Figure 2
From: Three-photon imaging of synthetic dyes in deep layers of the neocortex
Schematic of the OGB-1 AM injection method. (a) Step 1. The pipette entry point on the brain surface (red star) is determined by stereotaxic coordinates and anatomical landmarks. The surface vessels are visualized with a 40 × objective under bright-field illumination. (b) Step 2. The pipette tip is positioned directly above the entry point on the brain surface using green epi-fluorescent light after the objective is raised by 2 mm. (c) Step 3. The pipette and the objective are lowered vertically until the brain surface comes into focus. (d) Step 4. The pipette is driven down diagonally until its tip reaches the desired depth, here into cortical layer 5–6 located below layer 4 (L4). The tip is visualized throughout the travel via two-photon fluorescence of Alexa 633 under 1300 nm excitation. (e) Step 5. After dye injection and labelling of cells, the craniotomy is sealed with agarose and a 5 mm glass coverslip. The head is tilted back to horizontal orientation and a 25 × objective lens is positioned to perform three-photon imaging using 1300 nm excitation.
