Two-photon microscopy enhances neuroscience
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
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
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
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.)
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