C4. Functional Imaging
Initially, we focused on vectors encoding fluorescent proteins to mark individual cells for optical imaging. The cell specificity of the vectors, however, can be used to transfer new functional proteins ("optical switches") that will either control retinal cell activity with light, or report activity of retinal neurons by changes in light output. This nanotechnologic application of AOSLO will be an important focus in the renewal phase, but preliminary progress in this area is described here.
C4.1. Light-activated Ion Channels in Ganglion Cells (UCB-JF)
Fig. C20: Control of proteins with two classes of photoswitches. A) "Nano-pointers" attach to protein at one end and carry a ligand (agonist, antagonist, or active site blocker) at other end. Photo-isomerization of AZO linker moves ligand, permitting binding only in one state. B) "Nano-tweezers" attach at both ends. Photo-isomerization of AZO pulls/pushes protein forcing functional transition
We have collaborated to develop light-actuated devices that function as remote controlled photoswitches, which activate and deactivate proteins with high spatio-temporal resolution. A photoswitch covalently attaches to a target protein or peptide and either dangles a ligand from a nanometer length linker near a binding site on the protein and repositions it in response to light so that it binds conditionally, or applies force to drive a conformational change in response to light (Fig C20). In preliminary studies, we have transferred the light-gated SPARK inhibitory K+ channels with a pore blocker photoswitch (Banghart 2004), and light-gated LiGluR excitatory receptor-channel with an agonist photoswitch (Volgraf 2006) to retinal ganglion cells using viral vectors. These approaches set the stage for the development of photoswitches to control retinal ganglion cell activity with light, and development of retinal prosthetics to confer light-responses to non-light sensitive retinal neurons in animal models of human disease.
C4.2. Intrinsic Retinal signals
Various research groups (Tsunoda 2004, Abramoff 2006, Srinivasan 2006, Bizheva 2006, Nelson 2005) have found increases in scattered infrared light, termed 'intrinsic signals', from the retina in response to a visible light stimulus, which are analogous to those observed in 'optical imaging' of the visual cortex (Grinvald 1986). The origin of the signal is not well understood but is likely due to changes in blood flow, blood oxygen content, cell swelling or metabolic changes, or any combination thereof. The luminance and duration of the flash stimulus, and hence the flash strength, varies considerably from group to group. AOSLO, specifically the dual frame mode implemented at UCB-AR, is useful for investigating intrinsic signals because it offers superior lateral resolution, and is therefore better able to localize the signal in comparison to standard fundus images. Furthermore, while the axial resolution of AOSLO is a factor of 10 worse than that of OCT, it can still localize the signal to specific axial regions of the retina. In AOSLO pilot studies, we used a red 658 nm visible stimulus, and recorded scattered light changes with an 840 nm low-coherence laser diode. A 3 deg field size was used, and the subject fixated on the center left side of the raster scan. Subjects were typically dark adapted for 3 minutes before each movie was recorded. For each movie, we calculate the ratio of the average intensity of the stimulated portion of the retina to the unstimulated portion, after stabilization. On displaying this cumulative mean ratio in time we can see a clear increase in intensity following stimulation, then a return to baseline (Fig C21). The ratio image demonstrates how the scattering response is well-localized to the stimulated regions.
Fig C21: The left panel shows an overlay of the 658 visible stimulation pattern on a 3X3 deg AOSLO retinal image taken with 840 nm light. The center plot shows the normalized ratio of the stimulated to unstimulated retina, showing an increase in scattering in the stimulated region. The right image is a ratio of the retinal image prior to stimulation with the same region at the point of peak scattering change.
→ C5. Synergistic Progress of the BRP← C3. Adaptive Optics In Vivo Cellular Imaging