Optogenetics promises precise spatiotemporal control of neural processes using light. However, the spatial extent of illumination within the brain is difficult to control and cannot be adjusted using standard fiber optics. We demonstrate that optical fibers with tapered tips can be used to illuminate either spatially restricted or large brain volumes. Remotely adjusting the light input angle to the fiber varies the light-emitting portion of the taper over several millimeters without movement of the implant. We use this mode to activate dorsal versus ventral striatum of individual mice and reveal different effects of each manipulation on motor behavior. Conversely, injecting light over the full numerical aperture of the fiber results in light emission from the entire taper surface, achieving broader and more efficient optogenetic activation of neurons, compared to standard flat-faced fiber stimulation. Thus, tapered fibers permit focal or broad illumination that can be precisely and dynamically matched to experimental needs.
Subscribe to Journal
Get full journal access for 1 year
only $17.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Dawydow, A. et al. Channelrhodopsin-2-XXL, a powerful optogenetic tool for low-light applications. Proc. Natl. Acad. Sci. USA 111, 13972–13977 (2014).
Hochbaum, D.R. et al. All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins. Nat. Methods 11, 825–833 (2014).
Govorunova, E.G., Sineshchekov, O.A., Janz, R., Liu, X. & Spudich, J.L. NEUROSCIENCE. Natural light-gated anion channels: a family of microbial rhodopsins for advanced optogenetics. Science 349, 647–650 (2015).
Lee, J., Ozden, I., Song, Y.-K. & Nurmikko, A.V. Transparent intracortical microprobe array for simultaneous spatiotemporal optical stimulation and multichannel electrical recording. Nat. Methods 12, 1157–1162 (2015).
Canales, A. et al. Multifunctional fibers for simultaneous optical, electrical and chemical interrogation of neural circuits in vivo. Nat. Biotechnol. 33, 277–284 (2015).
Jeong, J.-W. et al. Wireless optofluidic systems for programmable in vivo pharmacology and optogenetics. Cell 162, 662–674 (2015).
Kim, T.I. et al. Injectable, cellular-scale optoelectronics with applications for wireless optogenetics. Science 340, 211–216 (2013).
Kwon, K.Y., Lee, H.-M., Ghovanloo, M., Weber, A. & Li, W. Design, fabrication, and packaging of an integrated, wirelessly-powered optrode array for optogenetics application. Front. Syst. Neurosci. 9, 69 (2015).
Zorzos, A.N., Scholvin, J., Boyden, E.S. & Fonstad, C.G. Three-dimensional multiwaveguide probe array for light delivery to distributed brain circuits. Opt. Lett. 37, 4841–4843 (2012).
Schwaerzle, M., Elmlinger, P., Paul, O. & Ruther, P. in Micro Electro Mechanical Systems (MEMS), 28th IEEE International Conference 162–165 (2015).
McAlinden, N., Gu, E., Dawson, M.D., Sakata, S. & Mathieson, K. Optogenetic activation of neocortical neurons in vivo with a sapphire-based micro-scale LED probe. Front. Neural Circuits 9, 25 (2015).
McCall, J.G. et al. Fabrication and application of flexible, multimodal light-emitting devices for wireless optogenetics. Nat. Protoc. 8, 2413–2428 (2013).
Szabo, V., Ventalon, C., De Sars, V., Bradley, J. & Emiliani, V. Spatially selective holographic photoactivation and functional fluorescence imaging in freely behaving mice with a fiberscope. Neuron 84, 1157–1169 (2014).
Pisanello, F. et al. Multipoint-emitting optical fibers for spatially addressable in vivo optogenetics. Neuron 82, 1245–1254 (2014).
Pisanello, M. et al. Modal demultiplexing properties of tapered and nanostructured optical fibers for in vivo optogenetic control of neural activity. Biomed. Opt. Express 6, 4014–4026 (2015).
Aravanis, A.M. et al. An optical neural interface: in vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology. J. Neural Eng. 4, S143–S156 (2007).
Yizhar, O., Fenno, L.E., Davidson, T.J., Mogri, M. & Deisseroth, K. Optogenetics in neural systems. Neuron 71, 9–34 (2011).
Stujenske, J.M., Spellman, T. & Gordon, J.A. Modeling the spatiotemporal dynamics of light and heat propagation for in vivo optogenetics. Cell Reports 12, 525–534 (2015).
Oldenburg, I.A. & Sabatini, B.L. Antagonistic but not symmetric regulation of primary motor cortex by basal ganglia direct and indirect pathways. Neuron 86, 1174–1181 (2015).
Greenberg, M.E., Ziff, E.B. & Greene, L.A. Stimulation of neuronal acetylcholine receptors induces rapid gene transcription. Science 234, 80–83 (1986).
