Spatial Light Modulator-Based Scanless Two-Photon Microscopy
We and others have developed another multibeam approach which uses SLMs in one-photon (Lutz et al., 2008) and two-photon regimes (Nikolenko et al., 2008). SLMs not only allow for truly simultaneous monitoring of fluorescence at multiple targets, but also allow for simultaneous photomanipulation of targets in a customizable and temporally dynamic manner. The core of this method is the SLM itself which acts as a programmable DOE to construct a custom light or beamlet pattern (Figures 3A,B), and can in fact dynamically change that pattern over time (at 60 Hz in our case). SLMs can create any pattern of light within the output resolution of the SLM itself (256 × 256 in our case) since they are made of dynamic controllable materials such as liquid crystal which allow the diffractive properties of each “pixel” of the SLM to be modified by electrical signals from a computer. It is important that as opposed to micromirror technology (Wang et al., 2007) in which patterns can be created by blocking out light at individual pixels to varying degrees (a purely subtractive process), the phase-only SLM (and DOE) preserves incident light by creating both destructive and constructive interference. For example, a beamlet pattern on a 256 × 256 output image in which only three pixels are allowed to be bright (i.e., three beamlets) keeps 50% of the input power in the output pattern, versus <1% with micromirror devices. An SLM approach was developed for one-photon microscopy by (Lutz et al., 2008), in parallel to the two-photon approach we developed (Nikolenko et al., 2008).
Figure 3  Spatial light modulator (SLM) two-photon imaging and photostimulation. (Ai) Arbitrary pattern of desired output from SLM in the shape of letters “HHMI”. (Aii) Pattern by SLM from a single circular input beam, projected through 60× 0.9NA objective into agar block filled with Alexa 488 fluorescent dye. (Bi) Similar to (Ai), however more complex desired output pattern of output from SLM in the shape of a patch clamped neuron. This image was thresholded and binarized to create a final output template. (Bii) SLM-created pattern projected into Alexa 488-filled agar block under 40× 0.8NA objective. (Ci) Basal dendrite from a layer 5 pyramidal neuron in an acute slice of mouse neocortex. The neuron is loaded with Alexa-488 and the slice bathed in MNI-glutamate. The SLM was used to create a diffraction pattern placing beamlets adjacent to individual spines of the dendrite (red dots). Pulses of excitation light delivered simultaneously at these points using the SLM triggered uncaging of MNI-glutmate. (Cii) Whole cell patch clamp recording at cellular soma of integrated potentials created by simultaneous uncaging at the spots indicated. Each horizontal trace represents a repetition of the stimulation and reveals the uncaging potentials recorded at the soma. (Di) Similar to (C), however now in a new neuron with uncaging targeted to locations near a dendritic shaft rather than near spines. (Dii) Patch clamp recordings of multiple trials of multi-point simultaneous uncaging revealing the uncaging potentials recorded at the soma. See Nikolenko et al. (2008) for more detailed description of the methods used for these recordings. Imaging-based uses of the SLM include simultaneous time lapse imaging of a fixed set of multiple targets. This is accomplished by delivering excitation light to all targets continuously while monitoring emission at all those sites at once using a camera. The number of excitation points which may be specified and still receive sufficient excitation power to give good signals is currently limited by the amount of power produced by modern lasers (roughly 20 neurons in brain slice experiments). Liquid crystal SLMs can update their output pattern every 16.7 ms (or more often, depending on the model of SLM) for faster than video rate updating of projected patterns. This temporal flexibility means the SLM may also be useful as a multiplexed random access scanner. In this mode it would update to a new set of multiple targets (rather than a single target) every 16.7 ms, thus allowing allow for random access scanning across large numbers of targets. This may be of particular interest in neuronal integration or circuit integration studies.
Perhaps the most unique application of the SLM, however, is for induction of photochemical effects such as uncaging of neuro-active compounds at multiple points simultaneously (Figures 3C,D). Caged neurotransmitters such as glutamate (Matsuzaki et al., 2001; Fino et al., 2009) and GABA (Rial Verde et al., 2008) are coming into increased use for manipulation of neurons (Araya et al., 2006; Nikolenko et al., 2008). The SLM allows for stimulation or inhibition of multiple neurites or neurons at once to achieve large-scale yet highly specific neuro-excitatory/-inhibitory effects One may be able to perform studies of whole neuron or whole circuit integrative properties via stimulation of specific targets chosen based on known properties of those targets (i.e., connectivity at spines, or firing properties of neurons) (Nikolenko et al., 2008).