The Three-Dimensional Signal Collection Field for Fiber Photometry in Brain Tissue

Abstract

Fiber photometry is used to monitor signals from fluorescent indicators in genetically-defined neural populations in behaving animals. Recently, fiber photometry has rapidly expanded and it now provides researchers with increasingly powerful means to record neural dynamics and neuromodulatory action. However, it is not clear how to select the optimal fiber optic given the constraints and goals of a particular experiment. Here, using combined confocal/2-photon microscope, we quantitatively characterize the fluorescence collection properties of various optical fibers in brain tissue. We show that the fiber size plays a major role in defining the volume of the optically sampled brain region, whereas numerical aperture impacts the total amount of collected signal and, marginally, the shape and size of the collection volume. We show that ~80% of the effective signal arises from 10⁵ to 10⁶ μm³ volume extending ~200 μm from the fiber facet for 200 μm core optical fibers. Together with analytical and ray tracing collection maps, our results reveal the light collection properties of different optical fibers in brain tissue, allowing for an accurate selection of the fibers for photometry and helping for a more precise interpretation of measurements in terms of sampled volume.

Keywords: collection fields; collection volumes; fiber photometry; optical fibers; optogenetics.

Figure 1

Computational models of light collection efficiency for optical fibers. (A) Reference system used throughout the manuscript. An example point source (green) is shown in the plane y = 0. (B) Analytical calculations of collection efficiency diagrams for light emitting from point sources locating in the xz (y = 0) plane for three different fibers. Data are shown for 0.22NA/50 μm, 0.39NA/200 μm, and 0.50NA/200 μm optical fibers, as indicated, immersed in a transparent homogeneous medium (n = 1.335). The horizontal dashed lines represent $z_0=a·{\left[2tan\left(\frac{\text{NA}}{n}\right)\right]}^{-1}$. (C,D) Ray tracing simulations of collection efficiency diagrams from a point source (λ = 520 nm) for same fibers as (B) immersed in a transparent homogeneous medium (n = 1.335) (C) or in a turbid medium (Henyey-Greenstein scattering, n = 1.360, l = 48.95 μm, g = 0.9254, T = 0.9989) (D). (E) Comparison of axial collection efficiency (x = 0, y = 0) for 0.22NA/50 μm, 0.39NA/200 μm, and 0.50NA/200 μm optical fibers at λ = 520 nm immersed in a homogeneous medium (blue curve and orange curve for analytical and numerical data, respectively) and in a turbid medium (yellow curve). The red arrows indicate the effect of light collection through the cladding. The horizontal dashed lines represent z₀.

Figure 2

Analytical and numerical estimation of collection volumes. (A) Volume iso-surfaces at different η estimated with the analytical model in transparent medium as a function of NA. Data are shown for η = 0.002, 0.005, 0.01, 0.02, 0.03 and for fibers with core/cladding diameters a/b = 200 μm/225 μm, 400 μm/425 μm (top and bottom panels, respectively). Missing data at low NA means null volume. (B) Same analysis of (A) shown for the ray-tracing model in transparent medium. The width of the curves represents the error in the volume estimation introduced by the domain discretization. (C) Same analysis of (B) shown for the ray-tracing model in a turbid medium to simulate brain tissue. Scattering was modeled with Henyey-Greenstein formulation (n = 1.360, l = 48.95 μm, g = 0.9254, T = 0.9989). The width of the curves represents the error in the volume estimation introduced by the domain discretization.

Figure 3

Measurements of light collection efficiency using 2-photon generated fluorescent point sources. (A) Schematic representation of the two-photon microscope used to measure the collection field of optical fibers in quasi-transparent fluorescent medium. The inset shows a magnification of the fiber facet surroundings. (B) Section y = 0 of the collection field of 0.22/50 μm, 0.39/200 μm, and 0.50/200 μm optical fibers, as indicated, in a 30 μM PBS:fluorescein solution, obtained through the fiber PMT as shown in (A). Isolines at 10%, 20%, 40%, 60%, and 80% of the maximum number of photons are shown (in black, blue, green, yellow, and red, respectively). (C) Analytical calculations of collection efficiency diagrams for the three fibers in (B) immersed in a transparent homogeneous medium (n = 1.335) assuming a gaussian source with lateral FWHM r_{x, z} = 3 μm, axial FWHM r_y = 32 μm. Isolines at 10%, 20%, 40%, 60%, and 80% of the maximum number of photons are shown (in black, blue, green, yellow, and red, respectively). (D) Comparison of normalized experimentally-measured (blue curve) and analytically-calculated (orange curve) axial collection efficiency profiles (x = 0, y = 0) for the same fibers in (B). Normalization is done with respect to the average of the data points within the firsts 80 μm. The horizontal dashed lines represent z₀. The width of the blue curves for the 0.39 NA/200 μm and 0.50 NA/200 μm fibers represents mean ∓ standard deviation over four different fibers.

