Figure 2. Through-cuticle imaging of the fly brain with 2P and 3P excitation.
(A) Schematic of the multiphoton microscope setup. Fly head is fixed to a cover slip and placed under the objective (HWP, half-wave plate; PBS, polarization beam splitter; PMT, photomultiplier tube). The imaging window on the fly head is shown in the picture (lower left). Scale bar = 200 µm. (B–D) The head-uncompressed and head-compressed imaging preparations. The first column shows the side image of the fly that is head fixed to the cover glass (scale bar = 1 mm). The second and third columns show the fly head visualized under a brightfield (top view) and fluorescent dissecting microscopes (widefield-fluo), respectively. Arrows and the rectangle area in widefield-fluo column indicate the imaging window (scale bar = 200 µm). (E–G) Cross-section imaging of the mushroom body Kenyon cells expressing CD8-GFP through the head cuticle at 920 nm (2P) and 1320 nm (3P) excitation. The Z projections of 2P (cyan, left) and 3P (green, right) imaging stacks. For each imaging preparation, the same fly head is imaged with 3P and 2P excitation (scale bar = 20 µm).
Figure 2—figure supplement 1. Head compression does not affect male courtship and removal of air sacs allows 2P and 3P imaging in a head-uncompressed preparation.
