Dynamic illumination of spatially restricted or large brain volumes via a single tapered optical fiber

Abstract

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.

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Figure 1: Emission properties of TFs.
Figure 2: Emission properties of TFs in brain slices.
Figure 3: In vivo examination of effective excitation in striatum.
Figure 4: Optogenetic manipulation of motor cortex with TFs and FFs.
Figure 5: Site-selective light delivery with TFs.
Figure 6: Selective light delivery with TFs in the open field.
Figure 7: Mapping subsecond structure of behavior during optogenetic manipulation of ventral or dorsal striatum.

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Acknowledgements

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).

Author information

Affiliations

Authors

Contributions

F.P., M.D.V., L.S. and B.L.S. conceived the TF technology; F.P., M.D.V., S.R.D. and B.L.S. supervised the research; F.P., G.M., M.P., I.A.O., L.S., J.E.M., R.E.P., A.D.P., T.M.H., M.S.E. and B.S. performed the research and analyses; F.P., G.M., and B.L.S. wrote the manuscript.

Corresponding authors

Correspondence to Ferruccio Pisanello or Bernardo L Sabatini.

Ethics declarations

Competing interests

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

Supplementary Figure 1 Simplified model of ray propagation and outcoupling in TFs.

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.

Supplementary Figure 2 Dependence of the output distance from taper tip on the input angle.

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.

Supplementary Figure 4 Approaches used to model and measure light output from the TFs.

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.

Supplementary Figure 5 Example of a typical low ψ taper with a modest nonlinear shape

Supplementary Figure 6 Characterization of fiber emission as a function of taper angle

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.

Supplementary Figure 9 Control conditions for c-Fos induction experiments.

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.

Supplementary Figure 10 Diminished tissue damage with TF vs. FF.

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).

Supplementary Figure 11 Complete dataset of SF and TF efficacy in primary motor cortex.

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’’.

Supplementary Figure 14 Effects of fiber movement and bending on site-selective light delivery.

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 information

Supplementary Text and Figures

Supplementary Figures 1–15 (PDF 2254 kb)

Supplementary Methods Checklist (PDF 177 kb)

Ray tracing simulation of site-selective light delivery

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)

Discrete switching between different taper emission segments

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)

Continuous movement of emitting segment

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)

Multiwavelength operation

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)

Influence of fiber shaking on light emission

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)

Influence of fiber bending on light emission

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)

Influence of simultaneous fiber shaking and bending on light emission

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)

Examples of pausing syllables

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)

Examples of body shake syllables

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)

Examples of spin syllables

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)

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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

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