Research ArticleOPTICS

Deep tissue optical focusing and optogenetic modulation with time-reversed ultrasonically encoded light

See allHide authors and affiliations

Science Advances  08 Dec 2017:
Vol. 3, no. 12, eaao5520
DOI: 10.1126/sciadv.aao5520
  • Fig. 1 Custom TRUE focusing and electrophysiological recording system.

    The custom TRUE focusing system combined a DOPC system with a patch clamp electrophysiology amplifier and headstage. Acute brain slices were held in a custom perfusion chamber that contained warmed, carbogenated aCSF. The TRUE light beam illuminated the tissue at an oblique 45° angle, and the borosilicate patch pipette electrode was used for neurophysiological measurements. (A) A DIC microscope was included for neuron visualization during patch clamping. (B) The TRUE focusing system allowed light to be sharply focused through the brain slice. (C) A close-up image of the TRUE focus on a patched neuron. Scale bars, 20 μm in (A) and 50 μm in (B).

  • Fig. 2 A comparison of TRUE focusing and conventional focusing.

    (A) The recording (A1) and playback (A2) procedures used to focus light through the slice onto its top surface with TRUE focusing. (B) Diagram of the experimental setup for measuring the light intensity distribution of the focus on the top surface of the brain slice achieved using a conventional lens illuminating the brain slice from below. A tube lens and a camera used together with the objective are not shown. (C) Images of the conventional and TRUE focus profile through living brain tissue slices (500, 800, 1000, 1500, and 2000 μm thick). (D) Full width at half maximum (FWHM) focal spot sizes for the conventional and TRUE foci as a function of tissue thickness. Error bars represent the SD of five measurements taken at different locations. Scale bar, 100 μm.

  • Fig. 3 Experiment design, opsin characterization, and demonstration of photocurrent and firing modulation via TRUE focusing.

    (A) An AAV vector was used to stereotaxically deliver the bReaChES transgene to the mPFC. (B) Characterization of normalized photocurrent response versus light intensity. The average maximum photocurrent across the 10 cells studied was 1047 pA. (C) Diagram illustrating the experimental scheme used to demonstrate the ability of TRUE focusing to elicit action potentials through 800-μm-thick living mouse brain tissue. (D) Representative traces demonstrating elicited photocurrent and membrane voltage changes achieved with and without TRUE focusing.

  • Fig. 4 Spatial resolution of optogenetic stimulation achieved by conventional versus TRUE focusing.

    Experimental configuration for photocurrent scan map generation using conventional focusing (A) and TRUE focusing (B). The normalized photocurrent enhancement as a function of lateral focal scanning position for conventional (C) and TRUE focusing (D). Scale bar, 100 μm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/12/eaao5520/DC1

    method S1. Calculation of the focal spot size of TRUE and conventional focusing.

    method S2. Viral injection surgery.

    method S3. Electrophysiological recordings.

    method S4. Daily alignment procedure.

    fig. S1. Setup.

    fig. S2. Electrical signal flow diagram.

    fig. S3. Ultrasound pulse-echo image of the tip of the glass pipette electrode.

    fig. S4. Electrophysiological photocurrent traces from neurons in 500- and 300-μm-thick acute brain slices.

    fig. S5. Electrophysiological photocurrent and membrane voltage traces comparing ultrasound on and off conditions.

  • Supplementary Materials

    This PDF file includes:

    • method S1. Calculation of the focal spot size of TRUE and conventional focusing.
    • method S2. Viral injection surgery.
    • method S3. Electrophysiological recordings.
    • method S4. Daily alignment procedure.
    • fig. S1. Setup.
    • fig. S2. Electrical signal flow diagram.
    • fig. S3. Ultrasound pulse-echo image of the tip of the glass pipette electrode.
    • fig. S4. Electrophysiological photocurrent traces from neurons in 500- and 300-μm-thick acute brain slices.
    • fig. S5. Electrophysiological photocurrent and membrane voltage traces comparing ultrasound on and off conditions.

    Download PDF

    Files in this Data Supplement:

Navigate This Article