Two photon imaging system

Horizontal slices of entorhinal cortex and hippocampus were prepared from 2- to 8-month-old mice of either sex. After anesthetization with isoflurane and decapitation, brains were removed and immersed in 0°C sucrose-substituted artificial CSF (ACSF) consisting of the following (in mm): sucrose 185, KCl 2.5, NaH₂PO₄ 1.25, MgCl₂ 10, NaHCO₃ 25, glucose 12.5, and CaCl₂ 0.5. Slices were cut to a thickness of 400 µm with a vibratome (model VT1200, Leica Microsystems). Slices were then incubated at 35°C for 20 min in ACSF consisting of the following (in mm): NaCl 125, NaHCO₃ 25, d-glucose 25, KCl 2, CaCl₂ 2, NaH₂PO₄ 1.25, and MgCl₂ 1. Afterward, slices were cooled to room temperature (20°C). After the incubation period, slices were moved to the stage of a two-photon imaging system (Thorlabs) with a mode-locked Ti:Sapphire laser (Chameleon Ultra II, Coherent) set to wavelengths between 915 and 950 nm, which was used to excite both Alexa Fluor 488 and tdTomato using a 20×, numerical aperture 1.0 (Olympus) objective lens. Laser scanning was performed using resonant scanners and fluorescence was detected using two photo-multiplier tubes (Hamamatsu) equipped with red and green filters to separate emission from Alexa Fluor 488 and tdTomato. The stage of the microscope contained recirculating ASCF, with all recordings conducted between 34°C and 36°C.

To guide electrodes during patch clamp recordings, mice that express the red fluorescent protein tdTomato were used to visualize layer 2/3 excitatory pyramidal cells in somatosensory cortex. C57BL/6J background, CaMKIIa-Cre mice (The Jackson Laboratory, stock #005359; Tsien et al., 1996 (link)) were crossed with the lox-stop-lox tdTomato reporter mice (The Jackson Laboratory, stock #007914; Zariwala et al., 2011 (link)). CaMKIIa promoter in transgenic mice has been established to drive specific expression of fluorescent protein in layer 2/3 pyramidal cells in S1, with expression in ∼32% of pyramidal cells in layer 2/3 (Wang et al., 2013 (link)).
The electrode pipette was visualized using the cyan-green fluorescent dye Alexa Fluor 488 hydrazide (Thermo Fisher Scientific), which was added to the intracellular electrode solution (0.3% weight/volume). Imaging was performed using a two-photon imaging system (Thorlabs) with a mode-locked Ti:Sapphire laser (Chameleon Ultra II; Coherent) set to wavelengths between 920 nm and 950 nm, which was used to excite both the Alexa Fluor 488 and tdTomato using a 20×, NA 1.0 (Olympus) objective lens. Laser scanning was performed using resonant scanners and fluorescence was detected using two photomultiplier tubes (Hamamatsu) equipped with red and green filters to separate emission from Alexa Fluor 488 and tdTomato.