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. 2018 Feb 13;115(7):E1374-E1383.
doi: 10.1073/pnas.1718721115. Epub 2018 Jan 29.

Wireless Optoelectronic Photometers for Monitoring Neuronal Dynamics in the Deep Brain

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Free PMC article

Wireless Optoelectronic Photometers for Monitoring Neuronal Dynamics in the Deep Brain

Luyao Lu et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Capabilities for recording neural activity in behaving mammals have greatly expanded our understanding of brain function. Some of the most sophisticated approaches use light delivered by an implanted fiber-optic cable to optically excite genetically encoded calcium indicators and to record the resulting changes in fluorescence. Physical constraints induced by the cables and the bulk, size, and weight of the associated fixtures complicate studies on natural behaviors, including social interactions and movements in environments that include obstacles, housings, and other complex features. Here, we introduce a wireless, injectable fluorescence photometer that integrates a miniaturized light source and a photodetector on a flexible, needle-shaped polymer support, suitable for injection into the deep brain at sites of interest. The ultrathin geometry and compliant mechanics of these probes allow minimally invasive implantation and stable chronic operation. In vivo studies in freely moving animals demonstrate that this technology allows high-fidelity recording of calcium fluorescence in the deep brain, with measurement characteristics that match or exceed those associated with fiber photometry systems. The resulting capabilities in optical recordings of neuronal dynamics in untethered, freely moving animals have potential for widespread applications in neuroscience research.

Keywords: neuroscience; optogenetics; photometry.

Conflict of interest statement

Conflict of interest statement: J.A.R. and M.R.B. are involved in a company, Neurolux, Inc., that offers different, but related, products to the neuroscience community.