Zhao, S. et al. Cell type–specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function. Nat. Methods 8, 745–752 (2011).
Wiltschko, A.B. et al. Mapping sub-second structure in mouse behavior. Neuron 88, 1121–1135 (2015).
Kravitz, A.V. et al. Regulation of parkinsonian motor behaviours by optogenetic control of basal ganglia circuitry. Nature 466, 622–626 (2010).
Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8, 1263–1268 (2005).
Zhang, F. et al. Multimodal fast optical interrogation of neural circuitry. Nature 446, 633–639 (2007).
Klapoetke, N.C. et al. Independent optical excitation of distinct neural populations. Nat. Methods 11, 338–346 (2014).
Cardin, J.A. et al. Targeted optogenetic stimulation and recording of neurons in vivo using cell-type-specific expression of Channelrhodopsin-2. Nat. Protoc. 5, 247–254 (2010).
Warden, M.R., Cardin, J.A. & Deisseroth, K. Optical neural interfaces. Annu. Rev. Biomed. Eng. 16, 103–129 (2014).
Eshel, N. et al. Arithmetic and local circuitry underlying dopamine prediction errors. Nature 525, 243–246 (2015).
Al-Juboori, S.I. et al. Light scattering properties vary across different regions of the adult mouse brain. PLoS One 8, e67626 (2013).
Hanks, T.D. et al. Distinct relationships of parietal and prefrontal cortices to evidence accumulation. Nature 520, 220–223 (2015).
Stark, E., Koos, T. & Buzsáki, G. Diode probes for spatiotemporal optical control of multiple neurons in freely moving animals. J. Neurophysiol. 108, 349–363 (2012).
Gilmartin, M.R., Miyawaki, H., Helmstetter, F.J. & Diba, K. Prefrontal activity links nonoverlapping events in memory. J. Neurosci. 33, 10910–10914 (2013).
Lambelet, P., Sayah, A., Pfeffer, M., Philipona, C. & Marquis-Weible, F. Chemically etched fiber tips for near-field optical microscopy: a process for smoother tips. Appl. Opt. 37, 7289–7292 (1998).
McGeorge, A.J. & Faull, R.L. The organization of the projection from the cerebral cortex to the striatum in the rat. Neuroscience 29, 503–537 (1989).
Afraz, A., Boyden, E.S. & DiCarlo, J.J. Optogenetic and pharmacological suppression of spatial clusters of face neurons reveal their causal role in face gender discrimination. Proc. Natl. Acad. Sci. USA 112, 6730–6735 (2015).
Lund, J.S. & Boothe, R.G. Interlaminar connections and pyramidal neuron organisation in the visual cortex, area 17, of the Macaque monkey. J. Comp. Neurol. 159, 305–334 (1975).
Saleh, B.E.A. & Teich, M.C. Fundamentals of Photonics 2nd edn. (Wiley Interscience, 2007).
Sparta, D.R. et al. Construction of implantable optical fibers for long-term optogenetic manipulation of neural circuits. Nat. Protoc. 7, 12–23 (2011).
F.P. acknowledges funding from the European Research Council under the European Union's Horizon 2020 research and innovation program (#677683); L.S., M.D.V. and B.L.S. are funded by the US National Institutes of Health (U01NS094190); and G.M., J.M., S.D. and B.L.S. are funded by the Simons Collaboration on the Global Brain. M.P. acknowledges funding from the Rotary Foundation and the Rotary International District 2120 (Global Grant GG1417647).
M.D.V., F.P., B.L.S., S.D. and L.S. are co-founders of Optogenix LLC, a company based in Italy that produces and markets the tapered fibers described here.
Integrated supplementary information
After total reflection at the core boundary with an angle α, a ray entering the taper (blue line) hits the taper sidewalls with an angle of incidence γj, that decreases as the number of hits j increases. The ray can outcouple in the surrounding medium when γj < θC (i.e. total internal reflection condition on the critical angle θC is not fulfilled). The values for the taper and the critical angle (Ψ = 20°, θC = 30°) were selected to improve the readability of the figure. In the picture the cladding is not shown and the refractive index n was assumed to be the same for both the core and the taper. This simplified analysis only applies to meridional rays.
Ray tracing was used to model the propagation of a ray (left) in a TF and determine the position at which it outcouples. Results for ray tracing simulation for TFs with 0.22NA/ψ = 2.2° core diameter 50 μm and cladding diameter 125 μm (red circles) and 0.39NA/ψ = 2.9° core diameter 200 μm and cladding diameter 225 μm (black squares) are shown (right).
