Traditional forms of microscopy used by neuroscientists have limited effectiveness for imaging intact neural tissues such as synapses. However, two-photon excitation microscopy, initially used to image dendritic spines in brain slices, has begun to enable imaging of whole tissues.

In this technique, two photons excite the specimen and cause a single fluorescent emission that is picked up by a photodetector and converted into an image. The detector reads all emissions — including scattered photons — as useful signal. With two-photon excitation microscopy, several types of fluorescent molecules can be imaged at once, which makes possible well-aligned color images.

In a primer regarding the two-photon technique, Karel Svoboda of Cold Spring Harbor Laboratory in New York and Ryohei Yasuda of Duke University Medical Center in Durham, N.C., note that the two-photon spectra can digress greatly from those predicted from single-photon excitation spectra. However, many bright one-photon fluorophores also tend to work well with two-photon systems.

They report that a blueshift has been observed in fluorophores used in two-photon experiments, as has an increase in the broadness of the spectra. No successful applications have yet been found for fluorophores synthesized specifically for two-photon excitation, although it is thought that suitable designer fluorophores could greatly increase the utility of two-photon microscopy.

Two-photon excitation microscopy also enables imaging deeper into tissue — as much as 1 mm under ideal conditions. Deep-tissue imaging with the technique permits high-resolution and high-contrast imaging.

Scientists hope to build an imaging unit using fiberlike lenses that could be attached to the head or inserted into the brain of animals to obtain deeper in vivo images in freely moving animals. It is also possible that the microscopy technique may be used to detect the activation of single neurons in the brain. (Neuron, June 15, 2006, pp. 823-839.)