- Video 1
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Figure 2. Three-dimensional volume rendering of microglial cells in relation to a branching blood vessel in a rat hippocampal tissue slice. The tissue was stained with FITC-IB4, a fluorescent marker of microglia and blood vessels. A through-focus stack of 93 confocal images was collected at 0.4 µm z-step intervals through a tissue depth of 37 µm. The volume was reconstructed using Voxblast (Veytek, Fairfield, IA).
- Video 2
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Figure 3. Time-lapse sequence showing contact between an axonal growth cone and a dendrite in a developing rat hippocampal slice. Neurons were labeled with a fluorescent membrane dye, DiI. To image growth of neuronal processes in three dimensions, stacks of 16 optical sections spanning 30 µm in the axial dimension (2 µm z-steps) were collected at time intervals of 6 min. Note the long, thin filopodia at the leading edge of the axonal growth cone (0 min), which advances (18 min) and bifurcates (30 min). The left branch of the growth cone contacts the adjacent dendrite (36 min), is stabilized, and the axon growth is subsequently reoriented in that direction (54 min). The images represent an axial projection of the 16 optical images in the through-focus stack. Elapsed time is shown in minutes.
- Video 3
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Figure 4. Imaging intracellular Ca2+ transients in rat brain tissue slices. Physiological time-lapse imaging reveals changing spatio-temporal patterns of intracellular calcium activity in brain tissue in response to potassium (K+) depolarization. The slice was loaded with fluo-4 AM (Molecular Probes), a membrane-permeant fluorescent indicator of intracellular calcium, and mounted in an open chamber for confocal imaging. Single image scans were collected at 7-s intervals to detect changes in fluorescence intensity, which reflect changes in intracellular calcium levels. The time-sequence shows a low baseline level of spontaneous calcium activity (0 min-1 min). After addition of 9mM potassium (K+) to the chamber, the rate of calcium activity is substantially higher (2 min; many small flashes represent calcium spikes in individual cells). Within minutes, the isolated cell activity diminishes and a new pattern of activity emerges corresponding to groups of 5-15 synchronously active cells within patches that are 100-200 µm in diameter (big flashes in upper right-hand corner at 4 min-7 min). The active cells are probably astrocytes, and the emergence of synchronously active groups of neighboring cells probably represents electrical (gap junction) coupling among astrocytes. Elapsed time is shown in minutes.