Supplementary Figure 3 Ray-tracing simulation of propagation and outcoupling in TFs of different NA and taper angles, as indicated.
a, Optical setup used to inject light in the TF, filling the whole numerical aperture of the fiber, and to image the fluorescence generated by light output into a fluorescein solution.b, Schematic representation (left) of light output from a TF, the irradiance profile measured by the linear detector placed alongside the taper (middle), and the Gaussian fit to the average line profile measured by the detector from which the length to half maximal intensity is calculated (L0.5) (right).c, Typical image of fluorescence produced by light emission from the TF into a fluorescein solution (left) and the measured fluorescence intensity profile obtained along the red line (right). L0.5 is calculated from the fiber tip as the width at which the intensity is half of the intensity represented by the dotted vertical line. The latter is the average of all data points exceeding 90% of the intensity detected in the pixel closest to the fiber tip.
a, First emission diameter of 0.39 NA and 0.22 NA TFs as a function of ψ. First emission diameter is evaluated with the fiber stub submerged into a fluorescein solution and measured as the diameter of the taper at the point at which the fluorescence intensity just beside the taper reaches a threshold level ~3 times the noise level. Error bars represent the uncertainty in measuring the taper diameter due to the pixel size of the image.b, Total output power for 0.39 NA and 0.22 NA TFs as a function of ψ.
Supplementary Figure 7 Estimated power density along the taper surface outcoupled by 0.39-NA TFs (core diameter, 200 μm; cladding diameter, 125 μm).
A total output power of 1 mW is distributed around the taper following the experimental emission profiles measured in a fluorescent bath as shown in Supp. Fig. 4.
Supplementary Figure 8 Estimated power density along the taper surface outcoupled by 0.22-NA TFs (core diameter, 50 μm; cladding diameter, 125 μm).
A total output power of 1 mW is distributed around the taper following the experimental emission profiles measured in a fluorescent bath as shown in Supp. Figure 4.
Schematic (a) and image (b) of c-fos immunolabeling in an animal that expressed ChR2-YFP in iSPNs but was not exposed to light. Minimal c-fos staining is present.Schematic (c) and image (d) of c-fos immunolabeling in an animal lacking ChR2-YFP but stimulated through a TF. Minimal c-fos staining is present.
a, Schematic showing the experimental design: a FF (right side of the brain) and a TF (left side of the brain) were implanted in the striatum of a wildtype animal, at 0.8 mm anterior and 2.0 mm lateral to bregma.b. Image of a coronal section of the mouse brain showing the damage caused by each fiber. The TF was covered in green fluorescing lipophilic dye to show the track.c. 6 coronal sections (from top left, starting with 1.3 mm anterior to bottom right, 0.4 mm anterior to bregma) from example mouse immunostained for the astrocyte marker GFAP after being implanted with a FF (right side of the brain) and a TF (left side of the brain) in the striatum.d, Quantification of fluorescence in tissue immunostained for the astrocyte marker GFAP from two mice (6 coronal sections each). Fluorescence is expressed for the TF and FF hemispheres are a fraction of the total fluorescence per tissue section – thus for each the total in the TF and FF regions sum to 1. The magenta lines indicate the means of the values across tissue sections of the TF and FF implanted hemispheres.e, Example coronal sections (0.8 mm anterior to bregma) immunostained for the microglia marker CD68.f, As in panel d but of tissue immunostained for the microglia marker CD68 from two mice (6 coronal sections each).
For each recorded unit an index of modulation of firing rate was calculated as:index = (fon-foff)/(fon+foff) where fon and foff are the firing rates of the neuron with the laser light on and off, respectively. In these recordings the vast majority of sampled neurons are excitatory and ChR2 is expressed in all GABAergic neurons. Therefore, the effect of light is expected to be inhibition of most units (index<0). The small number of units with index>0 may represent directly stimulated GABAergic interneurons. The values of index range from -1 to 1, with -1 indicating that all the spikes were with the light off and +1 indicating that all the spikes were with the light on. A value of 0 indicates no change in firing with light. Statistical comparison was done with Anova with Kruskal-Wallis Rank (i.e. non parametric) test and Dunn’s post-hoc multiple comparison test. *** indicates p<0.0001 (the limit of reporting of Graphpad). ** indicates p=0.0027. Comparisons at 200 μW were not significantly different (p=0.1704). Degrees of freedom are n-1, where n is the number of cells. Data for all cells are shown (n-values are 45, 49, 51, 51, 28, 28, 28, 28, 48, 49, 49, and 49 cells).
Supplementary Figure 12 Ray-tracing simulations for site-selective light delivery for a 0.22-NA, ψ =2.2° TF.
