The Journal of Neuroscience, March 31, 2004, ():
High-Resolution In Vivo Imaging of Hippocampal Dendrites and Spines
J. Neurosci. Mizrahi et al.
24: 3147
Supplementary material
Supplemental movies and figures
Files in this Data Supplement:
- supplemental movie 1
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Raw data Z series showing the GFP-positive dendrites and cell bodies in stratum oriens as imaged in vivo. The movie begins at the axonal sheets of the cingulum bundle and ends at the pyramidal cell body layer. Total of 49 images at 2.5 ?m Z steps (total stack depth, 120 ?m).
- supplemental movie 2
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High-magnification, raw data Z series showing that single spines are readily identified in vivo. Total of 11 images at 0.77 ?m Z steps (total stack depth, 7.7 ?m). Each image is shown twice (once in each direction).
- Supplemental Figure 1
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The surgical preparation for in vivo imaging of the hippocampus. a, The skull is exposed. b, A 2.5 mm craniotomy is opened. c, The dorsal hippocampus is exposed by aspiration of the cortex. d, A tube is inserted for mechanical support. e, The chamber is filled with agarose, covered with glass, and sealed with dental cement. dHip, Dorsal hippocampus. Scale bar, 1 mm.
- Supplemental Figure 2
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Correction of the movement artifact caused by the heartbeat. This example represents the largest movement artifact in our data set. a, Slow scanning (dwell time, 3.2 ?sec/pixel) of a dendritic branch. This slow scanning magnifies the movement artifact, which appears as a crisscross pattern within the image. b, Fast scanning (dwell time, 0.64 ?sec/pixel) of the same dendritic region as in a. Several fast scans (4-10) were used to reconstruct the morphology of the dendritic tree. c, An image of the dendritic tree shown in a and b after offline reconstruction. The result of the image processing is reduced movement artifacts and increased signal-to-noise ratio. Spines are denoted with arrows. Scale bar, 5 ?m.
- Supplemental Figure 3
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Morphology of CA1 pyramidal dendrites from fixed tissue. a, Low-magnification montage of a coronal slice from fixed tissue. GFP neuronal expression is evident at various degrees in different brain structures. Ctx, Cortex; PCL, pyramidal cell layer; DG, dentate gyrus. b, High-magnification montage (of the dotted region in a) of the CA1 pyramidal neurons. Top, Basal dendrites; bottom, apical dendrites. SO, Stratum oriens. Scale bar, 100 ?m. c, High-magnification image of CA1 basal dendrites from fixed tissue. The image was scanned with the same 40[times] (0.8 numerical aperture) water immersion objective that was used in the in vivo experiments. Scale bar, 5 ?m. d, e, Spine morphology of the boxed region in c using the 0.8 NA objective (d) and a 1.3 numerical aperture oil immersion objective (e). The images were processed in a similar way to the in vivo images and are qualitatively indistinguishable.
- Supplemental Figure 4
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Eight hours time-lapse imaging from the hippocampus of a urethane-anesthetized mouse. This representative example shows two spiny dendritic segments 8 hr apart (first imaging session, pseudocolored green; second imaging session, pseudocolored red). Each column represents five consecutive optical slices in the axial plane (1 ?m apart) for a total Z stack of 4 ?m. Despite changes in contrast levels and small x--y shifts, all spines can be identified at both time points. Scale bar, 2 ?m.
