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 (2,3).
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.
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 (2,4).
Increased spatial precision can be achieved with two-photon holographic stimulation, achieving spatial resolution on the order of nanometers (5), allowing activation of multiple single, discrete neurons simultaneously (1). 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 (1). 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 (6). 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.
- Packer, AM et al. (2012). Two-Photon Optogenetic of Dendritic Spines and Neural Circuits. Nature Methods, 9, 1202-1205.
- Reutsky-Gefen, I et al. (2013). Holographic Optogenetic Stimulation of Patterned Neuronal Activity for Vision Restoration. Nature Communications, 4, 1509.
- Papagiakoumou, E. (2013). Optical Developments for Optogenetics. Biology of the Cell, 105(10), 443-464.
- Nikolenko, V et al. (2010). A Portable Laser Photostimulation and Imaging Microscope. Journal of Neural Engineering, 7, 1-7.
- Papagiokoumou, E et al. (2010). Scanless Two-Photon Excitation of Channelrhodopsin-2. Nature Methods, 7(10), 848-854.
- Lutz, C et al. (2009). Holographic Photolysis of Caged Neurotransmitters. Nature Methods, 5(9), 821-827.
What Systems are Available for Cellular-Resolution Optogenetics? (Part 3) DMD Illuminator