3D scanning and uncaging two-photon microscopy

Two-photon (2P) microscopy is an extremely powerful technique that permits visualization and interrogation of complex neural tissue in 3D, with detailed subcellular resolution and minimal phototoxicity. When neurons receive and transform signals arising from hundreds and thousands of synaptic inputs, it is technically extremely challenging to investigate the underlying mechanisms at the necessary spatial and temporal resolution. When 2P microscopy is combined with 2P neurotransmitter uncaging, it becomes possible to precisely activate single and multiple synapses to investigate how dendrites transform synaptic inputs and how neurons and neural microcircuits process information.

This instrument will allow 2P-based structural and functional imaging and neurotransmitter uncaging in live (in vitro) brain tissue slices, combined with multi-electrode patch-clamp recording.

The setup is based on a custom-built 2P microscope (Independent Neuroscience Services, UK) with dual scan paths for resonance-galvo-galvo (RGG) and galvo-galvo scanning, epi- and transfluorescence GaAsP PMTs (Hamamatsu H11706P), fast transimpedance amplifiers (Thorlabs TIA60), and vDAQ acquisition hardware controlled by ScanImage (Vidrio Technologies / MBF Bioscience). Fast 3D scanning and uncaging are implemented by remote focussing units (RFUs) with voice coil actuators (PIMag, Physik Instrumente). 2P imaging and uncaging use a combination of a Mai Tai (eHP DS ; 690-1040 nm tuning range) and InSight X3 (680-1300 nm tuning range) lasers (SpectraPhysics).