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 (1). 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.
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 (2). 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 (3,4). DMDs have been widely used in in vitro, as well as in in vivo optogenetic experiments using organisms such as C. elegans (5), zebrafish (6), 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.
- Knapcsyk, M & Krishnan, A. (2005). High-Resolution Pulses Shaper Based on Arrays of Digital Micromirrors. IEEE Photonics Technology, 17, 2200-2202.
- Wilt, B et al. (2009). Advances in Light Microscopy for Neuroscience. Annual Review of Neuroscience, 32, 435-506.
- Blumhagen, F et al. (2011). Neuronal Filtering of Multiplexed Odour Representations. Nature, 479, 493-498.
- Munch, TA et al. (2009). Approach Sensitivity in the Retina Processed by a Multifunctional Neural Circuit. Nature Neuroscience, 12, 1302-1316.
- Leifer, AM et al. (2011). Optogenetic Manipulation of Neural Activity in Freely Moving Caenorhabditis elegans. Nature, 8(2), 147-152.
- Zhu, P et al. (2012). High-Resolution Optical Control of Spatiotemporal Neuronal Activity Patterns in Zebrafish Using a Digital Micromirror Device. Nature Protocols, 7, 1410-1425.