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Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain


Two-photon calcium imaging provides an optical readout of neuronal activity in populations of neurons with subcellular resolution. However, conventional two-photon imaging systems are limited in their field of view to 1 mm2, precluding the visualization of multiple cortical areas simultaneously. Here, we demonstrate a two-photon microscope with an expanded field of view (>9.5 mm2) for rapidly reconfigurable simultaneous scanning of widely separated populations of neurons. We custom designed and assembled an optimized scan engine, objective, and two independently positionable, temporally multiplexed excitation pathways. We used this new microscope to measure activity correlations between two cortical visual areas in mice during visual processing.

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Figure 1: Treapn2p system layout.
Figure 2: Focal excitation PSF profile of the Trepan2p system.
Figure 3: Two-photon imaging of neural activity across 9.6 mm2 of mouse cortex with single neuron resolution.
Figure 4: Temporally multiplexed, independently repositionable imaging pathways for simultaneous scanning two regions.


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We are grateful to Hongkui Zeng (Allen Institute) for kindly providing transgenic mice, Kei Eto (UNC-CH) for providing mice for imaging in pilot experiments, Janet Berrios (UNC-CH) for providing the YFP labeled brain section, Sally Kim and Ben Philpot (UNC-CH) for providing the Thy1-GFP O-line mouse, and Zemax LLC for providing upgraded software. This work was supported by the following: the National Institute of Child Health and Human Development (T32-HD40127) and Burroughs Wellcome Fund Career Award at the Scientific Interface (J.N.S.); a Helen Lyng White Fellowship (I.T.S.); a Career Development Award from the Human Frontier Science Program (00063/2012), and grants from the National Science Foundation (1450824), the Whitehall Foundation, the McKnight Foundation, the Klingenstein Foundation, the Simons Foundation (SCGB 325407SS), and the NIH (R01NS091335, R01EY024294) (S.L.S.). Custom-made image acquisition software (adapted by J.N.S.) is from code kindly provided by David Ferster, formerly of Northwestern University.

Author information

Authors and Affiliations



S.L.S. conceived the Trepan2p imaging system. J.N.S. and S.L.S. designed and engineered the system. J.N.S. developed the demultiplexing electronics, designed and optimized the optical systems, wrote the software, and constructed the system. M.W.K. consulted on optical optimizations. J.N.S., I.T.S., and S.L.S. performed the animal experiments. J.N.S. and S.L.S. analyzed and interpreted the data. J.N.S. and S.L.S. wrote the manuscript with input from all authors. I.T.S. and S.L.S. supervised the project.

Corresponding author

Correspondence to Spencer L Smith.

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Competing interests

UNC-Chapel Hill has filed a patent on portions of this instrumentation. The patent is not licensed. S.L.S. has a consulting relationship with two companies that have expressed interest in the technology described in this paper.

Integrated supplementary information

Supplementary Figure 1 Multiplexing and demultiplexing

(a) In the multiplexing scheme, laser pulses are split into two pathways, and Pathway 2 is delayed. The 6.25 ns delay in pathway 2 results in perfectly interleaved pulses upon recombination. Both pathways have different deflection angles (Ω1 or Ω2) on the galvanometer scanning mirrors, as dictated by SM1 and SM2. (b) High bandwidth electronics are used for demultiplexing and photon counting. (c) PMT pulses are equally divided into two pathways. The second pathway is time delayed by 6.25 ns. A veto pulse width of 6.25 ns is applied to the two detection pathways resulting in two demultiplexed output pulse streams. The veto signal prevents counting, and is active during the low state (i.e., when the veto signal is high, counting is allowed). These events are digitized either with a counter or a high speed digitizer (ADC) and assembled into images (Online Methods).

