Supplementary Figure 1: Schematic of 2P-ISIM AO optical setup.

Excitation from a femtosecond laser is passed through intensity control and shuttering optics (1/2 λ wave plate, polarizing beam splitting cube, and electro-optic modulator, EOM), spatially filtered (pinhole, 100 μm diameter), beam expanded 5-fold (lens pair L1 and L2, f1 = 40 mm, f2 = 200 mm; and iris) and directed onto a 2D galvanometric mirror (excitation scanner). The point between each galvo surface is made conjugate to a deformable mirror, via lens pair L3 and L4 (f3, f4 = 250 mm). Lens pair L5 and L6 (f5, f6 = 200 mm) images the deformable mirror onto the back focal plane of a 1.2 NA water objective, thereby creating diffraction limited excitation at the sample. Fluorescence is collected along the same path until a dichroic mirror, placed between the excitation scanner and L3, diverts it onto a second 2D galvanometric mirror (emission scanner). Lens pair L4 and L3 images the back focal plane of the objective onto this scanner, which serves to ‘rescan’ fluorescence emission before imaging onto an electron multiplying CCD (EM-CCD) via lens L7 (f7 = 250 mm), enabling super-resolution imaging. Alternatively, for wavefront measurement, the emission scanner is set to descan the fluorescence, and a flip mirror directs this descanned signal onto a Shack Hartmann sensor. Lens pair L8 and L9 (f8 = 250 mm, f9 = 100 mm) serve to image the point between galvo surfaces in the emission scanner onto the Shack Hartman sensor, thus mapping the back focal plane of the objective onto this sensor. Note that lens pairs L1 and L2; L3 and L4; L5 and L6; and L8 and L9 are placed in 4f imaging configurations, thereby ensuring that the back focal plane of the objective, galvanometric scanners, and deformable mirror are conjugate planes. Filters F1 and F2 serve to reject excitation from reaching the EM-CCD and SHS, respectively. See Methods, Supplementary Table 2 for more detail.