Dynamic Imaging of Nervous System Function
This section describes the principle and application of multiple dynamic imaging techniques.
Calcium imaging of hippocampal tissue slices has been ongoing for decades, with use of multiphoton microscopy in these studies reported since the 1990s. Conventional calcium imaging uses a calcium indicator dye loaded into cells. Calcium imaging is based on change in fluorescence emission spectra (e.g., OGB-1 and fluo3) or change in absorption spectra (e.g., fura-2) of calcium indicator dyes upon calcium ion binding.
Voltage sensitive dye imaging (VSDI) monitors neuronal activity though use of voltage-sensitive fluorescent or non-fluorescent dye molecules that are inserted into the cell membrane. Changes in membrane potential are in essence changes in the electrical environment in which the voltage sensitive dye (VSD)
One of the advantages of two photon over confocal microscopy is that, since infrared light interacts less with the tissue, one can image fluorescence in deeper structures; easily 200 microns from the surface and up to 1 mm depths in some cases. Another reason why one may want to use two photon microscopy is to avoid toxicity associated with use of UV light ....