Two-photon voltage imaging using a genetically encoded voltage indicator

Abstract

Voltage-sensitive fluorescent proteins (VSFPs) are a family of genetically-encoded voltage indicators (GEVIs) reporting membrane voltage fluctuation from genetically-targeted cells in cell cultures to whole brains in awake mice as demonstrated earlier using 1-photon (1P) fluorescence excitation imaging. However, in-vivo 1P imaging captures optical signals only from superficial layers and does not optically resolve single neurons. Two-photon excitation (2P) imaging, on the other hand, has not yet been convincingly applied to GEVI experiments. Here we show that 2P imaging of VSFP Butterfly 1.2 expresssing pyramidal neurons in layer 2/3 reports optical membrane voltage in brain slices consistent with 1P imaging but with a 2-3 larger ΔR/R value. 2P imaging of mouse cortex in-vivo achieved cellular resolution throughout layer 2/3. In somatosensory cortex we recorded sensory responses to single whisker deflections in anesthetized mice at full frame video rate. Our results demonstrate the feasibility of GEVI-based functional 2P imaging in mouse cortex.

Figure 1. Evaluation of Butterfly 1.2-reported neuronal electrical population activity recorded in 1P and 2P excitation mode.

(a) 1P-exited mCitrine image of a coronal brain section showing cortical layer 2/3 as a bright band. (b) Color-coded fractional fluorescence response (ΔF/F₀) in response to a 5 pulse (100 Hz) stimulus applied through a stimulation electrode placed in layer 4 (red asterisk). (c) Time course of Butterfly 1.2 response taken from the region of interest marked by black circle in (b). (d) Left: 2P-excited mCitrine image of an area lined out by red frame in (a). Middle: Fractional mCitrine fluorescence in response to the same stimulus as used before. Right: Response after bath application of the GABA_A-receptor antagonist Gabazine (10 μM). (e) Time course of 2P-excited mCitrine (yellow), mKate2 (red) and mKate2/mCitrine ratio (black) signals (4 trial and 32 trial averages) corresponding to the region of interest indicated in (d) in response to a 5 pulse stimulus. (f) Same in (e) after Gabazine (10 μM) application.

Figure 2. 2P imaging of single cell voltage response in-vitro.

(a) 2P mCitrine fluorescence image of a layer 2/3 pyramidal cell in a cortical brain slice 120 μm below slice surface. (b) Time lapse series of Butterfly 1.2 fluorescence images in response to a 5 pulse (100 Hz) extracellular current stimulus applied at time zero. Upper row: Absolute differential mCitrine response (ΔF/ΔF_min) at different times after stimulus presentation. Lower row: mCitrine fluorescence image (grey) overlaid with the color-coded mKate2/mCitrine response (ΔR/R₀) of those pixels yielding a mCitrine response amplitude (ΔF) of z-score of two over mean baseline fluctuation (ΔF₀) across the whole image area. (c) Time course of the trial number 1 to 10 taken from a region of interest surrounding a pyramidal cell body (indicated yellow in a and b). (d) Trail overview showing the color-coded mKate2/mCitrine ratio signal (ΔR/R₀) of the cell body (yellow circle in a and b) of all 32 trials. (e) Time course of the mCitrine (yellow), mKate2 (red) and mKate2/mCitrine ratio in the trial average.

Figure 3. 2P imaging of the population voltage responses in the barrel cortex in-vivo.

(a) Experimental setup permitting 1P and 2P voltage imaging of head-fixed mice. (b) Macroscopic mCitrine 1P fluorescence image showing the left cortical hemisphere of an 8 weeks old mouse. (c) mCitrine 1P image of the C1 barrel cortex after cranitomy. The yellow circle marks the area covered by the spiral laser scan in 2P imaging mode. (d) mCitrine 2P image of the field as in (c) 190 μm below pia. (e) Visualization of the C1 barrel response after craniotomy in 1P whole hemisphere imaging showing the mCitrine baseline fluorescence (gray) overlaid with the color-coded mKate2/mCitrine response at different times relative to stimulation time. (f) Time course of the mCitrine (yellow), mKate2 (red) and mKate2/mCitrine ratio signals recorded from the region of interest (yellow circle) indicated in (e). (g) Time lapse series of the Butterfly 1.2-reported cortical response to C1 whisker stimulation imaged in the 1P mode. Upper row: Absolute differential mCitrine response (ΔF/ΔF_min). Lower row: mCitrine fluorescence image (grey scale) overlaid with the color-coded mKate2/mCitrine response of pixels yielding a mCitrine response amplitude (ΔF) of z-score of 1.8 over mean baseline fluctuation (ΔF₀) across the whole image area after averaging 32 trials. (h) 2P excitation recording with the same field of view as in (g). (i) Time course of 2P mKate2/mCitrine signals recorded from the region of interest comprising all color-coded pixels of the peak response shown in h (lower row). Top: Overlay of first 10 trials together with the mean (red). Bottom: Overview of all 32 trials showing the ΔR/R₀ response according to the indicated color code.

Figure 4. Tactile responses in the barrel cortex at cellular resolution.

(a) 2P image of a layer2/3 pyramidal cell expressing Butterfly 1.2 in-vivio (top) with pixels of interest (POI) marked in green (buttom). (b) Time course of the 2P mKate/mCitrine ratio (ΔR/R₀) signal in response to D1 whisker (wD1; red) and C1 whisker (wC1; black) stimulus after averaging 16 trials, with error bars representing SEM and the time of the stimulus trigger signal indicated at the bottom. D1 response marked by asterisk. (c) Another field of view from the same animal preparation showing segments of dendrites with the color-coded ratio response (ΔR/R₀) superimposed onto the mCitrine image at pixels of highest brighness selected with a z-score of 1.3 from the mCitrine image, with the time measured from the onset of the stimulus trigger signal. (d) Time course of the response to D1 whisker (red) and C1 whisker stimulation (black) with pixels colored in (c) taken as the region of interest and the time of the stimulus trigger signal indicated at the bottom. Average over 16 trials with error bars representing SEM.