In the previous part, I already mentioned the basic tools for optogenetics experiments. Namely the full-field LED, the CW and the fiber-opto. These techniques are quite solid and trustful tools combined with conventional multiphoton microscopy to manipulate the activity of specific populations of neurons in the brain. However studying the activity patterns of individual neurons and specific circuits or targeting fine neuronal structures such as dendritic segments in deep brain structures are still challenging for neuroscientists, due to the light scattering within the tissue. In order to achieve the required spatiotemporal control for addressing complex scientific questions, we need high-end solutions like 2-photon photostimulation.
Using femtosecond laser source to modulate the neuronal population provides a huge benefit compared to conventional methods. One of the most important advantages is the possibility to activate even single cell or dendritic level. During scanning, the excitation volume is in the sub-µm3 range which makes this technique a perfect candidate also for uncaging experiments. Another advantage is the penetration depth of the femtosecond pulse laser, thus deeper neuronal ensembles can be easily reached even in cortical layer 5 or the CA3 region of the hippocampus or the dentate gyurs (DG).
Holographic optogenetics is one of the most advanced and popular technique nowadays, which can be combined with several in vivo applications and useful tools (e.g. electrophysiology). The main component of this method is the spatial light modulator (SLM). SLMs are transducers that modulate incident light in a spatial pattern corresponding to an electrical or optical input. The incident light may be modulated in its phase, intensity, polarization, or direction, and the light modulation may be achieved by a variety of materials exhibiting various electrooptic or magnetooptic effects and by materials that modulate light by surface deformation. Therefore, the SLM is the key component to form various 3D patterns for optogenetics tasks. In principle, any kinds of patterns can be achieved. There are almost no limitations regarding the number of ROIs and z-depth. Another key point is the timing. Three-dimensional scanless holographic photostimulation (SLM screen provides the pattern) enables to excite subpopulations of neurons simultaneously in any number of targeted planes. In given types of experiments the simultaneous stimulation of neurons is quite important. Although state of the art sensors are slow yet, a new faster one could be a game-changer.
Holographic optogenetics can be combined either with continuous wave (CW) laser source for excitation or femtosecond pulse laser source. Despite of using CW laser has several serious drawbacks, it is still very popular and useful because of the favorable price tag. Illumination by femtosecond pulse laser source is much more expensive, but if we choose a fixed wavelength laser (for example 1040 nm for C1V1, chrimsonR) for a specific experiment it will provide all the advantages of the 2P excitation. Moreover, it will cost effective as well! Even though holography is super sexy, do not forget - like anything else - it also has some disadvantages. For instance, the total excitation energy will be divided among your ROIs. That means, if the pattern contains 10 locations, each ROI will get only 10% of the total energy. That is a huge drop. Not to mention that the optical path ‘consumes’ a lot of intensity while the light is reaching the sample and if it happens in 3D you are not able to adjust the intensitiy level between the layers. To sum up, it is a very powerful and popular technique, but you will need a powerful laser source to reach a great performance.
In addition, one more very promising and interesting method should be mentioned. This is the so-called acousto-optical photostimulation. Only a few studies have been published yet, but it seems this approach will become widely used in neuroscience in the near future. In principle, this technique is based on the same acousto focusing method like the 2P imaging. Thanks to the sound-modulated focusing, it enables high-speed scanning across the sample. Since it is a scanning method, changing the intensity between the ROIs is also possible. Another benefit of such a scanning process, is that the light is not divided among the selected ROIs as in case of holographic optogenetics. However, it is important to mention that, increasing the numbers of the stimulation ROIs will result in a decrease of speed of the photostimulation. Do not forget, it is still not a commercially available method, but I think the price tag will be a ‘cutting edge’ astronomical price tag. :)
In summary, these techniques are very fascinating from the cheapest one to the most expensive high-end category. Every method could be a good and suitable solution if we ask the proper question. Here, @ #NRB we have tons of experiences, which we would like to share with you. Reader comments and questions will be more than welcome!
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