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FIGURE 5–6. Patch clamp electrophysiology and calcium imaging.

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FIGURE 5–6. Patch clamp electrophysiology and calcium imaging.By combining patch clamping with injection of selective dyes, the dynamics of calcium can be imaged in real time within isolated neurons. (A) Using infrared differential interference contrast (IR-DIC) microscopy, the image of a patch pipette can be observed attached to a neuron during a whole-cell electrophysiology experiment. Note the relatively large size of the pipette tip (coming from the left side of the image). Calibration bar = 2 m. (B) After filling the neuron with a calcium-sensitive dye, bis-Fura 2, the live neuron can be imaged with a fluorescence microscope. The dye takes about 10 minutes to fill the neuron after rupturing the patch membrane. Calibration bar = 10 m. (C) A unique property of the dye bis-Fura 2 is that it changes its fluorescence properties as it binds calcium. This can be observed by the changes in the fluorescence signal in response to a single (top) or five (bottom) action potentials. The fluorescence traces correspond to two regions, one close to the cell body (red box and red trace) and one farther out in the apical dendrite (orange box, orange trace). In this way, one can observe changes in calcium dynamics and how they correspond to activity states within single neurons.

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