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. 2017 Apr;14(4):388-390.
doi: 10.1038/nmeth.4183. Epub 2017 Feb 20.

In Vivo Three-Photon Imaging of Activity of GCaMP6-labeled Neurons Deep in Intact Mouse Brain

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In Vivo Three-Photon Imaging of Activity of GCaMP6-labeled Neurons Deep in Intact Mouse Brain

Dimitre G Ouzounov et al. Nat Methods. .
Free PMC article

Abstract

High-resolution optical imaging is critical to understanding brain function. We demonstrate that three-photon microscopy at 1,300-nm excitation enables functional imaging of GCaMP6s-labeled neurons beyond the depth limit of two-photon microscopy. We record spontaneous activity from up to 150 neurons in the hippocampal stratum pyramidale at ∼1-mm depth within an intact mouse brain. Our method creates opportunities for noninvasive recording of neuronal activity with high spatial and temporal resolution deep within scattering brain tissues.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1 |
Figure 1 |
Comparison of 2PM at 920 nm and 3PM at 1,300 nm for in vivo mouse brain imaging. (a) Near-simultaneous 2P and 3P imaging of a GCaMP6s-labeled L2/3 neuron in transgenic mouse (CamKII-tTA/tetO-GCaMP6s) cortex located 280 μm below the dura. Scale bars, 10 μm. (b) Activity traces of the neuron in a. The traces are representative of 19 recordings. (c) Measurement of signal-to-background ratio (SBR) of 2PM and 3PM by imaging fluorescein-labeled blood vessel 780 μm deep in mouse cortex (wild type, C57BL/6J). The two images have comparable numbers of signal photons, and they were displayed with the same contrast setting (top 0.2% saturation). Scale bars, 30 μm. (d) Quantitative comparison of SBR of blood vessels in c. Intensity profiles are drawn along the light yellow lines in c, averaged over 7.2-μm line thickness. SBR of 2PM approaches 1 at 780 μm depth, and SBR of 3PM is one to two orders of magnitude higher. The line profile represents similar measurements done on eight blood vessel sections from 720 to 780 μm in depth. (e) Measurement of SBR of 2PM and 3PM by imaging GCaMP6s-labeled neurons 780 μm deep in transgenic mouse cortex (CamKII-tTA/tetO-GCaMP6s). The out-of-focus fluorescence is measured inside a blood vessel, which is indicated by the “Background” arrow. Neuropil and the out-of-focus background are indicated by the “Neuropil + Background” arrow. The two images have comparable number of signal photons, and were displayed with the same contrast settings (top 0.4% saturation). Scale bars, 30 μm. (f) Quantitative comparison of SBR and neuropil of two neurons in e. Intensity profiles are drawn along the light yellow lines in e, averaged over 3.5-μm line thickness. The line profiles represent similar measurement done on 20 neurons from 720 to 780 μm in depth.
Figure 2 |
Figure 2 |
3PM of spontaneous activity in neuronal population labeled with GCaMP6s in the SP layer of the CA1 region of the mouse hippocampus. (a) 3D reconstruction of 3PM images of GCaMP6s-labeled neurons in the mouse cortex and the hippocampus (green, fluorescence; magenta, THG.). See Supplementary Video 1 for individual XY frames of the z-stack. (b) Selected XY frames at various depths in a. THG visualizes blood vessels and myelinated axons (green, fluorescence; magenta, THG). Scale bars, 50 μm. (c) Activity recording site in the SP layer of the hippocampus located at 984 μm beneath the dura with a field-of-view of 200 × 200 μm. Scale bar, 20 m. (d) Spontaneous activity recorded from the labeled neurons indicated in c. Additional traces from 30 active neurons are shown in Supplementary Figures 2–4 and Supplementary Video 2. The structural and functional imaging in this figure is representative of data from five animals.

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