Supplementary Figure 2 Crosstalk is minimal between the two pathways

(a) When measuring the crosstalk between the multiplexed pathways, the dynamic changes in the fluorescence intensity of GCaMP6s complicates analysis. Thus, we imaged neurons expressing YFP in an acute brain slice. Neurons were (top) imaged with Pathway 1 while blocking excitation in Pathway 2, and (bottom) vice versa. Any signal measured in the blocked pathway is due to crosstalk from the unblocked pathway. Crosstalk was measured with decreasing PMT gain and increasing levels of input power (maintaining constant signal amplitude. (b) Mean fluorescent intensity of individual ROIs (somas) between the two pathways was measured, and decreased for decreasing gain and increasing laser power (Online Methods). For the in vivo imaging described in this study, we used a gain setting of ~0.6 - 0.65 and laser power between 150 - 200 mW per pathway, which indicates < 5% crosstalk between pathways.

Supplementary Figure 3 Full prescription data for the steering mirror (SM) to X scanning mirror (SM-X) relay

The optical relay was constructed from commercial off-the-shelf (COTS) components and was designed to minimize aberrations at high scan angles. The polarizing beam splitter (PBS) was offset from the focal point of the relay to minimize photo-damage to the optical cement. The red outlined axial separations are used as compensators in system assembly.

Supplementary Figure 4 Full prescription data for the X scanning mirror (SM-X) to Y scanning mirror (SM-Y) relay

The optical relay was constructed from COTS components and was designed to minimize aberrations at high scan angles. The red outlined axial separations are used as compensators in system assembly.

Supplementary Figure 5 Full prescription data for the scan lens and tube lens optical subsystem

The scan and tube lens subsystem was constructed from COTS components. The terminal lens in this system has a 60 mm diameter in order to minimize vignetting at the extreme scan angles. The red outlined axial separations are used as compensators in system assembly.

Supplementary Figure 6 Full prescription data for the objective lens

The objective lens was constructed from COTS components and two custom cemented achromats. The objective has ~8.5 mm of working distance. The axial separation on surface 76 and decenter of custom lens 1 (red outline) are used as a compensators in the objective assembly. Focus compensation occurs at surface 83.

Supplementary Figure 7 Simulations of the Trepan2p optical performance and tolerance analysis

(a) The Trepan2p system was optimized for diffraction limited performance across a ~4 mm FOV in the simulated design and sufficient performance with in-house assembly. (b) The simulated tangential and sagittal field curvatures are less than 20 µm. The experimental field curvature is shown and was found by measuring the optimal Z focus of submicron beads. (c) F-theta distortions are minimal. The field curvature and F-theta distortion were sufficiently small to permit measurements of neural activity, and the minimal wavefront error was a major design criterion to ensure effective 2p excitation. (d) Monte carlo simulations (2500) for the tolerances given in the main text with no compensators in the objective (blue) and with all compensators (green). The value of the merit function is a combination of the mean RMS wavefront error across the entire FOV and the vignetting in the system. Shown are the merit function values for the give quantile for the two distributions. The nominal merit function value for a perfectly assembled system (Supplementary Figs. 3, 4, 5, 6) is 0.04730. Systems with similar excitation PSF measurements to our experimental measurements (Fig. 2) are in the range shown by the red area.

Supplementary Figure 8 Structural imaging with the Trepan2p

(a) Imaging across a wide range of depths: apical dendritic tufts (left) and somas (right) of Layer V pyramidal neurons (Thy1-GFP, line O) are resolved in vivo (Supplementary Video 2). (b) Dendritic spines in the apical tuft dendrites of Layer V cells (Thy1-GFP, line O) are visible at high magnification through the custom objective.

Supplementary Figure 9 Simultaneous imaging with two different excitation PSF

(a) Using a Nikon 16x (NA = 0.8) objective and under-filling the back aperture using Pathway 1 and over-filling the back aperture using Pathway 2, we controlled the axial excitation PSF (Online Methods). The lower resolution pathway was set to have a larger excitation PSF (14.0 µm) than our custom objective (~12 µm) for a conservative analysis. The higher resolution pathway's excitation PSF is 4.4 µm. (b) The same neurons in V1 were simultaneously imaged using both pathways. A slight XY offset (~40 µm, which is > than a cell body width) was used to ensure that crosstalk between the temporally multiplexed beams, however miniscule, would not influence the measurement. (c) Example traces (randomly selected) are shown from both the low resolution pathway (blue) and high resolution pathway (orange). The traces have been normalized to a maximum of 100% ΔF/F for display. The peak value for each neuron's raw ΔF/F trace was 58% ± 40% (mean ± SD) greater in the high resolution pathway. (d) The correlation between traces of identical cells imaged using both pathways is calculated. N = 456 cells over 5 different imaging regions.