Figure 4

Effective light collection volumes in quasi-transparent medium. (A) Cross-sectional views of the 3-dimensional reconstructions of the collection field for 0.22/50 μm, 0.39/200 μm, and 0.50/200 μm fibers in quasi-transparent solution. Iso-intensity surfaces defining the boundaries at which the number of collected photons falls to 10%, 20%, 40%, 60%, and 80% of its maximum are shown (in black, blue, green, yellow and red, respectively). The continuous and dashed circles in the xy plane represent the cladding and the core boundaries, respectively. (B) Volumes enclosed by the iso-intensity surfaces at 10%, 20%, 40%, 60%, and 80% of the maximum number of photons (left panel) and at η = 0.001, 0.002, 0.005, 0.01, 0.02, 0.03 (right panel) for 0.22/50 μm, 0.39/200 μm, and 0.50/200 μm fibers (yellow, orange, and blue curves, respectively). The dashed yellow curve represents the data for the 0.22/50 μm fiber multiplied by a factor 16 to adjust for the smaller cross-sectional area of this fiber. The width of the curves for the 0.39NA/200 μm and 0.50NA/200 μm fibers represents mean ∓ standard deviation over four different fibers. (C) Cumulative number of photons collected by the three fibers as a function of the distance from the fiber facet (left panel, number of photons are shown in a volume 900 μm × 600 μm × z) and as a function of the volume enclosed within the iso-surfaces at fixed η (right panel). The dashed yellow curve represents the data points relative to the 0.22/50 μm fiber multiplied by a factor 16. The width of the curves for the 0.39NA/200 μm and 0.50NA/200 μm fibers represents mean ∓ standard deviation over four different fibers.

Figure 5

Photon collection efficiency and effective collective volumes in brain slice. (A) Section y = 0 of the collection field of 0.39/200 μm and 0.50/200 μm optical fibers, measured in a 300 μm thick fluorescently stained brain slice using the 2-photon scanning system shown in Figure 3. Isolines at 10%, 20%, 40%, 60%, and 80% of the maximum number of photons are shown (in black, blue, green, yellow and red, respectively). (B) Comparison of normalized axial profiles (x = 0, y = 0) of experimental (in brain slices, blue curves) and numerical data (in turbid medium, orange curves) for 0.39/200 μm and 0.50/200 μm optical fibers. Normalization is done with respect to the average of the data points within the firsts 80 μm. The width of the blue curves represents mean ∓ standard deviation over four different fibers. (C) Cross-sections of the 3-dimensional reconstruction of the collection field of 0.39/200 μm and 0.50/200 μm fibers. Iso-intensity surfaces defining the boundaries at which the number of collected photons falls to 10%, 20%, 40%, 60%, and 80% of its maximum are shown (in black, blue, green, yellow, and red, respectively). The continuous and dashed circles represent the cladding and the core boundaries, respectively. (D) Volumes enclosed by the iso-intensity surfaces at 10%, 20%, 40%, 60%, and 80% of the maximum number of photons (left panel) and at η = 0.001, 0.002, 0.005, 0.01 (right panel) for 0.39/200 μm and 0.50/200 μm fibers (orange, and blue curves, respectively). The width of the curves represents mean ∓ standard deviation over three different fibers. (E) Cumulative number of photons collected by 0.39/200 μm and 0.50/200 μm fibers as a function of the distance from the fiber facet (left panel, number of photons are shown in a volume 900 μm × 600 μm × z) and as a function of the volume enclosed within the iso-surfaces at fixed η (right panel). The width of the curves represents mean ∓ standard deviation over three different fibers.

Figure 6

Scanning pinhole detection of the excitation light field for fiber optics. (A) Schematic representation of the optical path used to measure the photometry efficiency diagram of optical fibers in fluorescently stained brain slices. (B–D) Section y = 0 of the normalized light emission diagram β (B), the collection efficiency η (C) and the photometry efficiency ρ (D) of 0.39/200 μm and 0.50/200 μm optical fibers, as indicated, measured in a 300 μm thick fluorescently stained brain slice. In (D) isolines at 10%, 20%, 40%, 60%, and 80% of the maximum efficiency are shown (in black, blue, green, yellow, and red, respectively). (E) Comparison of normalized axial profiles (x = 0, y = 0) between 0.39/200 μm and 0.50/200 μm fibers for photometry efficiency (orange and blue continuous curve, respectively) and collection efficiency (orange and blue dashed curve, respectively). Normalization is done with respect to the average of the points within the firsts 80 μm. (F) Normalized photometry efficiency transversal profiles at different depths (z = 0 μm, 100 μm, 200 μm, 300 μm, y = 0) for 0.39NA/200 μm and 0.50NA/200 μm fibers (left and right panel, respectively).

Figure 7

Effective fiber photometry sampling volumes in tissue. (A) Cross-sections of the 3-dimensional reconstruction of the photometry efficiency diagram of 0.39/200 μm and 0.50/200 μm fibers (left and right, respectively). Iso-intensity surfaces at 10%, 20%, 40%, 60%, and 80% efficiency are shown (in black, blue, green, yellow, and red, respectively). The continuous and dashed circles represent the cladding and the core boundaries, respectively. (B) Volumes enclosed by the iso-intensity surfaces at 10%, 20%, 40%, 60%, and 80% of the maximum photometry efficiency (left) and at ρ = 0.001, 0.002, 0.005, 0.01 (right) for 0.39/200 μm and 0.50/200 μm fibers (orange and blue curves, respectively).