Uncaging
Uncaging means the activation of biochemically masked (‘caged’) molecules via photolysis, which mimics the physiological release of bioactive compounds. This technique is widely used in neuroscience, where the bioactive molecule is usually glutamate or another neurotransmitter. Using two-photon photostimulation with ultrafast pulsed IR laser light, very precise release of these compounds can be elicited in extremely small volumes. Two-photon imaging is a powerful opportunity to follow the changes evoked in dendrites or spines, even the distribution of receptors on the neurons can be investigated.
In vitro uncaging at well-defined points
FEMTOSmart Galvo equipped with a Multiple beam path extension
The accuracy of the excitation point, and the highly flexible scanning patterns, mean that the FEMTOSmart Galvo is the best choice for uncaging experiments. Multiple-point scanning (yellow points on the figure) is used for stimulation around spines, while multiple-line scanning (arrows) makes high-speed imaging along the dendrites possible. The secondary laser beam (Multiple beam path optional module), essential for the stimulation, is coupled to the existing light path. Thus the stimulation and the imaging can be performed near simultaneously using the galvo scanner. The microsecond-scale switching time between the stimulation and imaging is established by using of Pockels cell.
Specialized software functions for photostimulation mapping
Compounds for uncaging experiments
Stimulus mapping and analysis software module
This module of MES control software allows performing photostimulation mapping experiments at a range of locations and shapes. The locations can be picked manually one-by-one, along a line or in a raster, and various stimulation patterns can be selected like point, spiral, x, zigzag. It forms datasets quickly by evaluating fluorescence changes images at defined time intervals. Analysis tool for stimulation mapping can create (multichannel) map images formed by the elicited responses after a mapping experiment.
Compounds for uncaging experiments
DNI-GLU-TFA synthesized by FemtoChemistry
This dinitro-indoline-masked form of glutamate releases the bioactive glutamate more rapidly than any other commercially available compound. It was developed for high-quantum yield requiring less irradiation for release, so its effective concentration is lower than other caging scaffolds. DNI-Glu is a compound developed in-house, only available from Femtonics. See also Chiovini et al., Neuron, 2014.
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References
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