reliable way of drawing comparisons. Several publications report a requirement of less than 1mW on sample to achieve the desired activation levels of ChR2 in superficial cell layers in brain slices and monolayers of cultured cells, but other proteins require 10mW or a few tens of mW for activation. New developments are likely to result in lower required intensity levels.
Accurate control over intensity is also useful to limit photobleaching and phototoxicity, which can be further limited by using 2-P laser activation and limiting exposure to only the required sample field (e.g. spot illumination)
5) PULSING AND INTEGRATION
It’s important to be able to pulse the light source in a way that can best exploit the kinetics of the target proteins in order to achieve the desired effect on your sample. Flexibility is important on this front, since you will need to tune down pulse width to reach a minimum pulse duration for achieving adequate activation.
The more sophisticated systems projecting a spot or multiple spots onto the sample combine software-controlled complex pulsing protocols with spatial protocols, like pseudo-random grids and other user defined sequences. These spatial protocols can be essential for certain applications.
With the fast time constants of most opsins, light source timing needs to be real-time, accurate and repeatable. A further important consideration is having the software and hardware integrate well with external devices, like cameras, electrical stimulators and electrophysiology components. Having the option of real-time synchronization where the light source is used as the master or the slave adds essential flexibility for changing experimental questions.
NEXT WEEK’S BLOG POST
The selection of the best light delivery system for optogenetics requires careful consideration. In the next blog post we’ll discuss some of the available options out there and how it relates to the above factors. If you are interested in borrowing an LED-based optogenetics light source to test whether it’s appropriate for your setup, please contact uson firstname.lastname@example.org. Our friendly applications team would be happy to discuss options with you.
Please feel free to use this forum to discuss your experience about what works and what doesn’t.
LINKS AND SOURCES
- Targeted optogenetic stimulation and recording of neurons in vivo using cell-type-specific expression of Channelrhodopsin-2
- Optogenetic stimulation of the auditory pathway
- Light Sources for Optogenetics Experiments
- The Development and Application of Optogenetics
- Improved expression of halorhodopsin for light-induced silencing of neuronal activity
- Light Scattering Properties Vary across Different Regions of the Adult Mouse Brain
- Use of channelrhodopsin for activation of CNS neurons
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