Supplementary Figure 10 Additional example traces from the analysis in Supplementary Figure 9

Additional, randomly selected, normalized ΔF/F traces from the dual excitation PSF (14.0 µm and 4.4 µm) experiment. Traces were randomly selected from 456 cell recordings. The peak value for each neuron's raw ΔF/F trace was 58% ± 40% (mean ± SD) greater in the high resolution pathway.

Supplementary Figure 11 Identification of cortical areas across imaging modalities

Intrinsic signal optical imaging (ISOI) is a hemodynamic modality in which the reflectance of light off of the brain is modulated hemodynamic changes, which are in turn linked to stimulus-driven neural activity. ISOI can be used to map cortical activation at a resolution sufficient to delineate functional cortical area boundaries, and can be registered to maps of cortical vasculature (vessel image). (a) A vessel image was acquired prior to intrinsic signal optical imaging (ISOI) to map V1 and HVAs using retinotopic maps evoked by a single drifting (b) vertical (to map the azimuth of the cortical retinotopic maps in V1 and HVAs) or (c) horizontal bars (to map the elevation of the cortical retinotopic maps in V1 and HVAs). Phase of the evoked response is encoded in color and ranges from –π to +π, which corresponds to the two sides of the monitor (top and bottom when mapping elevation, or left and right when mapping azimuth). (d) These maps are used to locate the boundaries of V1 and HVAs. (e) These boundary lines were overlaid on the vessel map. The vasculature is imaged both in (f) the ISOI camera (the white line denotes the 3.5 mm FOV) and (g) the 2p imaging, and thus the vasculature is used to register between the two imaging modalities.

Supplementary Figure 12 Magnified sub-regions from 3.5 mm field of view

(a) The maximum projections and (b) ROI maps inlayed in Figure 3b,c have been enlarged for visibility.

Supplementary Figure 13 Fast imaging cycle times across a large field of view using arbitrary line scan in awake animals

(a) Line scan acquired at 96.1 Hz in an awake mouse expressing GFP in layer 5 neurons (Thy1-GFP, line O). The fluorescent structures in the left panel are ascending dendritic trunks from layer 5 neurons imaged at ~270 µm. Shown in red is the line scan path with the starting point and direction labeled in green. The open blue circles show the location of the traces shown in the right panel. The right panel is ΔF/F traces from ten structures in the line scan. Movement state of the mouse was tracked with simultaneous video monitoring and displayed above the traces. (b) Line scan acquired at 28.7 Hz in an awake mouse expressing GCaMP6s at a depth of ~210 µm. Shown in red is the line scan path with the starting point and direction labeled in green. The closed blue circles show the location of the traces shown in the right panel. The right panel are ΔF/F traces from ten cells in the line scan. (c) Fluorescent transients (ΔF/F) from 172 active neurons along the scan path in (b). Note that the amplitude of GCaMP6s signals is typically larger than the movement-induced transients in panel (a).

Supplementary Figure 14 Dual depth imaging across a 2.5 mm field of view

The majority of the primary and higher visual areas are contained in a 2.5 mm field of view (Supplementary Video 7). Both imaging pathways were utilized to image (0.8 frames/s) the field of view at different focal planes (DZ = -55 µm). Imaging depth was 245 µm for Pathway 1 and 190 µm for Pathway 2. Left: maximum projection of the time series; right: binary image showing active ROIs.

Supplementary Figure 15 Power transmission through the Trepan2p

After attenuating the overall power using the first polarizing beam splitting cube, the power at subsequent locations in the Trepan2p microscope was measured. The second half-wave plate was adjusted such that the final power out of the front of the objective was identical between the two pathways. The larger losses in Pathway 2 are due to the additional expansion (and subsequent clipping) the beam undergoes as it travels the additional 1.87 m path length. Overall, the system transmits 41% (20.5% per path) of the initial power.

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Stirman, J., Smith, I., Kudenov, M. et al. Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain. Nat Biotechnol 34, 857–862 (2016).

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