Introduction

Uncaging means activation of biochemically masked (’caged’) molecules via photolysis that mimics physiological release of bioactive compounds. This technique is widely used in the neuroscience where the caged molecule is usually neurotransmitter. Using two-photon excitation, precise release can be elicited in miniature volumes and uncaging is a powerful opportunity to investigate the distribution of receptors on the neurons or activate dendrites or spines.

PHOTOSTIMULATION AND IMAGING

Photolysis of caged glutamate can be performed with 720 nm ultrafast pulsed laser light. This stimulating laser beam is coupled to the existing light path with a dichroic mirror, and it is used simultaneously by the imaging IR laser. The light path is optimized for both wavelengths thanks for the full optical engineering. The stimulation and imaging can be performed along various patterns, which flexibility is established by galvanometer based scanner (see below). The imaging is interleaved with uncaging periods when the scanner jumps to the locations. The microsecond-scale switching time between the stimulation and imaging is established by using of Pockels cell.

Figure: Photostimulation of the MNI-Glu-TFA close to the dendritic segment of a hippocampal interneuron. The near simultaneous stimulation and the imaging are accomplished by point and line scanning, respectively. The stimulation is performed in the distributed points, and the evoked changes is followed along a line covering the dendrite. Ca2+ responses along the selected dendritic segment are analyzed as the ∆F/F ratio of OGB-1 fluorescence.

FemtoS-Galvo equipped with Multiple beam module

Uncaging in well-defined points of a specimen

The accuracy of the excitation point and the high flexible scanning patterns enables the FemtoS-Galvo equipped by Multiple beam module to be the best choice for performing uncaging experiments. Multiple point scanning performed by galvo scanner allows stimulation in points around dendrites or even around spines, multiple line scanning along the dendrite allows following the evoked changes with high speed and high SNR. (Femto2D-Galvo equipped with multiple beam module is still an existing opportunity.)

The accuracy of the uncaging and imaging is established by the followings:

  • the stimulating laser is focused on femtoliter excitation volume
  • the galvanometric scanner ensures microsecond-precision
  • MES control software allows flexible spatiotemporal scanning

Stimulus mapping and analysis software module

Specialized functions for photostimulation mapping

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.

References

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Spine neck plasticity regulates compartmentalization of synapses J Tønnesen, G Katona, B Rozsa, U V Nägerl, Nature Neuroscience(2014)

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