One interesting example is the measurement and analysis of calcium release from internal stores. These increases occur in the form of waves, sparks, and puffs whose properties cannot be easily inferred from electrical measurements.12 In particular, the puffs and sparks occur at unpredictable locations, making it necessary to look over an extended region to enable the detection (Fig. 1). These events are often very fast (rise times less than 10 ms, fall times less than 70 ms) requiring a high-speed detection system and fast responding indicators, especially if the measurement of diffusion from the spark source is important. If a neuron in a slice is filled with an organic indicator from a patch electrode and the cell (or part of the cell) is not too far below the surface of the slice, then some of the advantages of two-photon systems are not critical. For example, sections of thin dendrites and axons can be examined using an ordinary microscope and a CCD camera since the neuronal processes are thinner than the depth of focus of either system. In these conditions, one advantage of a CCD detector system is that the high-speed images can be made from spatially extended structures while detection at similar speeds with a two-photon system requires that the measurements use a line scan mode, restricting signals to very small areas or straight segments. This property has been useful in the detection and measurement of sparks and puffs in dendrites13,14 and in total internal reflection fluorescence measurements in cultured neuroblastoma SH-SY5Y cells.15 A line scan across the dendrite cannot be positioned at the right location to detect a spark since that location is not known in advance. Even if that location was known, a transverse line scan would reveal neither the spatial extent of the spark or puff nor the dynamics at different locations. In principle, a scan along a dendritic segment might capture a spark and its spatial extent, but such measurements have not been reported. Video rate confocal systems have been used to follow changes in dendrites16,17 but did not detect sparks or puffs, possibly because the frame rate and sensitivity were not adequate. These considerations would not apply to the detection of a change due to an action potential that backpropagated over the dendrites [backpropagating action potential (bAP)] since these signals are almost the same at most locations and, therefore, a transverse line scan is almost certain to detect this signal and to reveal the important physiological information (when and if the spike occurred and the duration of the change). One group18 did use two-photon microscopy to detect spark-like events in presynaptic terminals, imaging small regions in the terminal at 100-ms frame interval. Detection with this slow rate was successful in part because the sparks were so long-lasting in the terminal (over 1 s, possibly because a high concentration of indicator was used, which would severely buffer and slow the transients).