Online correction of licking-induced brain motion during two-photon imaging with a tunable lens

Abstract

Two-photon calcium imaging in awake, head-fixed animals enables the measurement of neuronal activity during behaviour. Often, licking for the retrieval of water reward is used as a measurable report of the animal's decision during reward-driven behaviour. However, licking behaviour can induce severe motion artifacts that interfere with two-photon imaging of cellular activity. Here, we describe a simple method for the online correction of licking-induced focus shifts for two-photon calcium imaging of neocortical neurons in the head-fixed mouse. We found that licking causes a stereotyped drop of neocortical tissue, shifting neurons up to 20 μm out of focus. Based on the measurement of licking with a piezo film sensor, we developed a feedback model, which provides a corrective signal for fast optical focus adjustments with an electrically tunable lens. Using online correction with this feedback model, we demonstrate a reduction of licking-related focus changes below 3 μm, minimizing motion artifact contamination of cellular calcium signals. Focus correction with a tunable lens is a simple and effective method to improve the ability to monitor neuronal activity during reward-based behaviour.

Figure 1. Licking-induced brain motion during two-photon imaging

A, schematic diagram of behaviour and microscope set-up. PMTs, photomultiplier tubes. B, schematic diagram of electrically tunable lens (ETL) and negative offset lens placement on top of the objective (top) providing remote focusing (bottom). C, in vivo images of cortical neurons expressing YC-Nano140 at different imaging depths obtained from a reference z-stack during anaesthesia (left) and during behaviour at the equivalent depths (right, averages of clustered images). Scale bar, 20 μm. D, example trial of cued licking behaviour and z-motion during the imaging time course. The measured z-position is plotted together with the voltage signal acquired from the lick sensor. Coloured dotted lines indicate the z-position corresponding to the images shown in (C). Individual licks were detected by application of a threshold (shaded red area) to the lick sensor trace. Periods of licking are indicated (grey region) together with inter-lick intervals (bottom).

Figure 3. Online correction reduces lick-induced brain z-motion

A, kernels used for motion correction. The original kernel (grey) is filtered into the resulting smoothed trace (blue) for electrically tunable lens (ETL) focusing. B, conditions during licking for triggering correction kernels. C, example of z-motion with online correction during imaging time course. Measured z-position plotted together with the applied z-correction from the resulting triggered kernels according to the detected licks. Periods of licking are indicated (grey region) with inter-lick intervals (bottom). D, average z-motion from one animal across several trials sorted according to one-bout (left) and two-bout (right) events. Trials are aligned to the first detected lick. Shaded region indicates SD. Blue trace represents kernels A and B superimposed over z-motion (n= 6 one-bout trials, n= 5 two-bout trials). E, z-correction in three different animals. Normalized histogram of z-positions during licking periods without (top) and with (bottom) online correction. Shaded region indicates ±3 μm.

Figure 2. Licking-induced brain z-motion is stereotyped

A, average licking rate from one animal for one-bout (left) and two-bout (right) events aligned to the first detected lick. Shaded region indicates SD. B, average z-motion from one animal across several trials sorted according to one-bout (left) and two-bout (right) events. Trials are aligned to the first detected lick. Shaded region indicates SD (n= 15 one-bout trials, n= 26 two-bout trials).

Figure 4. z-correction improves measurements of calcium signals

Example calcium traces from an imaged neuron: A, without correction; B, with correction. z-position (top) with coloured dotted lines indicating z-position corresponding to image of neuron (right). Periods of licking (raster plot and grey area) are shown, together with traces for changes in fluorescence intensity (ΔF/F) for cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), as well as the YFP/CFP ratio (ΔR/R) representing calcium signals. Histograms comparing the distribution of ΔF/F for CFP or YFP between licking (grey) and non-licking (red) periods are indicated. Asterisks indicate putative calcium transients. C, licking-related fluorescence changes expressed as the absolute difference in mean CFP intensity between licking and non-licking periods for trials with or without correction in three different animals. Black circles indicate the mean of individual cells shown in grey. A similar analysis for YFP intensity is shown in D (error bars = SEM) (*P < 0.05, **P < 0.01).