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The ability to illuminate select neurons within a population of optogenetic-expressing neurons is called cellular-resolution optogenetics (1).

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 (2).

What systems are available for cellular-resolution optogenetics?

Galvo-Scanning

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, or acousto-optic deflectors (AODs), which can allow scanning access to any single point in the field of view within microseconds (3). 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.

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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 (4,5).

In two-photon systems, fine enough spatial precision can be achieved to stimulate single cells, or even single dendritic spines (3,6). 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 (7,8).

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 (9). 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.

Next Post

What Systems are Available for Cellular-Resolution Optogenetics? (Part 2) Holographic Projection

References

  1. Shemesh, OA et al. (2017). Temporally Precise Single-Cell-Resolution Optogenetics. Nature Neuroscience, 20(12), 1796-1806.
  2. Ronzitti, E et al. (2017). Recent Advances in Patterned Photostimulation for Optogenetics. Journal of Optics, 19(11).
  3. Zhu, P et al. (2009). Optogenetic Dissection of Neuronal Circuit in Zebrafish using Viral Gene Transfer and Tet System. Frontier in Neural Circuits, 3, 21.
  4. Petreanu, L et al. (2009). The Subcellular Organization of Neocortical Excitatory Connections. Nature, 457, 1142-1145.
  5. Wang, H et al. (2007). High-Speed Mapping of Synaptic Connectivity Using Photostimulation of Channelrhodopsin-2 Transgenic Mice. PNAS, 104(19), 8143-8148.
  6. Packer, AM et al. (2012). Two-Photon Optogenetic of Dendritic Spines and Neural Circuits. Nature Methods, 9, 1202-1205.
  7. Rickgauer, JP & Tank, DW. (2009). Two-Photon Excitation of Channelrhodopsin-2 at Saturation. PNAS, 107, 15025-15030.
  8. Prafash, R et al. (2012). Two-Photon Optogenetic Toolbox for Dast Inhibition, Excitation and Bistable Modulation. Nature Methods, 9, 1171-1179.
  9. Lin, J et al. (2009). Characterization of Engineered Channelrhodopsin Variants with Improved Properties and Kinetics. Biophysical Journal, 97, 1803-1814.

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