Prairie technologies ultima multiphoton microscopy system

2-photon Ca²⁺ imaging experiments were performed either with a Prairie Technologies Ultima Multiphoton Microscopy System (Bruker) or with a Nikon A1R MP 2-photon scanning microscopy (Nikon), or with a Scientifica SliceScope 2-photon microscope system (Scientifica). The imaging laser’s wavelength was set at 820 nm and fluorescence was collected by a 60X objective (NA 1.0; Nikon), a 25X objective (NA 1.10; Nikon), or a 20X objective (NA 1.0; Olympus). Green (Fluo-4, 200 µM) and red (Alexa594 25 µM or TMR 100 µM) fluorescence were collected via 490–560/575/585–630 nm or 500–550/560/570–655 nm, or 500–550/565/590–650 nm filter cubes. The pre-chirped imaging laser (Chameleon Vision II, Coherent) intensity was ~9–12 mW (Prairie), ~8 mW (Nikon) and ~7 mW (Scientifica) at the surface of the specimen. Ca²⁺ signals in line-scan and frame-scan mode were acquired at a frequency of 100–300 Hz and 30 Hz (resonant scanner), respectively. Line-scan profiles illustrated in the figures were smoothed using a sliding average of five (Figures 3 and 10E) or nine points (Figures 4, 6, 9 and 10C). Pictured line-scan images were sequentially filtered with two orthogonal 1-D gaussian filters (width of 2 pixel in time, horizontal and 1 pixel in space, vertical, Figures 3, 6, 9 and 10). The ∆F/F frame-scan shown in Figure 5B (right) was filtered with a 2-D gaussian filter (width=2 pixels).

2-photon glutamate uncaging experiments were performed with Prairie Technologies Ultima Multiphoton Microscopy System (Bruker) equipped with two tunable Ti:sapphire lasers and two scan heads. The uncaging laser’s wavelength was set to 720 nm. The hippocampal slice was bathed with 5 mM MNI-glutamate (Tocris Bioscience) and the perfusion was stopped for up to 30 min. Stability of resting membrane potential, morphology and resting Ca²⁺ concentration (baseline fluorescence) were used to rule out damage to the cell potentially caused by stopping the perfusion. The pointing function of the second scan head was used to direct the uncaging laser to the target locations through the 'Triggersync' control software (Bruker). The software also controlled application of a brief laser pulse to uncage the MNI-glutamate (0.65 ms, Pockels cell modulation, intensity of ~35–45 mW at the surface of the specimen).