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13344251398
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- salt), and 0 1 to 0.25 mM FD (3 kD; Molecular Probes). Access resistances after break-in were 10 to 30 megohms Measurements were started 30 mm after break-in Action potentials and holding currents were monitored to assess cell viability.
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25
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0030532990
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The lasers were superimposed to submicron accuracy in the specimen plane and scanned by the same pair of galvomirrors, but they were under the control of separate shutters For experimentation at high magnification (time resolution, 2 ms per line, 8 14 μm line length), the dendrite was aligned with the scan direction with the use of an analog implementation of a rotation matrix (cos φ, sin φ. -sin φ, cos φ) acting on the scan voltages (W. Denk, unpublished data)
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spec, <200 mW; λ = 715 nm, which is close to the optimal excitation wavelength for fluorescein (C Xu and W W Webb, J. Opt Soc. B 13, 481 (1996)] The lasers were superimposed to submicron accuracy in the specimen plane and scanned by the same pair of galvomirrors, but they were under the control of separate shutters For experimentation at high magnification (time resolution, 2 ms per line, 8 14 μm line length), the dendrite was aligned with the scan direction with the use of an analog implementation of a rotation matrix (cos φ, sin φ. -sin φ, cos φ) acting on the scan voltages (W. Denk, unpublished data)
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13344287901
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note
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Single-exponential behavior is predicted for equilibration between one well-mixed compartment (the spine head) and a large reservoir (the parent dendritic shaft) that are connected by a thin constriction. Because of its small size, the spine head is well mixed by diffusion for times greater than ∼1 ms (30). Because of its large volume, the concentration in the shaft is almost unaffected by fluorophores diffusing into the head to replace those that have been bleached. Thus, when we bleached a spine, the fluorophore concentration in the adjacent shaft segment changed by ≤10%, and the change was largely attributable to bleaching from the tail of the point spread function, rather than to diffusional exchange - as was confirmed by repeating the experiment on a segment of shaft that lacked spines
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32
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13344255934
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4s) (K. Svoboda, D. W. Tank, W Denk, unpublished data) required longer exposure times (>12 ms). Although incidental in the photobleaching experiments, the longer wavelength of the monitor beam ensured that no additional release occurred during the decay period.
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34
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13344252161
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note
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h follows.
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35
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13344265132
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note
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To derive a crude estimate of spine neck length, we measured the smallest distance between the edges of the head and shaft, defined as the contour where the fluorescence intensity was one-half of the maximum. We then sorted our data into three categories according to head-shaft distance: <0 5 μm (Fig 3D), 0.5 to 1 0 μm (Fig. 3C), and >1 μm (Fig. 3B).
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36
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13344290091
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note
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o is a calibration intensity value measured inside a nearby dendritic shaft, large enough to contain most of the nonzero part of the point spread function
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38
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13344295637
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note
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3) and average spine density (1 3 versus 2.5 per micrometer of dendrite) (7, 36).
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40
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0028018010
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51
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We thank K. M. Harris, P. Mitra, and R. Yuste for helpful discussions
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We thank K. M. Harris, P. Mitra, and R. Yuste for helpful discussions
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