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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
500 µm
200 µm
a b
Nature Neuroscience doi:10.1038/nn.2879
800 µm below pia
Supplementary Figure 2
Mittmann_Wallace_Czubayko_Herb_Schaefer_Looger_Denk_Kerr
10 %∆F/F
10 s
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Nature Neuroscience doi:10.1038/nn.2879
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ratio
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Supplementary Figure 3Mittmann_Wallace_Czubayko_Herb_Schaefer_Looger_Denk_Kerr
Nature Neuroscience doi:10.1038/nn.2879
50 %∆F/F0
50 s
Supplementery Figure 4
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Nature Neuroscience doi:10.1038/nn.2879
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Supplementery Figure 5
Mittmann_Wallace_Czubayko_Herb_Schaefer_Looger_Denk_Kerr
Nature Neuroscience doi:10.1038/nn.2879