Figures

Fig. 1.
Fig. 1.
Miniaturized, ultrathin, lightweight wireless photometry systems for deep-brain Ca2+ measurements. (A) Schematic exploded-view illustration of a wireless, injectable, ultrathin photometry probe with a µ-ILED and a µ-IPD at the tip end. (B, Left) Optical micrograph of the injectable photometry probe. The tip has a total width of ∼350 µm and a thickness of ∼150 µm. The weight is 29 mg. (B, Right) Magnified colorized SEM image of the tip (orange, PI; yellow, interconnection; blue, µ-ILED; green, µ-IPD with an optical filter). [Scale bars, 2 mm (Left) and 200 µm (Right).] (C, Upper) Schematic illustration of a GaAs µ-IPD. (Lower) SEM image of a representative µ-IPD (lateral dimensions of 100 × 100 µm2 and thickness of 5 µm). Metal electrodes are colorized in yellow. (Scale bar, 50 µm.) (D) Schematic exploded-view illustration of a transponder. (E) Photographic image of the wireless detachable transponder on fingertip. (Scale bar, 1 cm.) (F) Images of the separated transponder and injectable (Left) and the integrated system in operation (Right). The transponder is connected only during signal recording. [Scale bars, 4 mm (Left) and 8 mm (Right).] (G) Image of a freely moving mouse with a photometry system (1 wk after surgery). (Scale bar, 7 mm.) (H) Schematic illustration of the electrical operating principles of the system: Read out and control occur with a detachable wireless transponder that also facilitates signal amplification and digitalization. The signal is transmitted via an IR-LED with a modulation frequency of 38 kHz for single-transponder operation and an additional 56 kHz in dual-transponder operation. A receiver system demodulates the signal and sends the received data to a PC for data storage.
Fig. 2.
Fig. 2.
Electrical and optical properties of wireless photometry systems. (A) EQE spectra of a µ-IPD with (red) and without (black) a narrow band absorber on top. The blue and green areas highlight wavelength ranges with strong emission from the µ-ILED and fluorescence of GCaMP6f, respectively. (B) Transmission spectrum of 7-µm-thick layer of SU-8 with 1.5 wt % absorber. (C) A system demonstration with the receiver system outfitted to a cage with an enclosure inside the test area. The receivers are marked with a red box. (Scale bar, 10 cm.) (D) A standard mouse home cage outfitted with the receiver system. (Scale bar, 10 cm.) (E) Demonstration of simultaneous recordings of two wireless photometry systems, where an external green LED is turned on for 1 s at different time periods on top of gray and red traced probes, respectively. (F) Two freely moving mice each implanted with a wireless photometry system and housing for simultaneous recordings in a behavior box. (Scale bar, 7.5 cm.) (G) Measurement of fluorescence intensity in calcium indicator (excitation maximum, 494 nm; emission maximum, 523 nm) solutions with different Ca2+ concentrations (from 0.1 to 20 µM) by the fiber-optic system (Upper; gray) and the wireless photometry probe (Lower; red). (H) Comparison of fluorescence signal fluctuations over time in a 10 µM Ca2+ solution normalized to the dynamic range shown in G between the fiber (gray) and wireless system (red). (I) Wireless probe responses to simulated fluorescence with defined green light intensities from an external green light LED source. Optical intensities are 4.5 × 10−3, 1.1 × 10−2, 2.9 × 10−2, and 5.7 × 10−2 mW/cm2 for the green, blue, red, and black curves, respectively. (J) Microscopic photograph comparison of the florescence profiles (outlined in white) of a conventional fiber photometry probe (Left) and the injectable photometry probe (Right). (Scale bars, 800 µm.) (K) Normalized emission intensity profile with 1% contour for the µ-ILED as a function of depth in brain tissue. (K, Insets) Emission intensity at positions 5 and 50 µm above the encapsulated µ-ILED. (L) Spatial distribution of fluorescence captured by the µ-IPD as a function of depth in brain tissue. (L, Insets) Fluorescence intensity at positions 5 and 50 µm from the top edge (the edge closest to the µ-ILED) of encapsulated µ-IPD.
Fig. 3.
Fig. 3.
Wireless photometry systems are compatible with awake, freely behaving mice. (A) Photographic image of a mouse in behavioral arena with wireless photometry system affixed. (Scale bar, 1 cm.) (B) Cartoon of social interaction task. (C and D) Mice implanted with miniaturized, lightweight wireless photometry probes display increased social interaction time (C) and number of interaction bouts (D) compared with mice implanted and tethered using traditional fiber photometry systems (n = 10, fiber; n = 9, wireless). *P = 0.01; ***P = 0.0003 [two-tailed t test, t(17) = 4.560]. (E) Representative traces of total activity in an open field arena for mice implanted with each device. (Scale bar, 10 cm.) (F and G) Mice implanted with wireless photometry probes display increased traveled distance, a measure of activity (F; n = 12 per group), and increased time spent in the center zone, a measure of anxiety-like behavior, in the open field compared with fiber-implanted mice (G; n = 12 per group). (F) *P = 0.02 [unpaired two-tailed t test, t(22) = 2.478]. (G) *P = 0.03 [unpaired two-tailed t test, t(22) = 2.262]. (H) Schematic and confocal image of the mouse brain at the point of observation of tissue damage for lesion and inflammation measurements from wireless neural probe. (Scale bar, 2 mm.) (I) Wireless photometry probes lesion less brain tissue compared with traditional photometry probes (n = 3 per group; three slices per mouse). ****P < 0.0001 (unpaired two-tailed t test). (J) Representative linescan of fluorescence intensity of glial cell markers from traditional and wireless photometry probes. (K and L) Representative confocal fluorescence images of horizontal amygdalar slices. Minimal inflammatory glial responses occur after implantation of both the wireless and fiber photometry probes, as shown by immunohistochemical staining of astrocytes (GFAP; red) and activated microglia (Iba1; green). (Scale bars, 100 µm.)
Fig. 4.
Fig. 4.
Wireless in vivo detection of calcium transient activities in the BLA. (A, Upper) Timeline of virus injection, device implantation, and behavior test. (A, Lower) Schematic of viral delivery and expression of AAV-DJ-CaMKII-GCaMP6f (shown in green) into the BLA and wireless delivery of 473-nm light; diagram of shock delivery and cartoon for a mouse with wireless device during behavior in the shock apparatus; and free-moving mice with a wireless device in the shock box. (B) SNR was calculated as the peak dF/F divided by the SD of the baseline dF/F. There is no significant difference between the two systems [n = 13, unpaired t test, t(11) = 1.632, P = 0.1309]. (C) A sample trace showing fluorescence change before and after shock of an animal using the fiber photometry system. (D) Heatmap (Upper) for seven trails of signals recorded by fiber optics before and after shock, aligned with trace plotted as mean (curves) ± SEM (shading around curves) (Lower). Fluorescence signals are normalized for each trail. Darker colors indicate higher fluorescence signal. (E) A sample trace showing fluorescence change before and after shock of an animal using the wireless photometry system. (F) Heatmap (Upper) for eight trails of signals recorded by wireless device before and after shock, aligned with trace plotted as mean (curves) ± SEM (shading around curves) (Lower). (G) Spike events frequency in the BLA before (baseline)/after shock in signals recorded by fiber (light green)/wireless (dark green) photometry systems. Postshock signals (filled bars) have significantly higher events frequency compared with preshock signals (open bars) recorded from fiber photometry system [n = 7; paired t test, t(6) = 6.978, P = 0.0004], and wireless photometry system [n = 8; paired t test, t(7) = 6.468, P = 0.0002]. (H, Upper) Fluorescence trace showing that injection of isoproteronol i.p. activates CaMKII+ BLA populations recorded by fiber photometry system. (H, Lower) Trace shows signals recorded by saline injection as control. Zoomed-in wave shapes from the dot frame are shown by side. (I, Upper) Fluorescence trace showing injection of isoproteronol i.p. activates CaMKII+ basolateral amygdalar populations recorded by wireless photometry system. (I, Lower) Trace shows signals recorded by saline injection as control. Zoomed-in wave shapes from the dot frame are shown by side. (J) Events frequency change (Δevents per min) before/after saline (open bars)/ISO (filled bars) for signals recorded from fiber photometry system (light green) and wireless system (dark green). Signal has a significantly higher Δevents per min for ISO injection in both systems [n = 14, unpaired t test, t(14) = 4.973, P = 0.0002 for fiber photometry system; n = 12, unpaired t test, t(12) = 5.915, P < 0.0001 for wireless photometry system].

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