Supplementary Figure 13 Three potential launching system configurations for controlling the zone of light emission from a TF.
a, A laser beam is injected into the fiber by a single lens. Translating one mirror in the direction perpendicular to optical axis of the fiber input facet modifies the input coupling angle. This system was used and described in more detail in Pisanello et al Neuron 2015.b The input laser beam is focused on the rotation axis of a galvo mirror, which deflects the beam onto a second lens that makes it parallel to optical axis. A third lens can then focus the laser into the optical fiber with a defined input angle.c, A system comprising two different optical paths similar to the ones displayed in panels a and b but working simultaneously with two different lasers. This configuration allows for delivering different wavelengths at different sections of the taper, also at different scanning rates. In all panels lens diameter is 2’’.
a, Optical setup used to inject light at defined angle θ.b-d, Variability of three metrics of light output (schematized in A) for shaken, bent, and bent and shaken patch fibers connected to a 0.39 NA/ψ=2.9° TF. The percent variability about the mean in peak intensity (red), full-width at half maximum (FWHM, green) and centroid (yellow) of the emission along the taper are graphed. Different colors along the time line depict the movement conditions detailed at the bottom of panels B-D and shown in Supp. Videos 5, 6, 7, respectively. ρ is the radius of curvature and the arrows (→) identify variations from one radius of curvature to another during the measurement.
Supplementary Figure 15 Characterization of outputs for the fibers and light input angles used for the experiments described in Figures 6 and 7 and used with a commercial launching system.
a and b, Total power of light output (a) and the distance of the centroid of light output from the fiber tip (b) for light injected at the full NA or each of the two angles used with 1mW input angle (mean ± std shown for n=8 fibers).c and d, Full NA launching into a TF using a commercial system. Image (left) of a Plexon LED driver and fiber launch system connected to a TF and the resulting fluorescence generated in a fluorescein bath (right).
Supplementary Figures 1–15 (PDF 2254 kb)
Each frame of the movie displays rays emitted from a 0.22NA/ψ=2.2° calculated by running the simulation at each of five different input angles θ (MOV 805 kb)
Using a mirror galvanometer (see optical path in Supplemental Figure 13B) to rapidly jump between input angles θ, light emission is quickly redirected to different taper segments (MOV 473 kb)
Using a mirror galvanometer (see optical path in Supplemental Figure 13B) to continuously scan across input angles θ at a variety of frequencies (see annotation in movie), light emission is continuously moved across the taper length (MOV 500 kb)
By combining different lasers beams, TFs can be used to deliver light at different wavelengths from independently-controlled taper segments. Experiment 1 shows steady emission of green light (λ=532nm) close to the taper tip and blue light (λ=473nm) continuously moving along the taper. In experiment 2, emission is moved along the taper for both wavelengths. (MOV 7538 kb)
The main frame shows light emission from a tapered fiber, while the inset displays a video of the patch fiber. For the first 30 sec the patch fiber is not moved, whereas for the subsequent 30 sec the patch fiber is hand-shaken as shown in the inset. This type of experiment was used to obtain the quantitative data shown in Supplemental Figure 14b. (MOV 8016 kb)
The main frame shows light emission from a tapered fiber, while the inset displays a video of the patch fiber. For the first 30 sec the patch fiber is held at a radius of curvature ρ=4.5cm. ρ is then changed to ρ=1.3cm and held for another 30 sec before being restored to 4.5 cm. This type of experiment was used to obtain the quantitative data shown in Supplemental Figure 14c (MOV 6733 kb)
The main frame shows light emission from a tapered fiber, while the inset displays a video of the patch fiber. For the first 30 sec the patch fiber is not moved and held with a curvature radios of ρ =3.3cm, whereas for the subsequent 30 sec the bent patch fiber is hand-shaken as shown in the inset. This type of experiment was used to obtain the quantitative data shown in Supplemental Figure 14d. (MOV 5895 kb)
Overlaid examples of individual locomotion pauses of varying lengths synchronized to the start of the syllables, as indicated by the appearance of the green dots. (MP4 46 kb)
Overlaid examples of individual “Body Shake” (#6) syllables of varying lengths synchronized to the start of the syllable, as indicated by the appearance of the green dots. (MP4 57 kb)
Overlaid examples of individual “Spin” (#7) syllables of varying lengths synchronized to the start of the syllable, as indicated by the appearance of the green dots. (MP4 88 kb)
About this article
Cite this article
Pisanello, F., Mandelbaum, G., Pisanello, M. et al. Dynamic illumination of spatially restricted or large brain volumes via a single tapered optical fiber. Nat Neurosci 20, 1180–1188 (2017). https://doi.org/10.1038/nn.4591
Ray tracing models for estimating light collection properties of microstructured tapered optical fibers for optical neural interfaces
Optics Letters (2020)
Current Biology (2020)
Nature Biomedical Engineering (2020)
Two-photon fluorescence-assisted laser ablation of non-planar metal surfaces: fabrication of optical apertures on tapered fibers for optical neural interfaces
Optics Express (2020)
Microfocusing sapphire capillary needle for laser surgery and therapy: Fabrication and characterization
Journal of Biophotonics (2020)