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Supplementary Methods for:

Two-Photon Calcium Imaging of Evoked Activity from L5 Somatosensory Neurons

in vivo.

Wolfgang Mittmann1,2

, Damian J. Wallace1, Uwe Czubayko

1, Jan T. Herb

3, Andreas T.

Schaefer3, Loren L. Looger

4, Winfried Denk

2, and Jason N. D. Kerr

1,5

1Network Imaging Group, Max Planck Institute for Biological Cybernetics,

Spemannstraße 41, 72076 Tübingen, Germany.

2Department of Biomedical Optics, Max Planck Institute for Medical Research,

Jahnstrasse 29, 69120 Heidelberg, Germany.

3Behavioural Neurophysiology, Max Planck Institute for Medical Research, Jahnstrasse

29, 69120 Heidelberg, Germany.

4Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia,

USA.

Nature Neuroscience doi:10.1038/nn.2879

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Supplementary figure legends: Supplementary Figure 1. Calcium indicator expression targeted to Layer 5 and imaged

using RAMM. (a) Two images of one section from one experiment showing GCaMP3-

loaded layer 5 neurons (left) and an overlay of the loaded neurons and DAPI-stain for the

same section (right). The DAPI staining of the section shows the loaded neurons as being

located just beneath the dense layer of cell bodies in layer 4. (b) Two-photon microscope

image (side projection) from a z-stack (2 m steps). L5 neurons labeled with GCaMP3

(green) and superficial astrocytes labeled with SR101 (red) in an anesthetized mouse.

Note that the deep-layer red is most likely due to fluorescence from bright astrocyte

labeling restricted to superficial layers.

Supplementary Figure 2. Functional calcium imaging at 800 m below pia

(a) MP image of layer 5 neuronal somata at 800 m below the pia. Ca2+ traces from the

outlined neurons are shown in b.

Supplementary Figure 3. So/Su ratios for 162 neurons as a function of depth of the

neuronal soma. Individual neurons (black circles) and mean for all neurons with a single

field of view (red bar) are shown. Numbered neurons highlighted in red at 680 m

indicated in Fig 2a, and those at 740 m indicated in Fig. 2b.

Nature Neuroscience doi:10.1038/nn.2879

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Supplementary Figure 4. Simultaneously imaging activity from large populations of L5

dendrites. (a) MP image of GCaMP3 labeled apical dendrites and L2/3 somas taken at

240 m below the pia. (b) 80 Ca2+ traces simultaneously recorded from dendrites shown

in a.

Supplementary Figure 5. Dendritic transients from thin tufted L5 neuron

(a) y-z side projection from a z-stack taken at 2 micron steps showing populations of L5

neurons and ascending apical dendrites containing GCaMP3 calcium indicator (b) with x-

y frames from three different depths indicated (white dashed lines on y-z projection) and

(c) location of soma (arrow). (d) Spontaneous calcium transients recorded from apical

dendrites at different depths from pia (indicated in a, b). (e) Reconstructed apical

dendrite showing common source of dendritic activity shown in d with corresponding

depths of transients depicted (dashed colored lines).

Supplementary Movie Z-stack from pia to layer 5b

Z-stack movie containing 256 x 256 pix el images, single channel, 2 mi cron steps, 500

images starting at the pial surface. L5 neurons (green) expressing the calcium indicator

GCaMP3.

Nature Neuroscience doi:10.1038/nn.2879

Supplementary Figure 1Mittmann_Wallace_Czubayko_Herb_Schaefer_Looger_Denk_Kerr

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Nature Neuroscience doi:10.1038/nn.2879

800 µm below pia

Supplementary Figure 2

Mittmann_Wallace_Czubayko_Herb_Schaefer_Looger_Denk_Kerr

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Nature Neuroscience doi:10.1038/nn.2879

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Supplementary Figure 3Mittmann_Wallace_Czubayko_Herb_Schaefer_Looger_Denk_Kerr

Nature Neuroscience doi:10.1038/nn.2879

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Nature Neuroscience doi:10.1038/nn.2879

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Nature Neuroscience doi:10.1038/nn.2879